Absolute Quantification Experiment Using Exicycler 96

Transcription

1 Exicycler 96 Real Time Quantitative Thermal Block Absolute Quantification Experiment Using Exicycler 96 Exicycler 96 Real Time Quantitative Thermal Block User s Guide

2 Warranty and Liability All Bioneer products are tested under extensive Quality Control procedures. Bioneer guarantees the quality under the warranty period. Any problems should be reported immediately. Liability is conditional upon the customer providing full details of the problem to Bioneer. Once the problem occurrs, customers must report to Bioneer within 30 days. QC Testing Each lot of Bioneer s product is carefully tested by the quality control team. Notice Patent Pending Exicycler is a trademark of Bioneer Corporation. Certain applications of this product are covered by pending or issued patents in certain countries. Because purchase of this product does not include a license to perform any patented application, users of this product may be required to obtain a patent license depending upon the particular application and country in which the product is used. For more information, please contact Legal manager, Bioneer Corporation, 49-3, Munpyeong-dong, Daedeok-gu, Daejeon , Republic of Korea. Appearance and specification are subjected to change without notice. Copyright Feb by Bioneer Corporation. All rights reserved. Rev

3 Table of Contents Foreword... 2 How to use The Manual... 2 Suggestion on How to Use Manual... 2 Requirements for Proper Use... 2 Method of Expressing the Sentence... 2 Explanation on Caution and Warning... 2 For Safe Use... 3 To get more Information... 3 Information for Service and Support... 3 Introduction and Detection Chemistry... 4 Introduction... 5 Terminology used in Real Time Quantitative Analysis and Absolute Quantification... 6 Additional Requirement for Absolute Quantification Assay... 8 Detection Chemistry... 9 TaqMan Probe... 9 Intercalating Dye Molecular Beacons Design of Primer and Probe for Absolute Quantification Design of Primer and Probe Usage of Exicycler Primer 3 Software or Primer and Probe Design Tool Consideration in The Design of Primer and Probe for Absolute Quantification Guidelines in the design of primer and probe Absolute Quantification (AQ) Reverse Transcription Reaction for Absolute Quantification Experiment One- or Two-step RT-PCR Products that can be used for Two-step RT-PCR RNA Preparation Products that can be used for RNA Extraction RNA Purity and Requirement for Reverse Transcription Reaction cdna Synthesis for Absolute Quantification Amplification Reaction Reverse Transcription Reaction Preparation Reaction Temperature and Time for Reverse Transcription Reaction Absolute Quantification Experiment Method and Reaction Plate Preparation Absolute Quantification Absolute Quantification Experiment Selection of PCR Method for Absolute Quantification Absolute Quantification Experiment Design... 31

4 Preparation of Plate Information for Absolute Quantification Configuration of The Element Required for The Preparation of Plate Information Mixing of Reaction Element to be used in Absolute Quantification How to use Exicycler Run Program for Absolute Quantification Experiment Execution of Exicycler TM Run Program How to use Exicycler Analysis Program for the Analysis of Absolute Quantification File Open for Absolute Quantitative Analysis Fluorescence Graph (Flu. Graph) Tap Screen STD Curve Tap Screen Melting Graph Tap Screen Plate Info Tap Screen Well vs. Ct Tap Screen Report Form Tap Screen Data Conversion of Absolute Quantitative Analysis Result Appendix 1. How to Set Baseline and Threshold Baseline and Threshold Setting Automatic Setting of Baseline and Threshold Manual Setting of Baseline and Threshold... 82

5 Foreword How to use The Manual Suggestion on How to Use Manual This manual was made to help the researchers in their preparation of Absolute Quantification experiment using Exicycler 96 when they want to quantify the copy number of a virus, bacteria, or etc. Therefore, this manual has been prepared assuming that the user/researcher intending to use Exicycler 96 has fundamental knowledge in handling DNA and RNA samples Nucleic Acid Amplification Reaction (PCR). Requirements for Proper Use To properly use this manual, the user has to: Have enough skills in handling the operating system such as Windows XP. Have fundamental knowledge in handling DNA and RNA samples for PCR. Be familiar with the general use of Hard Drives including saving, copying, pasting, etc. Have experience on network setting method and usage in order to utilize the data obtained from Exicycler 96 through the network such as computer system, etc.. Method of Expressing the Sentence The letters that should be directly entered or selected by the user were expressed in bold type. For example, enter Test and then click OK button to move to the following stage. The sentences that have important meaning or to be emphasized were expressed in italics. For example, whenever an analysis is made, it is saved in a new name. The selection of the menu used in S/W and the menu to go directly were expressed in continuous movement using bold letters accompanied by > symbol. For example, File> Config> Scan. Explanation on Caution and Warning Caution and Warning used below appear across Exicycler 96 and the manual. Meaning of each represents the following, which require a certain level of concern and care. Reference: It provides the contents that may be helpful or relevant, but does not constitute any significant matter in the use of the product. Important: It provides necessary information on product operation, kit usage, or safe use of the samples such as buffer, etc. Danger: It shows potentially dangerous situation, but in relatively minimal state. It is intended to remind the user of non-safe/dangerous operation. 2

6 Warning: It indicates potentially hazardous state, especially when it may cause death or severe injury when problem occurs. For Safe Use Please refer to the manual on Exicycler 96 Real Time PCR Installation and Maintenance To get more Information To obtain additional information on Exicycler 96 System, please visit the Bioneer website ( or Information for Service and Support Bioneer uses a nationwide telephone number ( ) to minimize inconvenience to customers, as well as to provide quick A/S service for the product. In addition, the Bioneer website ( or facilitates a Q&A board so that the customers requirements and questions may be addressed. 3

7 Exicycler 96 Real Time Quantitative Thermal Block Introduction and Detection Chemistry 4

8 Introduction The Exicycler 96 Real-Time Quantitative Thermal Block combines a thermal block in a standard 96-well format and an innovative fluorescence detector. The detector monitors the fluorescence emitted as an indicator of amplified nucleic acid product during each PCR cycle in real time. The thermal block is built in the lower part of the Exicycler 96, which carries out a thermal cycling. The detector is located in the upper part of the Exicycler 96, which measures the fluorescence emitted from samples in the thermal block in real time and transfers data to the computer for analysis. Data transferred from the detector is analyzed with the Exicycler Analysis Software. The Exicycler 96 Real-Time Quantitative Thermal Block provides a maximum ramping rate of 2.5 /sec, and features various funtions such as gradient, time increment, temperature increment, and ramp rate control for myriad applications. The Exicycler 96 Real-Time Quantitative Thermal Block detector consists of an light source part and a detection part. The light source part is an energy source to excite fluorescent dyes. A short arc lamp that has a long lifetime is bright, and is implemented. A white-light source is divided into particular wavelength groups by band path filters. The band path filters that are set in the Exicycler 96 covers within 480 to 680 nm and works as a set consisting of an excitation and an emission filter. Five of the filter sets are provided for various applications. Therefore, adding additional band path filters is not required. The detector, a hight sensitive 2D CCD camera, provides high sensitivity and detects fluorescence signals from a 96-well plate in the thermal block all at one time. This simultaneous detection has a great advantage over sequential detection. The innovative detector invented by Bioneer has also reduced well-to-well variation and has minimized dye-to-dye interference, therefore providing more accurate results. Bioneer s state-of-the-art technologies applied to the Exicycler 96 are to generate a uniform light surface over the thermal block, to detect multiple fluorescent signals emitted from various dyes through the light surface, and to separate the signals within a selective wavelength range of each dye. The Exicycler 96 Real-Time Quantitative Thermal Block features an auto loading function for automation reducing errors and Self-diagnosis for diagnosing systematical conditions of the Exicycer 96 for users convenience. The Exicycler 96 Software is composed of three main programs. 1) A set up program for calibration, diagnosis and confirmation of the instrument s information. 2) An operation program for creating a protocol, assigning a probe and plate, saving & displaying data while operating the Exicycer 96. 3) An analysis program for data analysis. The analysis program includes Absolute Quantification, Relative Quantification, Multi-Relative Quantification, SNP genotyping, Existence/ Nonexistence. Analysis and is applicable to Gene expression, Quantification of cell and virus, and SNP genotyping. 5

