miracle mirna qpcr Instruction Manual Store at -20 Version 3.0 (Aug.2013)

miracle mirna qpcr Instruction Manual Store at -20 Version 3.0 (Aug.2013)

Contents I. PRODUCT INTRODUCTION 1 A. OVERVIEW 1 B. KIT CONTENTS 1 1. REAGENT KITS 1 2. PRIMER SETS 3 3. MIRACLE ON CALL SCREEN MIRNA QPCR PANEL 3 C. PRODUCT DESCRIPTION 5 1. MIRACLE MIRNA QPCR SYSTEM 5 2. RNA SPIKE IN 6 3. MIRACLE ON CALL SCREEN MIRNA QPCR PANEL 6 II. PROTOCOL 8 A. INDIVIDUAL MIRNA QUANTITATION 8 1. MIRACLE RT REACTION SETUP 8 2. MIRACLE QPCR REACTION SETUP 9 B. ON CALL SCREEN MIRNA PANELS 10 1. MIRACLE RT REACTION SETUP 11 2. MIRACLE QPCR REACTION SETUP 12 3. DATA ANALYSIS 13 4. QUALITY CONTROL 14 III. QUALITY CONTROL 15 A. SPECIFICITY TEST 15 B. SENSITIVITY TEST 16 V. APPENDIX 18 A. RELATED PRODUCTS 18 B. TECHNICAL SUPPORT 18 VI. LICENSING AND WARRANTY STATEMENT 19

I. Product Introduction A. Overview This manual provides details and information necessary to use the miracle mirna qpcr system which comprised of different types of reagent kits; including: miracle cdna Synthesis Kit miracle qpcr mirna Master Mix miracle RT qpcr Detection Kit miracle mirna qpcr Gene Primer Set miracle On Call Screen mirna qpcr Panel The system is designed for sensitive and accurate detection of mirna by quantitative real time PCR using SYBR Green. To ensure optimal results, please read the entire manual before using the reagents and material supplied with kits. B. Kit contents 1. Reagent Kits miracle cdna Synthesis Kit (Product # 110008 and 110009) These kits contain all reagents required for first strand cdna synthesis for 20 50 reactions. Contents: Contents Poly A Polymerase mirscript RTNase Description Catalyzes the addition of Poly A to the 3 end of mirna Contains reverse transcriptase and RNase Inhibitor Quantity Catalog#110008 Catalog#110009 20 µl 50 µl 20 µl 50 µl 5 PolyA/RT Mix Contains the ratp, dntp, MgCl 2, RT primer etc. 100 µl 250 µl RNA Spike in Nuclease free Water Used exclusively for the Control primer set included in the qpcr mirna Master Mix DNase, RNase, and nuclease free, deionized water 20 µl 50 µl 1 ml 1 ml miracle qpcr mirna Master Mix (Product # 110015 and 110016) These kits contain all reagents required for PCR amplification of micrornas. The reagents are provided in amounts sufficient for 200 600 reactions of 20 μl. 1

Contents: Contents 2 miracle qpcr Reaction Mix 50 ROX Dye Reverse PCR Primer (50 μm) Spike in Control Primer (2 μm) Description Contains the DNA polymerase, dntp and reaction buffer used in qpcr reactions Used in qpcr instruments requiring ROX for calibration The Universal mirna reverse qpcr primer which combines with mir specific forward primer Used exclusively for detection of the RNA spike in provided with the cdna synthesis kit Quantity Catalog#110015 Catalog#110016 2 1 ml 3 (2 1 ml) 1 80 µl 3 80 µl 1 20 µl 3 20 µl 160 µl 3 160 µl miracle RT qpcr Detection Kit (Product # 110001 and 110002) These kits contain all reagents for RT and PCR amplification of micrornas. The reagents are provided in amounts sufficient for 20 50 RT and 200 500 qpcr reactions of 20μL. Contents: Contents Poly A Polymerase mirscript RTNase 5 PolyA/RT Mix Nuclease free Water 2 miracle qpcr Reaction Mix RNA Spike in Spike in Control Primer(2 μm) 50 ROX Dye Reverse PCR Primer (50 μm) Description Catalyzes the addition of Poly A to the 3 end of mirna Contains reverse transcriptase and RNase Inhibitor Contains the ratp, dntp, MgCl 2, RT primer etc. DNase, RNase, and nuclease free, deionized water Contains the DNA polymerase, dntp and reaction buffer used in qpcr reactions Used exclusively for the Control primer set included in the qpcr mirna Master Mix Used exclusively for detection of the RNA spike in provided with the cdna synthesis kit Used in qpcr instruments requiring ROX for calibration The Universal mirna reverse qpcr primer which combines with mir specific forward primer Quantity Catalog#110001 Catalog#110002 20 µl 50 µl 20 µl 50 µl 100 µl 250 µl 1 ml 1 ml 2 1 ml 5 1 ml 20 µl 50 µl 160 µl 400 µl 80 µl 200 µl 20 µl 50 µl 2