9 Terminology used in Real Time Quantitative Analysis and Absolute Quantification Baseline: Of Nucleic Acid Amplification Reaction, the initial cycle that occurs with actual amplification reaction but with rare change in fluorescence value measured. Threshold: A certain level of fluorescence value that is used to determine Ct value from the result of real time gene amplification. It is necessary to designate this threshold having equal fluorescence value for both unknown sample and standard sample to determine Ct value from the result of real time Nucleic Acid Amplification. Exicycler 96 software either automatically or manually sets the threshold. In general, such threshold is set higher than the level of fluorescence value that falls under the baseline. On the contrary, it should be located at far lower level in the exponential phase instead of plateau phase on Nucleic Acid Amplification curve. Threshold Cycle (Ct): PCR cycle that falls under the threshold as mentioned above in the fluorescence amplification curve. Target sequence or target template: Subject nucleotide sequence to be detected or quantified. Endogenous control: It refers to a specific Nucleic Acid that measures the expression level. In case of Relative Quantification experiment, it means the Nucleic Acid that is consistently maintained regardless of the external physical and chemical treatments. Generally the housekeeping gene is used, but other genes having constant expression level can also be used. Reference dye: Fluorescent dye for internal reference that is used when analyzing the result obtained from the reporter dye. Such reference dye is used to correct the difference of real time Nucleic Acid reaction system in its surface light source. It can also be used to reduce error of the fluorescence value that appears in the volume or concentration of the reaction sample, but it may include error arising from the correction of fluorescence value. Exicycler 96 Real-Time Quantitative Thermal Block utilizing the Light Pipe technology produces homogeneous two-dimensional surface light source where no correction with reference dye is required. Therefore, it excludes error arising from the correction of fluorescence value using the reference dye. Reporter dye: Fluorescent dye that is used to show in fluorescence size the volume of nucleic acid amplified through Nucleic Acid Amplification Reaction. In case of TaqMan probe, fluorescent dye attached to 5 falls under the reporter dye. Delta F ( F): The increment of fluorescent value created from a certain Nucleic Acid Amplification Reaction (df= F baseline). Standard sample: Sample with its concentration known that is used to construct the standard curve. Unknown sample: Sample that is used to judge whether a certain sequence is included or not, or to quantify the target sequence. Negative control (NTC): Sample used to find out if contamination occurs during the preparation of 6

10 quantitative experiment. It includes all the elements to amplify the target Nucleic Acid, but excludes template. Negative control is used to judge the validity of the Nucleic Acid Amplification result. In this case, fluorescence value should not be detected from quantitative experiment result. IPC (Internal Positive Control): Probe and primer as a pair are added to the construction of reaction monitoring the PCR process. When such test result comes out negative, IPC is used to check whether it arises from the failure of PCR reaction or not. IPC-: Sample that is used to find out whether the reaction of IPC sample is normal or not (blocked IPC). The following graph represents the concept of the terminology used in quantitative analysis through the figure of Nucleic Acid Amplification curve. Fluorescence (F) F max Sample Delta F Threshold Baseline F min Ct 7

11 Additional Requirement for Absolute Quantification Assay cdna synthesis Kit TaqMan PCR Premix Optical 96 Well Plate Optical sealing film Labeled Primer and Probes Microcentrifuge Tube Centrifuge Microcentrifuge Disposable glove Nuclease-free water Pipette tip with filter plugs Pipette Vortexer 8

12 Detection Chemistry It is necessary to select chemistry that will be used in fluorescence detection. Exicycler 96 supports a variety of detection chemistries that can detect Nucleic Acid Amplification Reaction in real time basis. Above all, three typical methods are explained below. For the detection chemistry where TaqMan probe or SYBR Green I is used, one or two-step RT- PCR method can be adopted. TaqMan Probe In the test where TaqMan probe is used, pair of primers and an internal oligo is required. A pair of primers is designed to include the range to be amplified, while TaqMan probe is designed to be combined with the part amplified. TaqMan probe method takes advantage of 5 3 nuclease activation of DNA Polymerase where Taq polymerase is adopted in general. TaqMan probe is designed in the shape of dual labeled oligo with reporter dye (5 ) and quencher dye (3 ) attached at both ends. For the reporter dye, FAM, JOE, TET, Texas Red, Cy3, Cy5, etc. are used, while for quencher dye, TAMRA, DABCYL and BHQ (Black hole quencher) are used. The combination of reporter dye and quencher dye should be determined depending on the range of excitation/emission wavelength for the dye selected. (Reporter dye emission wavelength Quencher dye excitation wavelength) Before the amplification reaction starts, the reporter dyes of 5 and 3 are located near the quencher dye, and therefore the fluorescence does not appear because the reporter dye fluorescence is absorbed by quencher dye. Nevertheless, upon Taq polymerase causing the extension reaction, the probe is cut away, and separation starts between reporter dye and quencher dye. The reporter dye in free state veers away from Quencher dye, then, shows fluorescence, which gradually increases following the repetition of amplification reaction. Therefore, in case of TaqMan probe method, fluorescence should be measured at the extension step. TaqMan probe is combined with target sequence in specific form, which can be applied to gene expression analysis, virus infection and quantitative tests, etc. 9

13 Denaturation F Q Annealing Taq F Q Extension F Q F Q Taq Note: The following are the advantages when adopting TaqMan probe: Increases probe specificity. Multiplex is available. Optimization experiment (optimized quantification) is available. Uses 5 -nuclease feature out of PCR reaction. 10

14 Intercalating Dye SYBR Green I (Molecular Probes Inc.) is normally used to perform real time Nucleic Acid Amplification Reaction using the characteristic of intercalating dye. When SYBR Green I is combined with the minor groove of DNA double helix strand, fluorescence increases by approximately 200 times. For this reason, as Nucleic Acid Amplification Reaction is carried out, the fluorescence of SYBR Green increases consistently that enables quantitative analysis. Because of such Intercalating dye, any other probe design is not necessary. That is, where SYBR Green I is added to the existing Nucleic Acid Amplification test, the real time Nucleic Acid Amplification Reaction can be tested in simple manner. Nevertheless, SYBR Green I can be combined with all DNA having double helix strand such as primer dimer, non-specific band, secondary structure, as well as amplified products. Therefore, the fault of this is to separate amplified products from non-specific amplified products through a melting curve to avoid test error. SYBR Green I is sold in 10,000, and DMSO (Dimethyl sulfoxide) is used if diluted into 1,000 and 100. In case of 1 working solution, it should be diluted with 5% DMSO (Dimethyl sulfoxide) or 10mM Tris buffer (7.5~8.0) before use. SYBR Green I and DMSO are harmful to the human body, and therefore the test should be performed by wearing vinyl gloves at all times during the test. Denaturation F F F F Annealing F Taq F F F F Extension F F F Taq Note: The following are the advantages when adopting SYBR Green I dye: The entire double helix strand DNA can be amplified. After PCR reaction, the characteristic of the amplified products can be identified through the analysis of a melting curve. The sensitivity enables the detection of amplified products by adjusting the length of PCR products. 11

15 Note: The problems that may occur when using SYBR Green I dye are the following: SYBR Green I tends to be combined with double helix DNA in a non-specific manner. To solve the problem arising from non-specific products, melting curve or agarose gel analysis is necessary. 12

16 Molecular Beacons The molecular beacon method is similar to TaqMan probe method. However, its shape of internal oligo is slightly different from TaqMan probe. Molecular beacons, like TaqMan probe, are designed in the shape of dual labeled oligo with reporter dye and quencher dye attached to 5 and 3 respectively. Nevertheless, unlike TaqMan probe, it should be designed into stem-loop structure by adding arm to the internal oligo. In case the molecular beacon probe is in hairpin structure, reporter dye and quencher dye in 5 and 3 are located in the vicinity, so fluorescence does not appear. When the probe is combined with target sequence at the annealing step, hairpin structure is released where both ends are split away from each other, and then reporter dye shows fluorescence. Thereafter, when the probe is split but not cut away at the extension step, it becomes a hairpin structure again, where fluorescence does not appear. In case of Molecular beacon method, unlike the fluorescence of reporter dye cut away from TaqMan probe being accumulated and increased, fluorescence increases with the change in probe structure repeated. Molecular beacon probes adopt reporter dye similar to TaqMan probes. Quencher dye discharges heat instead of fluorescence where Black hole quencher (BHQ) or DABCYL that has less noise. Combination of reporter dye and quencher dye depends on the range of excitation/emission wavelength of the dye selected. (Reporter dye emission wavelength Quencher dye excitation wavelength) For the molecular beacon probe, its probe design is a little bit complicated, but a variety of application tests such as gene type analysis and SNP (single nucleotide polymorphism) using a melting curve can be carried out. Denaturation F Q Annealing Taq F Q Extension Taq F Q 13

17 Note: The following are the advantages when adopting the molecular beacon probe: Increases probe specificity. Multiplex is available. Optimization experiment (optimized quantification) is available. The characteristic of amplified products through melting analysis after PCR reaction is identified. 14

18 Exicycler 96 Real Time Quantitative Thermal Block Design of Primer and Probe for Absolute Quantification 16

19 Design of Primer and Probe Bioneer recommends the following methods to design the probe and primer that fall under the subject sequence to be amplified. Usage of Exicycler Primer 3 Software or Primer and Probe Design Tool It provides the function of helping the design of primer and probe for gene analysis. There is an existing design software commercially sold, but the primer design tool included in Bioneer homepage bioinformatics tool can be used free of charge as it was developed based on Primer 3 open source. Such design tools have different design algorithms, and therefore it is advantageous to acquire a certain result to use the same software if possible, in case of designing several types of primer and probe. 17