2. Primer sets miracle mirna qpcr Gene Primer Set(Catalog# 100001 107999) These primer sets are designed to quantitatively measure mirnas. And these qpcr primers which are validated by qpcr reactions using their specific cdnas as templates are provided in the following table. The primer sets are supplied in sufficient amounts for 200 reactions of 20 μl. Contents: Contents Description Quantity miracle mirna qpcr Gene Primer Set Positive cdna Control Validated mirna qpcr primer Synthesized with miracle cdna synthesis kit for validating the mirna qpcr primers 2μM, 10 Concentrated, 20 μl 200 rxns 20 μl 3 rxns miracle mirna qpcr Reference Gene Primer Set(Catalog# 108xxx) The reference Gene Primer set is designed for use with the mirna primer sets above as reference genes for normalization. The primer sets are supplied in sufficient amounts for 200 reactions of 20 μl. 3. miracle On Call Screen mirna qpcr Panel Content s : Catalog# Product Name Number of mirnas Number of plates 120002 miracle On Call Screen human brain cancer mirna qpcr panel (4 sets) 84 1 x 96 well 120003 miracle On Call Screen human breast cancer mirna qpcr panel (4 sets) 168 2 x 96 well 120004 miracle On Call Screen human leukemia mirna qpcr panel (4 sets) 168 2 x 96 well 120005 miracle On Call Screen human lung cancer mirna qpcr panel (4 sets) 168 2 x 96 well 120006 miracle On Call Screen human ovarian cancer mirna qpcr panel (4 sets) 168 2 x 96 well 120007 miracle On Call Screen human colorectal cancer mirna qpcr panel (4 sets) 84 1 x 96 well 120008 miracle On Call Screen human endometrial cancer mirna qpcr panel (4 sets) 84 1 x 96 well 120009 miracle On Call Screen human gastric cancer mirna qpcr panel (4 sets) 80 1 x 96 well 120010 miracle On Call Screen human bladder cancer mirna qpcr panel (4 sets) 79 1 x 96 well 3

120011 miracle On Call Screen human hepatocellular carcinoma mirna qpcr panel (4 sets) 168 2 x 96 well 120012 miracle On Call Screen human melanoma mirna qpcr panel (4 sets) 84 1 x 96 well 120013 miracle On Call Screen human pancreatic cancer mirna qpcr panel (4 sets) 84 1 x 96 well 120014 miracle On Call Screen human prostate cancer mirna qpcr panel (4 sets) 84 1 x 96 well 120015 miracle On Call Screen human head and neck cancer mirna qpcr panel (4 sets) 84 1 x 96 well 120016 miracle On Call Screen human lymphoma mirna qpcr panel (4 sets) 84 1 x 96 well 120017 miracle On Call Screen human inflammatory mirna qpcr panel (4 sets) 84 1 x 96 well 120018 miracle On Call Screen human heart disease mirna qpcr panel (4 sets) 84 1 x 96 well 120019 miracle On Call Screen human immunopathology mirna qpcr panel (4 sets) 84 1 x 96 well 120020 miracle On Call Screen human muscle disease mirna qpcr panel (4 sets) 84 1 x 96 well 120021 miracle On Call Screen human toxicology related mirna qpcr panel (4 sets) 84 1 x 96 well 120022 miracle On Call Screen human IPS (Stem cell) mirna qpcr panel (4 sets) 168 2 x 96 well 120023 miracle On Call Screen human serum and plasma mirna qpcr panel (4 sets) 168 2 x 96 well 121001 miracle On Call Screen mouse breast cancer mirna qpcr panel (4 sets) 84 1 x 96 well 121002 miracle On Call Screen mouse brain cancer mirna qpcr panel (4 sets) 84 1 x 96 well 121003 miracle On Call Screen mouse ovarian cancer mirna qpcr panel (4 sets) 84 1 x 96 well 121004 miracle On Call Screen mouse prostate cancer mirna qpcr panel (4 sets) 84 1 x 96 well 121005 miracle On Call Screen mouse colorectal cancer mirna qpcr panel (4 sets) 84 1 x 96 well 121006 miracle On Call Screen mouse hepatocellular carcinoma mirna qpcr panel (4 sets) 84 1 x 96 well 121007 miracle On Call Screen mouse lung cancer mirna qpcr panel (4 sets) 84 1 x 96 well 121008 miracle On Call Screen mouse melanoma mirna qpcr panel (4 sets) 84 1 x 96 well 121009 miracle On Call Screen mouse pancreatic cancer mirna qpcr panel (4 sets) 84 1 x 96 well 4

121010 miracle On Call Screen mouse head and neak cancer mirna qpcr panel (4 sets) 84 1 x 96 well 121011 miracle On Call Screen mouse leukemia mirna qpcr panel (4 sets) 84 1 x 96 well 121012 miracle On Call Screen mouse inflammatory mirna qpcr panel (4 sets) 84 1 x 96 well 121013 miracle On Call Screen mouse heart disease mirna qpcr panel (4 sets) 84 1 x 96 well 121014 miracle On Call Screen mouse immunopathology mirna qpcr panel (4 sets) 84 1 x 96 well 121015 miracle On Call Screen mouse serum & plasma mirna qpcr panel (4 sets) 84 1 x 96 well 120001 miracle On Call Screen human cancer mirna qpcr panel(4 sets) 420 5 x 96 well : Shipped at room temperate; Stable for at least 6 months when stored at 2 8 C C. Product Description Mature mirnas are naturally occurring, 22 nucleotide, noncoding RNAs that mediate post transcriptional gene regulation. Unlike mrnas, mirnas are not polyadenylated in nature. 1. miracle mirna qpcr System Mature mirnas are polyadenylated by poly(a) polymerase and reverse transcribed into cdna using oligo dt primers. Polyadenylation and reverse transcription are performed in parallel in the same tube. The oligo dt primers(reverse PCR Primer) have a 3' degenerate anchor and a universal tag sequence on the 5' end, allowing amplification of mature mirna in the real time PCR step. miracle mirna PCR Primer Set(miRNA specific forward primer), used in combination with the miracle qpcr mirna Master Mix, enable quantification of mature mirna by real time PCR. The combination of polyadenylation and the universal tag addition ensures that miracle mirna qpcr Gene Primer Set do not detect genomic DNA( Figure.1) Figure 1. Schematic outline of the miracle mirna qpcr System. A poly A tail is added to the mature mirna template. cdna is synthesized using a poly T primer with a 3 degenerate anchor and a 5 universal tag. The cdna template is then amplified using mirna specific forward and Reverse PCR primers. SYBR Green is used for detection. 5