20 Consideration in The Design of Primer and Probe for Absolute Quantification When using exclusive software for designing primer and probe, it is recommended to use the parameter requested in the software as it is. Nevertheless, please refer to the following for the parameter values required to be changed according to the experiment purpose. Furthermore, even in case of selecting SYBR Green I method for its detection chemistry, it is recommended to design both primer and probe (ex, TaqMan ) for use. It is because when changing detection chemistry in the future, the probe can only be used in combination that would be an excellent method of increasing specificity. In this case, the primer selected can be properly used in both SYBR Green I experiment condition and TaqMan probe experiment condition. Guidelines in the design of primer and probe Size of Amplified products The Size of Amplified products is selected by the length of 50~150 base pair. Amplified products in small sizes are advantageous to obtain the amplification with high efficiency. As the size of amplified products gets larger, its efficiency becomes lower. Therefore, it also can be applied to Existence/Nonexistence Assay or SNP Genotyping Assay, but cannot be applied to quantitative experiment such as relative quantification. In case that the size of amplified products cannot be reduced to 150 base pair or less, relative quantification method can be applied. G/C content The primer and probe are designed with the sequence of G/C ratio in the range of 30~80% selected. Furthermore, it is designed in such a manner that the G base may not appear in four or more consecutively. If those with high G/C ratio are selected of target sequence, amplification efficiency becomes less during thermal cycling. Furthermore, as the sequence with high G/C ratio is likely to generate non-specific reaction, there is a small probability that a problem may occur when SYBR Green I experiment method is selected. Melting Temperature (Tm) Melting temperature (Tm) is used as a thermal cycling parameter when designing primer or probe. Tm of the primer is designed depending on the condition to be tested in general. When designing the other relevant primer with Tm used as the same parameter, however, it is advantageous to maintain experiment conditions in a consistent manner. In addition, it is possible that the primer designed with same Tm value can be tested under the same thermal cycling condition. For this purpose, it is generally designed so that Tm value of the primer should be at 58~60 degrees and the probe be approximately 10 degrees more than Tm of the primer. 18

21 Probe sequence At the 5 end of the probe with the reporter dye attached, the G base should not be located. The quenching effect of G base on this position still remains after the probe is cut away. This causes reduction of the level of fluorescence value detected from reporter dye, which can also influence the result of the analysis. Primers sequence When designing the primer, two or more C or G bases should not be located within five (5) bases at 3 end. It may cause the increase of the probability of non-specific products being constructed. It is recommended to design G/C ratio at 3 should not be so high. Miscellaneous When selecting the probe for detection chemistry, the position of probe is recommended to be in the vicinity of primer. When using TaqMan probe, such design can maximize the activation of 5 nuclease. It should be designed in such a manner that avoids the repetition of equal sequence or the order where the same base is being repeated. If it is difficult to design the probe and primer that fall under the target sequence, or trouble arises from the design experience, please contact Bioneer s Customer Call Center to place an order for probe and primer together with target sequence. 19

22 Exicycler 96 Real Time Quantitative Thermal Block Absolute Quantification (AQ) 20

23 Absolute Quantification Workflow Exicycler 96 Real Time Introduction Quantitative Thermal Block Absolute Quantification Chapter 1 Experimental Design Chapter 2 Reaction Plate Preparation Chapter 3 Exicycler Operation Chapter 4 Real Time Amplification Chapter 5 Analysis Exicycler Data Chapter 6 21

24 Exicycler 96 Real Time Quantitative Thermal Block Reverse Transcription Reaction for Absolute Quantification Experiment 22

25 One- or Two-step RT-PCR Real time Nucleic Acid Amplification can be divided into two methods: the One-Step Reaction where reverse transcription reaction and Nucleic Acid Amplification Reaction are carried out in the same reaction unit; and Two-Step Reaction where they are carried out separately. Sample is varied depending on whether it will be done in one-step or two-step reaction. Two-step RT-PCR reaction constitutes the process where the entire RNAs are produced into cdna through the reverse transcription reaction, and then the process of Nucleic Acid Amplification (PCR) where they are amplified using cdna. This method is used for the purposes of detecting several transcripts using single cdna template, and of performing later experiment by storing the combined cdna. This two-step RT-PCR reaction has also advantage in that UNG (uracil N-glycosylase) 효소 can be applied to minimize the trouble arising from contamination. Therefore, the explanation related to absolute quantification will be carried out based on two-step reaction. Sample Tissue RNA qpcr Mixture qpcr Amplification Results RT Reaction One-step RT-PCR is the method where reverse transcription reaction and PCR reaction are carried out under the same buffer condition. Its advantage is that the reaction can be prepared in an easy and convenient manner as the same reaction unit can be used. However, its disadvantage is that it cannot remove the contaminant introduced externally/from the outside using enzyme such as UNG (uracil N-glycosylase). RT Mixture Sample Tissue RNA qpcr Mixture RT Reaction qpcr Amplification Results 23

26 Products that can be used for Two-step RT-PCR AccuPower RT PreMix (K-2041) AccuPower DualStar TM qpcr PreMix (K-6100, 96 reaction, 8-well strip, 20 ul reaction) AccuPower DualStar TM qpcr PreMix (K-6110, 96 reaction, 8-well strip, 50 ul reaction) AccuPower DualStar TM qpcr PreMix (K-6103, 96 reaction, 96-well plate, 20 ul reaction) AccuPower DualStar TM qpcr PreMix (K-6113, 96 reaction, 96-well plate, 50 ul reaction) AccuPower Greenstar qpcr PreMix (K-6200, 96 reaction, 8-well strip, 50 ul reaction) AccuPower Greenstar qpcr PreMix (K-6203, 96 reaction, 96-well plate, 50 ul reaction) AccuPower Greenstar qpcr PreMix (K-6210, 96 reaction, 8-well strip, 20 ul reaction) AccuPower Greenstar qpcr PreMix (K-6213, 96 reaction, 96-well plate, 20 ul reaction) 24

27 RNA Preparation Bioneer provides the product (kit) that extracts RNA from a variety of tissues, microbes, viruses, plants, blood, etc. Products that can be used for RNA Extraction Tissue RNA PrepMate TM (K-3080) Viral RNA PrepMate TM (K-3060) Blood RNA PrepMate TM (K-3070) AccuPrep Viral RNA Extraction Kit (K-3033) RNA Purity and Requirement for Reverse Transcription Reaction RNA used for Absolute Quantification experiment requires A 260/280 to be 1.8 or more and should be shown in normal pattern when electrophoresis is carried out in agarose gel (ex, Total RNA). When extracting RNA from blood, etc., special attention is required so that no material that may block reverse transcription reaction or PCR reaction is included. In case of AccuPower RT PreMix (K-2041) sold by Bioneer, total RNA per cdna tube can be used up to 0.5 ~ 1 ug. It is necessary to use sufficient amount of RNA in order to synthesize cdna required for Nucleic Acid Amplification Reaction. The amount of RNA required for synthesis and the amount of cdna synthesized depend on the kit used in reverse transcription reaction. Please refer to the manufacturer s manual for further information. 25

28 cdna Synthesis for Absolute Quantification Amplification Reaction In case of cdna synthesis for absolute quantification reaction, and for reverse transcription reaction condition, please refer to the manual included in the kit used. In this manual, explanation will be made based on AccuPower RT PreMix (K-2041) sold by Bioneer. The following are prepared based on AccuPower RT PreMix (K-2041) manual that basically covers the method of synthesizing cdna using total RNA. Please refer to the following process to conduct the reverse transcription reaction that is the first phase of two-step RT-PCR method. Reverse Transcription Reaction Preparation For reverse transcription reaction, RNA and primer should be mixed as shown in the table below: Concentration of template RNA and primer Reaction volume 20 ul reaction 50 ul reaction Template RNA Primer Total RNA ug ug RNA Poly(A) RNA ug ug Oligo dt ug (100 pmoles) 1.0 ug (200 pmoles) Sequence Specific pmoles pmoles Mixture in liquid type should be left at 70 C for five (5) minutes, and then moved into ice. Then, mixture should be moved into AccuPower RT PreMix (K-2041) where DEPC-DW to be filled up based on the amount of reaction. As AccuPower RT PreMix (K-2041) is supplied in frozen and dried state (pellet formed at the bottom of tube), pellet should be completely melted prior to the following process. Reaction Temperature and Time for Reverse Transcription Reaction Reaction temperature and time is the condition used when reverse transcription reaction is carried out with AccuPower RT PreMix (K-2041). Reverse transcription reaction condition depends on the product used. Therefore, when using the other reverse transcription reaction kit, please refer to the manufacturer s manual to change reaction temperature and time. 42 C, 60 min. (cdna Synthesis) 94 C, 5 min. (RTase Inactivation ) 26

29 After the reverse transcription reaction, the following process (Nucleic Acid Amplification Reaction) should be carried out using the cdna 2~5 ul synthesized. For processing nucleic acid amplification reaction, please refer to the following chapter. The cdna synthesized like this can be used immediately for Nucleic Acid Amplification Reaction, but can be stored under the temperature of -15 ~ -25 for the next experiment. To minimize the problem arising from repeated freeze-thaw of cdna, the synthesized cdna should be stored separately. 27

30 Exicycler 96 Real Time Quantitative Thermal Block Absolute Quantification Experiment Method and Reaction Plate Preparation 28

31 Absolute Quantification Absolute quantification means a series of processes where quantification is made in absolute volume of single sequence existing within unknown sample. Absolute quantification is carried out using real time Nucleic Acid Amplification Reaction unit (real time PCR), where PCR process can be read through real time Nucleic Acid Amplification. In addition, quantification result can be observed during the process of Nucleic Acid Amplification Reaction (PCR). In real time Nucleic Acid Amplification reaction, the absolute quantification is conducted in such a manner that the degree of nucleic acid amplification is measured at every Nucleic Acid Amplification Reaction cycle. Accordingly, in real time Nucleic Acid Amplification, all the results of reaction are analyzed at the time when the amplified products can be measured for the first time during the cycle. 29

32 Absolute Quantification Experiment Absolute quantification experiment consists of the initial step where cdna is produced from DNA or RNA, the subject matter of absolute quantification (reverse transcription), the step where target sequence is amplified using real time Nucleic Acid Amplification unit, and the step where the result of absolute quantification is analyzed and completed. In this absolute quantification inspection method, target sequence included in unknown sample is calculated in quantitative manner using standard curve. Therefore, the result from absolute quantification experiment is analyzed with the same standard as that used in the standard curve applied. Exicycler 96 system saves the entire test data obtained from absolute quantification reaction in a file type in the name of *.ex3. file, which includes a variety of information such as tested time, protocol information, plate information, temperature graph, etc. Accordingly, one can get the same result when sending out the data file only with it copied or analyzing it in another computer. Data file can be analyzed in a variety of types through Exicycler 96 analysis software. 30

33 Selection of PCR Method for Absolute Quantification In the absolute quantification experiment, general singleplex PCR method is basically used. Accordingly, a pair of primers is used only for Nucleic Acid Amplification Reaction, where for the measurement of fluorescence, TaqMan Probe or SYBR Green I is included. This Chapter mainly covers the explanation on the requirement and factors for absolute quantification experiment. Absolute Quantification Experiment Design It is one of the very important processes to properly arrange the factors that constitute the design and absolute quantification of the test to obtain the result of Absolute Quantification experiment on the plate. It does not matter if the sample is in small quantity, but in order to obtain a consistent result when the number of the sample increases or from repeated tests, it is necessary to minimize the occurrence of the problem that may arise from the same work. For the absolute quantification experiment, the information of reaction plate prepared becomes the basis of the plate information for use of Exicycler, and the result of absolute quantification is analyzed based on the plate information. Absolute quantification experiment should be designed in order to obtain result from at least three repeated tests if you intend to obtain statistical significance, as well as exact result. For the correct statistical result in the analysis, it is recommended to design absolute quantification experiment after determining the number of the desired samples subjected to statistical analysis. Absolute quantification experiment can be designed with a single test if necessary, but it may reduce the accuracy of the analyzed result. Preparation of Plate Information for Absolute Quantification Plate information is used to save the basic information required for Absolute Quantification experiment that is adopted to construct the reaction plate. In absolute quantification, the plate information should be constructed with standard sample and unknown sample, negative sample, etc. In case of standard sample, information of quantitative value or copy number should be prepared. In addition, the plate information file should include the name of sample and the information on the kind and position of the detecting probe used in the real time Nucleic Acid Amplification Reaction. Plate information is the essential element required for the analysis of result in the real time 31

34 Nucleic Acid Amplification Reaction. If the plate information is incorrectly prepared, the analysis of the Absolute Quantification experiment result may be incorrect or even no result could come out. Configuration of The Element Required for The Preparation of Plate Information Detecting probe: Required to enter the probe information that is to be used to detect the real time gene amplification reaction. Enter the information of the reporter used on the basis of detection chemistry selected in the real time gene amplification reaction. Unknown sample: Enter the information of the well where the sample, which has the desired sequence to be quantified, is included. Negative sample: Enter the information of the well where the sample, which has all the elements used in the real time Nucleic Acid Amplification Reaction except the target sequence, is included. Sample name: Enter the information on the name of the sample against the desired sequence. Standard sample (STD): The standard sample includes the target sequence, and the correct volume should be known. In the absolute quantification experiment, the standard sample should be included against the target sequence in the desired reaction plate to be tested. 32

35 Absolute Quantification Reaction Preparation Absolute quantification reaction plate can be prepared in the following design: The following absolute quantification reaction plate, as an example of the reaction plate for absolute quantification, is not to be absolutely used, but can be used according to the needs of the user. Standard Unknown Samples The factors required for absolute quantification should be arrayed on the reaction plate as above. The above example was intended for two repeated tests for four standard samples, and designed to conduct four repeated tests to quantify the unknown sample. According to the needs of the user, the frequency of repeated tests and the number of standard samples that can be used with it may decrease or increase. In case of designing the reaction plate as above (four repeated tests), you can obtain quantification result for a total of four samples. (In case of four times of repetition for four samples, a total of 40 wells are required). The following table shows the information on the position and concentration of the standard sample and unknown sample used in the example of absolute quantification experiment. No Well Position Probe Type Concentration Sample Name 1 D3, D4 SYBR_AQ STD 1,000,000,000 std1 2 D5, D6 SYBR_AQ STD 100,000,000 std2 3 D7, D8 SYBR_AQ STD 10,000,000 std3 4 D9, D10 SYBR_AQ STD 1,000,000 std4 5 E3 ~ E6 SYBR_AQ Sample Unknown Sample1 6 E7 ~ E10 SYBR_AQ Sample Unknown Sample2 7 F3 ~ F6 SYBR_AQ Sample Unknown Sample3 8 F7 ~ F10 SYBR_AQ Sample Unknown Sample4 When the array of the factors required for absolute quantification reaction plate as above is determined, the plate information should be prepared according to the plate information preparation method. 33

36 Mixing of Reaction Element to be used in Absolute Quantification For the synthesis of real time Nucleic Acid Amplification Reaction where the prepared cdna or target sequence DNA is used, please refer to the manual included in the kit. This manual is prepared based on AccuPower Greenstar TM qpcr PreMix (K-6210) being supplied by Bioneer. The following shows the method where real time Nucleic Acid Amplification Reaction is conducted using DNA (template DNA) where target sequence is included, or cdna prepared through reverse transcription process, which is based on the manual for AccuPower Greenstar TM qpcr PreMix (K-6210). Please conduct Nucleic Acid Amplification process, the second step of RT-PCR method, by referring to the following steps: 1. Mix cdna (template DNA), primer, DW, etc. with AccuPower Greenstar TM qpcr PreMix (K- 6210) under the following conditions: Reaction components in AccuPower Greenstar TM qpcr PreMix (K-6210) Components Volume/ reaction Final concentration Forward primer Variable 0.5 um Reverse primer Variable 0.5 um Nuclease-free water Variable Template DNA 5 ~ 10 ul 500 ng/reaction Total volume 20 ul 2. Completely stir the mixed liquid as above, and then divide it into the reaction containers (plate or tube). 3. On the proper location of the well where reaction mixed liquid is contained, divide with plate DNA (template DNA) to 500 ng/reaction, provided that such division should be done so that each element can be arrayed properly in the designed position for absolute quantification experiment. 4. When conducting synthesis through two-step RT-PCR, the amount of raw cdna liquid (volume) should not exceed 10% of the entire reaction liquid used in Nucleic Acid Amplification Process. 5. Prepare the protocol information and plate information required for real time Nucleic Acid Amplification reaction referring to the method of using Exicycler TM 96 Run software for Absolute Quantification experiment. Temperature condition for the Nucleic Acid Amplification Test to be used for Protocol information is as follows: 34

37 Exicycler TM 96 conditions Step Time Temp. Additional comments Initial activation step 15 min 95 C HotStart Taq DNA Polymerase is activated by this heating step. 3-step cycling Denaturation: ~1 min 95 C Annealing: ~1 min 58~60 C Approximately 5 C below Tm of primers. Extension: ~1 min 72 C Scan - Hold Data acquisition Cycle number 30 or more Final extension 10 min 72 C Hold temperature (if required) 1 min 60 C Hold 60 C for starting melting analysis. Melting curve (if required) 1~ 10 sec. hold per steps 60~95 C Increase temperature with 1.0 C or below steps. Re-annealing (optional) 2 min 25 C Re-annealing is important if the samples are to be analyzed on an agarose gel 6. Place the reaction plate or tube prepared in Exicycler TM 96 block on the relevant well, and perform the real time Nucleic Acid Amplification Reaction. 35