2. RNA Spike in RNA Spike in is to use as reverse transcription controls, which can be used to monitor the efficiency of the RT reactions and the PCR. Some sample types may contain compounds that inhibit the RT or the PCR even though the RNA has been purified using the best procedures. And it s may influence the efficiencies of the RT or PCR between compared samples. One way to control for differences in efficiencies is by adding a known RNA Spike in to the sample prior to cdna synthesis. Proceed with first strand synthesis and subsequently real time PCR as described in the protocols of the current instruction manual. 3. miracle On Call Screen mirna qpcr Panel miracle On Call Screen mirna qpcr Panels are mature mirna specific forward primers and reverse primers that have been arrayed in biologically relevant Cancer related and Disease related panels. These PCR Panels are provided in ready to use 96 well plate. miracle On Call Screen mirna qpcr Panels, which are available for several species, provide guaranteed high performance and are fully customizable. Mature mirna expression profiling is now within reach of every laboratory because of the ease, convenience, and consistent performance of On Call Screen mirna qpcr Panels. Each 96 well plate contains up to 84 sets of PCR primers (forward: mirna specific primer; reverse: universal primer), which are pre deposited in each well. Each plate also designated 12 control wells for monitoring the efficiency of every step of the experiment from RT to qpcr reaction. Panel format options Provides five qpcr array formats (A1, A2, B1, B2, R1) suitable for use with the following real time cyclers. Real time Cycler Real time Cycler Model Panel Format Applied Biosystems 5700, 7000, 7300, 7500, 7700, 7900HT (Standard 96 well block), ViiA TM 7 (Standard 96 well block) A1 7500 (Fast block), 7900HT (Fast block), StepOnePlus TM, ViiA TM 7 (Fast block) A2 Agilent (Stratagene) Bio Rad Mx3000P, Mx30005P Mx4000 icycler iq, MyiQ, iq 5 CFX96, DNA Engine Opticon, DNA Engine Opticon 2, Chromo4 A1 B2 B1 B2 Eppendorf Mastercycler ep realplex 2, 2S, 4, 4S B1 Roche LightCycler 480 (96 well block) R1!Important Notes Upon receipt, please check and make sure that the correct array format was ordered to ensure the compatibility with your qpcr instrument. 6

Panel layout: Figure 2. Illustration of miracle On Call Screen mirna qpcr Panel layout (96 well plate) mirna primer sets: Wells 1 84 are designated wells for pre deposited mirna primer sets. NC: Negative controls, which only have the pre deposited reverse PCR primers HK1 6: Six pre deposited housekeeping snrnas (HK1 6) primer sets, which can be used as endogenous positive controls as well as for array normalization. RT: RNA Spike in reverse transcription controls, which can be used to monitor the efficiency of the RT reactions. These pre deposited primer sets specifically amplify the cdna template reversed transcribed from the spike in exogenous RNA in the sample. PCR: Positive PCR controls, which are used to verify the PCR efficiency by amplifying the pre deposited DNA template with its specific pre deposited primer sets. 7

II. Protocol A. Individual mirna Quantitation This protocol is used for the conducting the RT and qpcr using the validated mirna Gene Primer set. Additional required materials Nuclease free PCR tubes or plates for use with individual assays Nuclease free, aerosol barrier pipette tips Nuclease free, low nucleic acid binding (siliconized) micro centrifuge tube Sealing foils for PCR plates Micro centrifuge and plate centrifuge Heating block, thermal cycler 1. miracle RT Reaction Setup!Important Notes It is important to start with total RNA that includes the small RNA fraction. a. Thaw template RNA on ice. Thaw 5 PolyA/RT Mix and Nuclease free water at room temperature (15 C ~25 C). b. Gently mix mirna reverse transcription reagents by flicking to dissolve all reagents thoroughly. Briefly centrifuge to collect residual liquid from the sides of the tubes and then place on ice. c. Prepare mirna reverse transcriptase reaction solution. Place RNase free reaction tubes on ice and then add the following reagents to a final volume of 25 µl. Reagent Volume (μl) Quantity Total RNA 2 µg or small molecule RNA 100 ng Poly A Polymerase 1 mirscript RTNase 1 5 PolyA/RT Mix 5 1 RNA Spike in optional replace with H 2 O if omitted (1) Nuclease free Water To final 25 The amount of total RNA can be between 1ng ~ 5µg. If using purified small molecule RNA, the amount can be between 0.1ng ~ 1µg. d. Prepare reverse transcription reaction. 8

Mix the prepared reaction mix gently, but thoroughly incubate at 37 C for 60 minutes after a brief centrifugation. Incubate at 85 C for 5 minutes to inactivate the enzyme. The resulting reverse transcription reaction product should be diluted 5 50 times with sterile H 2 O before using for the next qpcr experiment or it can be directly stored at 20 C. 2. miracle qpcr Reaction Setup a. Dissolve 2 miracle qpcr Reaction Mix by gently inverting. Briefly centrifuge and place on ice. If required, dissolve 50 ROX Dye. b. Dilute the 50 µm Reverse PCR Primer to 2 µm with sterile ddh2o before using for the next qpcr experiment(( e.g. add 480 μl nuclease free water to 20 μl Primer ). c. Prepare qpcr solution on ice. See the following example. Reagent Volume (μl) Final concentration 2 miracle qpcr Reaction Mix i 10 1 miracel mirna qpcr Gene Primer (2 µm) ii 2 0.2 µm Reverse PCR Primer (2 µm) 2 0.2 µm First strand cdna (diluted 1:5) iii 2 50 ROX Dye iv 0.4 (0) 1 Nuclease free Water Not using ROX Reference Dye 4 Using ROX Reference Dye 3.6 Final volume 20 Notes i. Use the 2 miracle qpcr Reaction Mix as half of the total reaction volume and adjust other reagents accordingly. If the total reaction volume is changed, maintain each component in proper proportion. ii. Primer concentration should be in the range of 0.2 to 0.4 μm. In general, a PCR reaction using 0.2 μm primers produces good results. If using Spike in control primer, please also add 2 μl primer to the 20 μl qpcr reaction mix. iii. The first strand cdna should be diluted before using for the PCR reaction in order to avoid interference to the qpcr from the reverse transcription system. iv. ROX Reference Dye is used in Real Time qpcr instruments that require ROX for calibration, such as the ABI qpcr instrument. Details as per each instrument manufacturer s manuals and protocols. d. Thoroughly mix the qpcr reaction solution, add to PCR tubes, and briefly centrifuge to make sure that all the reagents are in the bottom of the tubes. 9

e. The following standard 3 step method for the qpcr reaction is recommended (these experiments were designed based on Bio Rad iq5 qpcr instrument). Cycles Steps Temperature Time Detection 1 Initial denaturation 95 C 10 min No Denaturation 95 C 10 sec No 40 Annealing T m 2 C 20 sec No Extension 72 C At least 10 sec Yes Notes i. When using SYBR Green dye to monitor the qpcr reaction, a melting curve analysis should be performed immediately after qpcr cycling. For instructions, please find them in the documentation for your qpcr instrument. The following is an example adapted from the Bio Rad iq5 real time detection system. The conditions for your instrument may differ. Temperature Range Heating Rate Constant Temperature Detection 65 C ~ 95 C 0.5 C/ time 6 sec/ time Yes 30 C 30 sec No ii. The DNA polymerase used in the miracle qpcr mirna Master Mix is a chemically especially modified hot start enzyme. Incubation for 10 minutes at 95 C will sufficiently activate the enzyme. iii. Specific properties of a mirna lead to special properties of the designed primer. Therefore the annealing temperature needs to be strictly controlled in order to avoid non specific amplifications. For validated miracle mirna primers purchased from Genetimes Technology, please refer to the optimal conditions for the experiment. iv. The Reverse PCR primer for reverse transcription is 53 nucleotides, therefore the resulting PCR amplification fragment is about 75bp (assuming the sequence of mirna is about 22 nucleotides), which requires at least about 10 seconds extension time. From the melting temperature of the products, the T m value is generally determined to be between 75 C ~ 83 C. If the melting temperature exceeds this range, other assaying methods such as electrophoresis are suggested for the specific properties of the product. v. The main conditions for the above reactions are for use with the iq5 qpcr instrument from Bio Rad. If the different brand qpcr instrument is used, please reference the instrument manual and adjust the extension time and melting curve conditions accordingly. B. On Call Screen mirna Panels This protocol is used for the conducting the RT and qpcr using the following products: Cancer Related Panel Disease Related Panel Customized Panel Additional required materials Nuclease free, aerosol barrier pipette tips Nuclease free, low nucleic acid binding (siliconized) micro centrifuge tube Sealing foils for PCR plates Swing bucket centrifuge for 96 plates Heating block,96 well thermal cycler, compatible with mirna panels ordered 10