38 Exicycler 96 Real Time Quantitative Thermal Block How to use Exicycler Run Program for Absolute Quantification Experiment 36

39 Execution of Exicycler TM Run Program 1) Turn on the main power switch of Exicycler TM 96, and press the POWER button on the front panel to start Self Test. Upon completion of Self Test, perform Exicycler Run program after ensuring that LED at the front panel changes into green. When Exicycler Run program is being executed, the software starts checking the status of the system. 2) When Exicycler TM 96 is in normal condition, it creates a pop up window as shown on the right. Important: The program starts checking the system when it is being executed. In this case, if the power of Exicycler 96 is off, it displays the following message on the screen. When such message is shown, please power on the Exicycler 96 product and try to run the program again. When the following message is shown, we cannot ensure the ordinary operation of Exicycler 96. Important: When the following message is shown after running the program, it is the case where there is no calibration data. It may occur in case of using the product without any calibration data after purchase, or calibration data is lost because of careless use. When the following message is shown, immediately stop using the program and implement the calibration first. Important: When the program is run, the following message box may be shown. The following warning message appears when the system is not properly turned off or the lamp is turned off abnormally because of power failure. In this case, special attention is required as the lamp life may be reduced, resulting to shortened lifespan of the device. 37

40 3) When Exicycler 96 is in normal condition, it is changed into the main screen of the program as shown below. The method of preparation based on the protocol in the example file Sample_AQ1.ex3 is as follows: b. Experiemt Information a. menu c. Protocol Information d. Well Information e. Temperature Profile a. Menu: You can run it by selecting the relevant menu such as File, Setup, Run, Window, Help, etc. b. Experiment Information: When performing the test, you can verify each file name, user name, running time, and closing time. c. Protocol Information: After producing the protocol suitable for each test, you can verify the protocol when conducting its execution. d. Well information: You can verify how the sample or type is designated in 96 wells. e. Temperature Profile: It shows the test protocol designed by the user in graphical format, and then you can verify which step of the protocols designed by the user is under progress. 4) In order to create the probe designed by the user, click Setup > Probe at the top menu. 38

41 Important: When Exicycler Run software is installed, nine (9) probes are installed by default. Probe functions to designate which fluorescent dye and quencher to be used in the probe designed by the user according to the target DNA to be detected by the user. In the analysis software after the test, all the calculations are made in the unit of probe, and therefore, user is required to create the probe designed by him/her using this menu. When registering the probe, the user is required to register only once when conducting the test using Exicycler 96. When the user conducts repeated tests using such probe, the user does not need to re-register until the relevant probe is deleted. 5) The probe list that can be currently used is displayed as follows. In order for user to prepare it anew, please click the Add button. 6) Assume the name of the probe that is to be used by the user as SYBR_AQ. Important: Enter SYBR_AQ in the Name edit box. As SYBR_Green will be used in Dye edit box, please select the relevant fluorescent dye from the combo box. In case of SYBR_Green, select None from the Quencher combo box according to the kind of quencher. Color to be expressed is determined in the graph according to the probe created by the user. This is designated by the relevant edit box being clicked as the user pleases. Upon completion of such setting, click OK button. 39

42 7) Be sure that the probe designated by the user is created in the Probe Information window, and close the window after clicking Save button 8) Click File > Design Experiment menu to prepare Protocol information; then the Quick Start sub-screen appears. a. Master, Protocol, Plate b. User c. Protocol Information d. Temperature Profile e. Protocol setting a. Master, Protocol, Plate: To prepare the name of each file to save them. b. User: To select the User, prepare the file for the relevant user, and manage them. c. Protocol Information: To show the protocol designed by the user by stages. 40

43 d. Temperature Profile: To show the temperature of the test protocol in graph type e. Protocol setting: To enter the step of test protocol. 9) Click Incubate tab, enter the set temperature (ex, 94.0) in Temperature item, and then enter the desired time to be reacted (ex, 10 min). a. Incubate: Designate the temperature and time maintained in the thermal block. Time Increment: Option that causes the increase as much as the time designated for the time temperature being maintained at every cycle. Temperature Increment: Option that causes the increase of the set temperature as much as designated at every cycle. Ramping Rate: Option that controls the change in the speed of temperature in the thermal block. b. Scan: Measures the fluorescence occurring from the sample c. Goto: Designates the frequency of repetition as well as starting position of the repeated cycle Melting: Protocol that is used to verify the nonspecific band when SYBR Green I dye is used, where the time to be maintained can be designated according to the starting and closing temperature, increment of temperature, and every temperature. d. Gradient: Means to seek the optimized annealing temperature, which is useful for the tester at the beginning stage to seek experiment conditions. e. Store: Function that is used for saving the sample upon completion of the test. It is not recommended to be used when the weather is humid, for instance during the rainy season, as the moisture may solidify. f. Pause: Used when the user intends to proceed to the next step after conducting a specific activity such as verification of sample during the test process. In case of Exicycler 96, if the user inserts Pause function, the door is automatically opened, and stands by until the user presses the Run ( ) button. g. Hottop: Used to set the temperature of hot top that contacts the tube cap or sealing film and its usage. 41

44 Caution Scan condition tab Used to measure the fluorescence in the Optical Zone, it is generally present at a certain temperature condition. When measuring the fluorescence, it is measured with the set temperature of the former step maintained. It takes approximately 25 seconds to measure. Thermal cycling Condition should be fixed in consideration of the scan time being added (approx 25 sec.). In case the scan step is excluded from experiment conditions display window, be careful that general thermal cycling is carried out only when fluorescence data is not collected. Gradient condition tab Reaction temperature and time can be entered. In case setting the temperature at both ends of each row, the temperature calculated in order in each row is applied. The function of the Gradient is used to optimize the temperature at the annealing step. Enter the minimum and maximum temperatures to be set as the gradient in From and To. The time where the gradient to be applied should be entered In Time, the available range of entry is 1~99:59:59. Melting condition tab Used to measure the change in the fluorescence value according to the change in the temperature. Click Melting condition tab, then the details can be set. Enter, in From and To, the desired temperature to start melting analysis and the desired temperature to close melting analysis. In the Between item, enter the temperature interval to be measured. The set range is 0.5~10. Enter, in Time item, the time when the interval of temperature to be maintained applied in the Between item. The set range is 1~99:59:59. - When Intercalating dye is used, it is possible to verify whether it is amplified product, primer dimmer or non-specific band. If TaqMan probe is used, no melting analysis is necessary Store condition tab It is used to maintain the temperature for safe storage of the sample by setting 96 well thermal blocks at a certain temperature after the completion of PCR reaction. When Store starts, the hot lid is automatically turned off, and then it maintains the set temperature until it is stopped when the STOP button is pressed. In case Store condition (step) is not included in the experiment conditions display window, if thermal cycling is completed, the 96 well thermal blocks are automatically opened. But if Store condition (Step) is included, Exicycler 96 maintains the set temperature before the STOP button is pressed for its stoppage (96 well thermal blocks are not automatically opened). 42

45 Hottop check box In case heating or cooling 96 well thermal blocks, it functions as the button to set the part of hot top that contacts tube cap or sealing film. In case the check box is not selected, the hot top is not heated. The basic value is 100, and the set range is 90~ ) Click Append button to add protocol step in the experiment conditions display window. a. Append button adds the step designated by the user b. Update button is used when the user revises the protocol and updates it. c. Delete button is used when the specific step of the protocol is erased. 11) Click Incubate tab to add the temperature condition, and then click Append button. If necessary, repeat the process of No. 9 through 10 (ex: sec, sec). Note: If there was an error in the temperature experiment condition entry in the display window, the relevant experiment condition can still be revised. Just click the update button. 43

46 12) Click Scan tab at the temperature where the fluorescence value is to be measured, and then click Append button to add it on the experiment conditions display window. Note: If shown as the following figure, the scan temperature reaches 60. To scan another temperature, it is necessary to scan after inserting the relevant temperature step before scanning. 13) Click Goto tab, enter the number of first step of the desired sector for thermal cycling (ex, 2), and then enter the number of repeittion to be cycled (ex, 40). 14) Click Append button. When shown as the following figure, perform the scan operation for a total 40 of times. 15) Click Incubate tab and enter the temperature condition, and then complete the entry by clicking Append button (ex: 72 2min 30sec, 60 2min 30sec). 44

47 16) Click Melting tab to see the change in the fluorescence value arising from the change in temperature, and then enter 60 in the From column and 94 in To column. 17) Enter 0.5 in Between column, and 1 in Time column. Click Append button and add it to the experiment conditions display window. Note: Melting analysis is available through the process of Nos. 16 and 17. If the data is entered as shown in the figure, raise the temperature with the interval of 0.5 (Between) from 60 (From) to 94 (To), and then measure the fluorescence value for one second (Time). 18) Click Incubate tab to cool down the plate. Enter the temperature condition, and then complete the entry by clicking Append button (ex: 20 2min 30sec). Note: In case the Store stage is not included in the protocol information, Exicycler 96 completes the final stage of thermal cycling condition and automatically opens the door. In case, however, the stage of Store is included, it maintains the sample at the set temperature in the final stage, continuing the operation. Therefore, in order to extract the sample from the test where Store stage is included, use the Stop button to stop the operation of Exicycler

48 Important: In order to set the temperature of Hot top, the Hottop check box should be selected. The basic value is set at 100. If you release the check box, the hot top is not heated. 19) Enter the name of the file to be saved in Protocol Input Column (ex, AQ1), click button, then the prepared protocol is saved accordingly. Note: Save the completed protocol information by clicking button. Click button at Quick Start, then you can load the protocol information file already prepared. 20) Click the plate tab at the lower left area upon completion of the entry of protocol. It is then changed into plate window. a. Well Position Windows b. Well Information Windows c. Probe Information Windows d. Protocol/Plate Tab e. Sample Name Assign Add probe a. Well Position window: Place where the desired well to be tested is designated. b. Well Information window: Window where the information on the name, probe, etc. of the sample designated in the well is determined. c. Probe Information window: Window where the probe, sample type, concentration, etc. is designated in the well. d. Protocol/Plate tab: Used when selecting Protocol or Plate tab. 46

49 e. Sample Name: Button that is used to enter the Sample name. Assign: Button that is used to designate the information set in the Well Add Probe: Used to add the probe to be used in the test. Important: After loading the sample or control that will be used in the specific well by the user, it is necessary to designate the relevant information for the well. In order to obtain the correct result from the analysis software, it is necessary to properly set the information on the relevant probe and control for each test. 21) Click Add probe to save the probe to be used in the actual test. Add probe 22) Check the desired probe to be used in the Add Probe window, and then click OK button. a. Probe Name: The name of the probe defined by the user b. Fluorescent Dye: The name of fluorescent dye used in the probe designed by the user c. Quencher: The name of the quencher used in the probe defined by the user 47

50 23) The probe that will be used in the test is displayed as shown in the figure. 24) Select the position of the sample or standard to be designated in the well position window. Drag! Click! Note: Each well can be selected using the left mouse button. In order to select several wells simultaneously, click and drag using the left mouse button or select the well by pressing the Ctrl key. To select the well in the column unit, click A through H, and to select in row unit, click 1 through 12. To select the entire plate, press the arrow mark at the upper left part. 25) For setting the type, select the check box in front of the Probe Name, choose STD at the combo box, and then click Assign button. Sample STD NTC IPC- IPC 48

51 a. Sample: It means the unknown sample to be quantified using Exicycler 96. b. STD: The abbreviation of standard, it means the standard sample with known concentration. c. NTC: Abbreviation of No Template Control, which means the sample that not only includes template, but also includes all the reagents required for real time quantitative PCR. d. IPC: Abbreviation of Internal Positive Control, which is used to verify the failure of PCR reaction when the test result came out negative after PCR process is monitored. e. IPC-: Sample where the reagent prohibiting the IPC reaction instead of template in the relative test is included that does not involve any amplification phenomenon at all. This is not used in SNP Genotyping test. 26) Click D3 and D4 wells, and then enter the concentration and the name. For example, if these are 10^9 copy, select STD for the type, and enter 1,000,000,000 for these concentration, and then click Assign button. Enter the sample name on the edit box at the left of Sample Name button with std1 and then click Sample Name button. Well Position Window Probe Information Well Information 27) Using the same method, assign the D5~D10 wells to be standard as std2 ~ std4. 28) E3 Click E3 and E4 wells, and assign the Type as Sample and the name. Enter the sample name on the edit box at the left of Sample Name button with Sample1 and then click Sample Name button. 49

52 29) Using the same method, assign Sample2 ~ Sample 4. 30) Verify that the setting desired to be tested is true through the well Information window. Well Information 31) Enter the desired plate file name (ex, AQ1) in the Plate Entry and click information prepared. button to save the Plate 32) After Protocol information and Plate information are completed and saved, enter the desired Master file name (ex, AQ1) in Master Entry, and click button, and then save the prepared Master information. Note: Save the completed Master, Protocol, and Plate information by clicking button. Click button at Quick Start, then you may bring the prepared file by selection. When loading Master information, protocol information and plate information set at the time saved are automatically loaded together. 50

53 33) Click OK button at upper right part to display all the prepared information on the screen of the main program. 34) Check once more if the green lamp representing stand-by keeps on flashing the LED display window at the front of Exicycler ) Open the drawer of 96 well block by pressing DOOR button for about one second or longer at the front panel of Exicycler 96. After producing the test A1 Position plate, insert the plate so that A1 position of the plate can be placed at the left inner part of the equipment as shown in the figure. A1 Position 51

54 36) Make sure that the prepared information is correct, and then click Run > Run of Top Menu, or click shortcut icon. 37) Enter the file name (ex, Sample_AQ) of the data to be saved. When the entry is not made, use the filename created at the user s option from the software. Upon completion of entry, click OK button 38) Click OK button, and the following messages are displayed in order: a. The message on the right appears as the door automatically closes when the thermal block that runs with it is opened. b. The following message appears during the time the system checks whether the lamp can be turned on or not. 52

55 c. When the lamp can be turned on, Exicycler ignites the lamp. d. Exicycler 96 checks if the lamp is properly turned on or not and then initializes the system. 39) When Exicycler 96 operates in normal condition, the progressive bar of pop up window as shown above is executed to 100% Scan Window and then returned to its main screen. Melting Window Temperature Profile a. Scan Window: When PCR is performed, amplification curve of the sample can be shown in realtime basis on this window. b. Melting Window: This window appears when user inserts the melting step on the protocol where SYBR Green I is used. It shows the fluorescence data in real time basis when it is melted. c. Temperature Profile: This window displays the temperature condition on the protocol prepared by the user showing which step is currently performed by the protocol in real time basis. d. Experiment Information: This window shows a variety of information related with the tests currently run. e. Protocol Information: This window shows the protocol information used in the current test that highlights the step that is currently performed. f. Well Information: This indicates the information of the well configured by the user. When converting the configuration into Probe or Type in the lower part of Graph Color Setting combo box, you may see the information currently assigned to the well by colors. 53

56 Click the check box in front of the probe designated by the user below it, where a variety of probes are used, then you may see a certain probe selectively from Scan1 graph. That is, select the well from Well Information, and then the probe without fail in order to see the amplification curve of the relevant well. 40) To stop the thermal cycling, click Stop button or select Run>Stop menu. Click! 41) Click Pause button so as to stop Thermal cycling temporarily. Click! Note: When running Exicycler 96 with Bath opened, it automatically closes the bath and then starts thermal cycling. Important: In case the run time of Experiment Information window does not increase in excess of approximately five minutes even after the progressive bar of the pop up window is carried out up to 100%, turn the Exicycler off. Then, after waiting for more than five minutes, turn it on again and run the program to operate Exicycler 96. When the same problem continues, please call for After Service. Important: When turning Exicycler 96 off and then on again, please stand by for approximately five minutes for the stabilization of the lamp before resuming operation. For stabilization of Exicycler 96 lamp and protection of the system, do not operate Exicycler 96 immediately. It should be run after the stand-by of approximately five minutes. 54

57 Note: When the hottop temperature of Exicycler 96 is set, have the hot lid heated with bath temperature maintained at 25 degrees. When the hot lid reaches the temperature set and the brightness of Lamp is stabilized, it starts thermal cycling according to the test condition input. Note: Upon completion of the test, *.ex3 file is created on the path of the data of the designated user folder. *.ex3 file can be used when analyzing it using the analysis program. 55

58 Exicycler 96 Real Time Quantitative Thermal Block How to use Exicycler Analysis Program for The Analysis of Absolute Quantification 56

59 File Open for Absolute Quantitative Analysis When real time gene amplification test using Exicycler is completed, the ex3 file obtained can be analyzed using Exicycler Analysis program. In Exicycler analysis software, the absolute quantitative analysis consists of tap screens such as Flu. Graph, Plate Info, STD Curve, Melting Graph, Well vs Ct, Report Form. 1) Run Exicycler Analysis program. 2) Click File > Open menu. 3) Select *.ex3 file from open sub-screen. 4) Click open button. Note: Basic folder of *.ex3 that is the data file of Exicycler consists of C:\ExiData\Guest. When the user is configured, *.ex3 file is located in the user folder below C:\ExiData. 57