10μl to 1,000μl adjustable single channel micropipettes with disposable tips 5μl to 20μl adjustable multichannel micropipette, disposable tips, and reservoir, but recommend Liquid handling robot for pipetting 1. miracle RT Reaction Setup!Important Notes It is important to start with total RNA that includes the small RNA fraction. a. Adjust each of the template RNA samples to a concentration of 500 ng/ μl(total RNA) or 250 ng/ μl (mirna) using nuclease free water. b. Thaw the reagents of miracle cdna synthesis kit, mix by gently flicking the tube, briefly centrifuge to bring the contents to the bottom of the tubes and then place on ice. c. If performing RT on multiple RNA samples, it is recommended to prepare an RT master mix of the Poly A Polymerase, mirscript RTNase, 5xPolyA/RT Mix, Water, and RNA spike in (in the proportions indicated in the following Table) The following procedure is recommended: Prepare the required amount of RT master mix and place it on ice. Dispense RT master mix into nuclease free tubes. Dispense template RNA in each tube. Component 1 96well Plate/Sample 2 96well Plate/Sample 5 96well Plate/Sample Volume(μl) Volume(μl) Volume(μl) Total RNA(500 ng/μl) 4 4 12 PolyA Polymerase 1 1 3 mirscript RTNase 1 1 3 5 polya/rt Mix 5 5 15 RNA Spike in 1 1 3 Nuclease free Water 13 13 39 Total Volume 25 25 75 To increase the rate of positive detection, an input of 0.5~1.0μg of small RNA or 1 2 μg of total RNA is recommended for the standard mirna reverse transcription reaction (25μl). d. Perform reverse transcription reaction: Mix the prepared reaction mix gently by pipetting up and down. Incubate at 37 C for 60 minutes. Stop the reaction by incubating at 85 C for 5 minutes. Store the cdna products at 20 C or dilute the cdna products 10 times with sterile water and use it for the subsequent qpcr reactions. 11

2. miracle qpcr Reaction Setup!Important Notes 1. Be sure the On Call Screen mirna qpcr plate is compatible with your qpcr instrument before beginning this protocol. 2. Before use, remove any condensation which has accumulated on the plate sealing surface and centrifuge briefly to collect the contents at the bottom of the plate wells 3. Strictly follow standard procedures for PCR to avoid nucleic acid contamination and non specific amplifications. a. Thaw the reagents of miracle qpcr Master Mix. Protect the 2 miracle qpcr Reaction Mix vials from light. Invert the tubes to mix gently but thoroughly. Briefly centrifuge to bring the contents to the bottom of the tubes and then place on ice. Before removing the plate seal, briefly spin down the plate(s) in a plate centrifuge. Carefully remove sealing film before use 96 Well qpcr. b. Prepare qpcr solution on ice Components 1 well N well i Volume (μl) Volume (μl) 2 miracle qpcr Reaction Mix 10 10 (N 110%) mirna cdna(10 times dilution) 1 1 (N 110%) 50 X Rox Dye ii 0.4 0.4 (N 110%) Nuclease free Water Not using Rox Reference Dye 9 9 (N 110%) Using Rox Reference Dye 8.6 8.6 (N 110%) Total Volume 20 20 (N 110%) i. On Call Screen mirna qpcr Panel is used to detect multiple mirnas simultaneously in the same sample. Ensure sufficient mix is available by preparing enough for the number of reactions to be used with a 10% additional volume for pipetting loss. ii. 50 Rox Dye is added only for qpcr instruments that require ROX for calibration. c. Mix the qpcr solution thoroughly and centrifuge briefly. Accurately transfer exactly 20 μl reaction mix to each well. Change tips after each transfer to avoid cross contamination. d. Tightly seal the qpcr reaction plate with a new sealing film, Ensure that the film seals smoothly to prevent refraction of light. Centrifuge briefly to remove bubbles. e. Run qpcr. The following three step PCR program is recommended for running qpcr. 12