60 5) When the Select quantitative Type sub-screen appears, select quantitative type as Absolute Quantification 6) Click OK button to load the data for the analysis program. 7) Click Flu Graph tap screen. 8) Select the probe to be analyzed. When the probe is not selected, the relevant Flu. Graphics is not displayed 58

61 9) Select the desired well to analyze the result. 10) Then, the graph that falls under the selected well appears. Important: When the analysis program is run, the baseline of Flu. Graph and the threshold of Ct are calculated automatically. After running the analysis program, be sure to check if the relevant threshold value for the selected probe name is properly configured. 59

62 Fluorescence Graph (Flu. Graph) Tap Screen On the Fluorescence graph tap screen, the test result can be seen or the parameter that influences the analysis of the result can be changed. It is possible to determine the baseline sector for fluorescence graph and the threshold value by probes and change the baseline subtraction method too. In order to change the configuration of the graph displayed, you may change the basic configuration or figure that falls under X axis or Y axis on the Display Setup sub-screen that appears as a result of clicking Setting > Display menu. The required interval of configuration differs from the desired result to be analyzed. In addition, on the Flu Graph tap screen, you can find the calculated Fn value. In order to see the result of test by type for each well, if you select it by Type or Probe on Graph color setting sub-screen, the color designated by item is indicated. In order to change the Y axis scale of the graph indicated on Flu Graph tap screen into the log scale, select the Log Scale check box of Show Setting indicated on Analysis Setting sub- screen. In order to change the set value displayed on Flu Graph tap screen, observe the following process: 1) Click Setting > Display menu 2) When Display Setup sub-screen appears, change the set value located in Flu Graph tab. 3) Click Update button; the change is then applied. 60

63 How to use Well Info Sub-screen In order to select all 96 wells, click the corner at the upper left part of the plate. To select each individual well, click the left mouse button. To select several wells, click and drag the left mouse button, or select the column or row of the well while pressing the Ctrl key. If you want to exclude the selected well from analysis, click the right mouse button to select Omit. Then X mark is indicated on the well, when the final result of judgment is not used. The well removed from the analysis using Omit menu can be included in the result of analysis again by clicking the right mouse button. How to use Analysis Setting Sub-screen The Analysis setting sub-screen is intended to set the basic parameter to extract Ct value from the measured result of fluorescence. The initial set value is all set as Auto, but can be used to set the threshold and baseline for the test result as manual. Method for baseline subtraction is set as Trend average. In case Probe setting is set as All, it is impossible to set threshold and baseline of each probe. In order to set threshold and baseline by probe, it is necessary to change Probe item into the individual probe item used in the test. In order to activate the setting of Threshold that falls under the probe selected, the initial set value is established as Auto. In case Threshold item is set as Auto, Baseline sector cannot be set separately. In order to set the baseline sector individually, it is necessary to change Threshold item into Manual. In order to set Baseline sector as manual, set Baseline item as Manual. In order to change the Baseline sector, use the mouse to click and drag Start baseline ( ) and End baseline ( ) displayed in the graph s X axis. The threshold value by probe is indicated along with the threshold line on Flu graph tap screen. To change the value of threshold level, click and drag the threshold line by using the mouse or click OK button after entering the desired threshold value to change the Input Threshold Value sub-screen that appears by clicking the right mouse button. Threshold indicated on Flu graph tap screen is displayed on the screen only when selecting the 61

64 Threshold Level check box on Analysis Setting sub-screen. In order to change Y axis scale indicated on Flu Graph tap screen into the log scale, select Log Scale check box of Show Setting displayed on Analysis Setting sub-screen. In order to change the analysis parameter, observe the following process: 1) Select Probe item of Ct Setting on Analysis Setting sub-screen from All to the desired probe to be changed (ex, SYBR_AQ). 2) When Threshold item is activated, change it from Auto into Manual. The following is the method of changing threshold when Threshold item is set as Manual. First of all, to make it easier to set the threshold value, select Log Scale check box in Show Setting. i. Click threshold line using the mouse. ii. Click and drag the mouse, and then release the mouse button on the desired position to be set. 62

65 iii. Or, click threshold line using the right mouse button; then the Input Threshold Value subscreen appears. iv. Enter the desired threshold value to be set. v. Click OK button to complete the configuration of threshold line. 3) In order to set the sector of the baseline, change Baseline item from Auto into Manual. The following is the method of setting the baseline sector when the Baseline item is set as Manual: i. Icon showing baseline start and baseline end appears on the graph X axis (Cycle). ii. iii. Click Baseline End icon using the mouse. Click and drag the mouse, and release the mouse button on the desired position to be set. Start End 63

66 iv. Click Baseline Start icon using the mouse. v. Click and drag the mouse, and release the mouse button on the desired position to be set. Start 4) In order to change the method of subtraction for correcting the baseline, change Substation Method item. Subtraction method consists of the following: None: The baseline is not corrected Sum Average: Correct the baseline by using the average value of the sector where the baseline is set. 64

67 Trend Average: Corrects the baseline by seeking the change in the baseline by each probe in the sector where baseline is set. 5) If multiple kinds of probes are used, repeat the process of Nos. 1) through 4). 6) As mentioned before, when the setting of the Analysis setting item is changed, don t fail to click Analyze button every time the configuration is completed by probe to reflect the changes. 65

68 How to use Graph Color Setting Sub-screen Graph color setting sub-screen indicates the configuration according to the kind and type of each probe used in the test in the relevant colors. When the set value is Probe, the number and kinds of the probes used are displayed on the subscreen, and on Well info sub-screen. In addition, the color of the graph is displayed in unique color by probes. When selecting Probe in Color item, the color by type designated on the well is not displayed. When changing the set value into Type, the type of the probe designated to each well is only expressed. The unique color as per the Type is applied to the remaining tap screen too, and the color of the graph is expressed in the same color, when the color according to the probe used is not indicated. 66

69 67

70 STD Curve Tap Screen This is the screen that displays the standard curve used for the analysis of absolute quantification test result. The standard curve displayed on STD Curve tap screen is produced on the basis of the parameter set on Flu. Graph tap screen. The standard sample is included in ex3 data file, and in case the parameter used is set as Auto, the result of the standard curve automatically analyzed is displayed. The trend line function and R^2 value, which is useful in determining the straightness of standard curve, and the PCR efficiency of the standard curve are indicated together. In case the parameter is set as Manual on Flu. Graph tap screen, all the results displayed on STD Curve tap screen depend on the result of configuration by the user. Color of the standard curve is the same as that of the designated probe, and the color of the dots representing the result of calculation located on the standard curve is the same as that of the sample type designated. 68

71 How to use Well Info Sub-screen Click the corner of the upper left part of the plate to select all 96 wells. Each individual well can be selected by clicking the left mouse. To select several wells, click and drag the left mouse button or select the column or row of the well while pressing the Ctrl key. When you intend to exclude the selected well from analysis, click the right mouse button and select Omit, and X will be indicated on the well. This is not used in the final result of judgment. However, such well can be included in the result of analysis again by clicking the right mouse button and selecting Include menu. How to use Analysis Setting Sub-screen On Standard curve tap screen, Analysis Setting sub-screen is intended to change the probe for selecting standard curve what you want to see. Using Show Setting menu, you can see one standard curve selectively when the experiment data has several probe information for making standard curve each. 69

72 How to use Graph Color Setting Sub-screen Graph color setting sub-screen indicates the configuration according to the kind and type of each probe used in the test in the relevant colors. When the set value is Probe, the number and kinds of the probes used are displayed on the subscreen, and on Well info sub-screen. In addition, the color of the graph is displayed in unique color by probes. When selecting Probe in Color item, the color by type designated on the well is not displayed. When changing the set value into Type, the type of the probe designated to each well is only expressed. The unique color as per the Type is applied to the remaining tap screen too, and the color of the graph is expressed in the same color, when the color according to the probe used is not indicated. 70

73 Melting Graph Tap Screen This is the screen where the user can analyze if the desired target sequence is correctly amplified when the dye (such as enabling SYBR Green I) is used. The graph displayed on Melting Graph tap screen indicates fluorescence (F) according to the change in temperature. Nevertheless, for more detailed analysis, the graphs of primary (F ) and secondary (F ) differentiation can be expressed individually or jointly. How to use Well Info Sub-screen Click the corner of the upper left part of the plate to select all 96 wells. Each individual well can be selected by clicking the left mouse. To select several wells, click and drag the left mouse button or select the column or row of the well while pressing the Ctrl key. When you intend to exclude the selected well from analysis, click the right mouse button and select Omit, and X will be indicated on the well. This is not used in the final result of judgment. However, such well can be included in the result of analysis again by clicking the right mouse button and selecting Include menu. 71

74 How to use Analysis Setting Sub-screen In case multiple kinds of probes are used in the absolute quantification experiment, the probe can be seen selectively from Analysis Setting sub-screen. When selecting a certain probe from Probe, the melting graph of the relevant probe is displayed. In this case the melting graph of all the probes used, and in case All was selected, are displayed. In addition, in case you intend to indicate primary (F ) and secondary (F ) differentiation graphs for detailed analysis, you can selectively see the information displayed on the screen by selecting or removing the relevant check box. How to use Graph Color Setting Sub-screen Graph color setting sub-screen indicates the configuration according to the kind and type of each probe used in the test in the relevant colors. When the set value is Probe, the number and kinds of the probes used are displayed on the subscreen, and on Well info sub-screen. In addition, the color of the graph is displayed in unique color by probes. When selecting Probe in Color item, the color by type designated on the well is not displayed. When changing the set value into Type, the type of the probe designated to each well is only expressed. The unique color as per the Type is applied to the remaining tap screen too, and the color of the graph is expressed in the same color, when the color according to the probe used is not indicated. 72

75 Plate Info Tap Screen Plate Info tap screen provides the function that enables to change Plate Information prepared for the absolute quantitative analysis. When the information designated in the well for the analysis of absolute quantification result is wrong, a problem/error may arise in the final result of analysis. The change in name and type of sample can be revised on Plate Info tap screen from the data file obtained from the test result. Nevertheless, it is impossible to add a new probe unused. In case Plate information is abnormally revised, a problem may arise in the result of analysis, or serious analysis error may occur in the analysis program. Special attention is required in this matter. In order to change the type of probe or sample assigned to each well, follow the process below. 1) Select the desired well to be changed. 2) Select the check box of the probe indicated at the lower part of the screen. Remove the probe check box, and delete the information of the probe assigned to the well. 73

76 3) Select either of Sample, STD, and NTC in the Type item of the sample against the well selected. Important: When the information designated in the well for the analysis of absolute quantification result is wrong such as missing the STD or NTC samples or not amplifying them, a problem/error may arise in the final result of analysis. 4) Click Probe assign button and designate the item configured. 5) Update All Well Information button so that all the changes can be applied to the final result of judgment. How to use Well Info Sub-screen Click the corner of the upper left part of the plate to select all 96 wells. Each individual well can be selected by clicking the left mouse. To select several wells, click and drag the left mouse button or select the column or row of the well while pressing the Ctrl key. When you intend to exclude the selected well from analysis, click the right mouse button and select Omit, and X will be indicated on the well. This is not used in the final result of judgment. However, such well can be included in the result of analysis again by clicking the right mouse button and selecting Include menu. 74

77 How to use Analysis Setting Sub-screen On Plate Info tap screen, Analysis setting sub-screen is intended to change the name of the sample. To change the name of the sample, enter the desired name to be changed in Sample Name column, and click ReName button. If you want to apply all the changes to the result of final analysis, click Update All Well Information button. How to use Graph Color Setting Sub-screen Graph color setting sub-screen indicates the configuration according to the kind and type of each probe used in the test in the relevant colors. When the set value is Probe, the number and kinds of the probes used are displayed on the subscreen, and on Well info sub-screen. In addition, the color of the graph is displayed in unique color by probes. When selecting Probe in Color item, the color by type designated on the well is not displayed. When changing the set value into Type, the type of the probe designated to each well is only expressed. The unique color as per the Type is applied to the remaining tap screen too, and the color of the graph is expressed in the same color, when the color according to the probe used is not indicated. 75

78 Well vs. Ct Tap Screen Of the same samples or repetitive samples, Well vs. Ct tap screen can be used to find out the sample with large error of Ct. As each well position is indicated on X axis while Ct value on Y axis, it is easy to verify Ct deviation that occurs in the same samples. When the well with large error is found, select it by clicking the left mouse button, and then select Omit by clicking right mouse button. Then, an X mark is indicated on the well, which will be excluded from the final result of analysis. The well removed from the analysis using Omit menu can be included again in the result of analysis by clicking the right mouse button and selecting Include menu. How to use Well Info Sub-screen Click the corner of the upper left part of the plate to select all 96 wells. Each individual well can be selected by clicking the left mouse. To select several wells, click and drag the left mouse button or select the column or row of the well while pressing the Ctrl key. When you intend to exclude the selected well from analysis, click the right mouse button and select Omit, and X will be indicated on the well. This is not used in the final result of judgment. However, such well can be included in the result of analysis again by clicking the right mouse button and selecting Include menu. How to use Graph Color Setting Sub-screen Graph color setting sub-screen indicates the configuration according to the kind and type of each probe used in the test in the relevant colors. When the set value is Probe, the number and kinds of the probes used are displayed on the subscreen, and on Well info sub-screen. In addition, the color of the graph is displayed in unique color by probes. When selecting Probe in Color item, the color by type designated on the well is not displayed. When changing the set value into Type, the type of the probe designated to each well is only 76

79 expressed. The unique color as per the Type is applied to the remaining tap screen too, and the color of the graph is expressed in the same color, when the color according to the probe used is not indicated. Omit well setting A great number of errors may occur compared to the other wells if amplification is not sufficient due to the experimental error in the specific well, or because of any other factors. Generally, the well related with such problem shows Ct value other than the repetitive wells that are mutually associated. If the analysis is carried out using Ct value obtained here, error should be inevitably included in the final result. Accordingly, in order to perform more accurate Absolute quantification, it is necessary to carefully remove the well with large error from repetitive group. On Well vs. Ct tap screen, the well having such large error should be analyzed, and then omitted by directly selecting it if necessary. To omit the well having large error, follow the process as below. 1) Select Well vs. Ct tap screen. 77

80 2) After selecting the well where the sample required for Ct error analysis is included, analyze the samples having similar Ct results according to the repetition frequency from the graph into the same group. Then, check if there exists any well to be removed from analysis in the same group. 3) When a well is required to be removed from the analysis, select the relevant well first with the left mouse button, click the right button, and then click Omit. 4) Then, an X mark is indicated on the relevant well, which is not used in the final analysis of result. Note: The well removed in the analysis using the Omit menu can be included in the final analysis of result by clicking the right mouse button again and then selecting Include button. 78

81 Report Form Tap Screen It indicates the result of analysis for the well selected in a specific form of result. To the entire analysis result, the criteria set up on Report tap screen are applied. The results of analysis from the absolute quantification experiment are expressed with well, sample name, sample type, probe information used, quencher information, Ct, mean, deviation, concentration, and etc. The quantitative result obtained here can be saved in converted data that can be accessed in other software such as MS Excel. In order to change the array of the data, please click the title of the item indicated. The title of the item indicated is changed into descending or ascending order at every click. 79

82 Data Conversion of Absolute Quantitative Analysis Result The result obtained from absolute quantification experiment can be exported into the data opened in Excel. 1) Click the target tap screen to export in csv file type or jpg image type. 2) Select the menu File > Export > Excel or File> Export > Image. 3) Enter the name of file converted. 4) Click Save button. Note: In some instances, the tap screen with the Export function is not available. 80

83 Appendix 1. How to Set Baseline and Threshold Baseline and Threshold Setting Automatic Setting of Baseline and Threshold The Exicycler software calculates the value of baseline and threshold that falls under each probe, assuming that the entire test results show general amplification curve. General amplification curve has following characteristics. Plateau phase Linear phase Exponential phase Background Plateau phase Linear phase Exponential phase Background In a normal experiment, the general amplification curve above can be shown. External contamination or pipetting error, however, shows a curve different from the general amplification curve shown above. The result including such problems may produce abnormal baseline and threshold when automatically analyzed in the analysis software. Therefore, Bioneer recommends you to reconfirm that the values of baseline and threshold are properly set up after the entire 81

84 automatic analysis. Manual Setting of Baseline and Threshold The values of baseline and threshold that fall under each probe are decided automatically by the Exicycler software, but please refer to following explanation to manually set up proper baseline and threshold. The following amplification curve shows the result that appears when the setting of baseline and threshold are changed. Adequate Baseline Baseline sector is set up at proper position where amplification curve increases. No adjustment is required. Low Baseline The position where amplification curve increases is located far from the sector where the baseline is set up. Using Baseline End and Baseline Start icons, please set up the baseline sector at a higher level. 82

85 High Baseline Within the baseline sector set up, the amplification curve starts to increase. Using Baseline End and Baseline Start icons, set up the baseline sector at a lower level. Adequate Threshold Threshold is located at the exponential phase of amplification curve. When raising or lowering the threshold at the current status, the repeated standard deviation increases. Low Threshold The threshold is located below the exponential phase of the amplification curve. The standard deviation is higher than the adequate threshold. Please move the position of threshold up to the exponential phase. 83

86 High Threshold The threshold is located above the exponential phase of the amplification curve. The standard deviation is higher than the adequate threshold. Please move the position of threshold down to the exponential phase. 84