Cycles Steps Temperature Duration Detection 1 Initial Denaturation 95 C 10 min i No 40 Denaturation 95 C 10 sec. No Annealing 60 C ii 20 sec. No Extension 72 C iii 10 sec. Yes i. The DNA polymerase used in the 2 miracle qpcr Reaction Mix is a special chemically modified hot start enzyme. The indicted initial denaturation is sufficient to activate the enzyme. ii. The annealing temperature of the cross linked primers in On Call Screen Human Single Nucleotide Mismatch mirna qpcr Panel are designed and optimized. For comparing the mirnas with single nucleotide difference, a higher annealing temperature (65 C) might be necessary. iii. The extension time indicated above is suitable for Bio Rad s iq5 real time PCR instrument. Adjust the time duration according to the documentation provided with your instrument. When using SYBR Green dye to monitor the qpcr reaction, a melting curve analysis should be performed immediately after qpcr cycling. Temperature range Heating rate Constant temperature Detection 66 C~95 C 0.5 C/unit time 6sec./unit time Yes 30 C 30sec. No 3. Data Analysis a. Define the baseline The baseline is the noise level in early cycles. Each real time PCR instrument has algorithms to perform the baseline setting. This may be a fixed number of cycles for all samples or adaptive for each sample, depending on the type of instrument that is being used. If the lowest Ct is less than the upper limit of the baseline setting, then the baseline should be manually adjusted. Use the Linear View of the amplification plot to determine the earliest visible amplification, and then set the baseline from cycle 2 to 32 cycles before the earliest visible amplification. Normally it is between 2 to 10 cycles. Do not use cycles greater than 15. Ensure that baseline settings are the same across all PCR runs in the same analysis to allow comparison of results. b. Setting threshold Correct placement of the threshold is the next crucial step in data analysis. To adjust the threshold properly, set the threshold value within the exponential phase of all amplification plots when viewed using the logarithmic scale for the y axis. Generally, the expression level of each reference gene should be higher than most other genes. c. Obtain the Ct or Cp values The Ct is defined as the cycle when sample fluorescence exceeds a chosen threshold above background fluorescence. This is also known as the Cp or crossing point. d. Export the data. Most qpcr instruments provide a function for exporting Ct or Cp values to Excel. e. Analyze the qpcr results using the ΔΔCt method of relative quantification and interpretation of the control wells. 13

f. All Ct values reported as greater than 35 or as N/A (not detected) are considered as not detectable. 4. Quality Control 1. Examined amplification and melting status of each gene using the qpcr instrument software. Each reference gene, RTC and PPC should exhibit only one melting peak per reaction. 2. Examined Ct values of the positive PCR control wells (PCR). If the RNA sample is of high quality, the cycling program has been correctly run, and the thresholds have been correctly defined, the value of Ct of PCR should be 20±2 across all arrays or samples. 14

B. A. miracle mirna qpcr Instruction Manual III. Quality control A. Specificity test With 200 ng total RNA mixture from human brain and heart as template, the miracle qrt PCR Detection Kit and the miracle mirna qpcr Gene Primer Set were used to detect 30 mirna and an internal reference snrna U6. Results from qrt PCR and electrophoresis showed neither non specific amplification products nor primer dimer formation. Figure 3. Amplification and melting curves of 31 mirna and the internal reference snrna U6, in which double channel detection was used for the positive control, and single channel detection was used for the NTC (No Template Control). Figure 4. Agarose gel electrophoresis (3% agarose gel) of the amplification products of 31 mirna and the internal reference snrna U6, in which double channel detection was used for the positive control, and single channel detection was used for the NTC (No Template Control). 15

B. Sensitivity test Starting with different amounts (5μg, 1μg, 200ng, 20ng, 2ng, 100pg) of human brain total RNA, the miracle RT qpcr Detection Kit was used to detect the expression level of hsa mir 124. The results showed that linear amplification can be detected between 5μg ~ 100pg of total RNA. R 2 =0.997 E=103.9% Figure 5. The amplification curve and standard curve generated from different amount of human brain total RNA as template, and using miracle RT qpcr Detection Kit to detect hsa mir 124 expression level. 16

IV. Troubleshooting Problems Signal appears in No RT control Signal appears in No Template control No fluorescent signal is detected during the RT No fluorescent signal is detected during the PCR Suggestion Contamination of the template RNA with genomic DNA. Perform DNase treatment of the RNA sample. Contamination of reagent. Perform new reagent for reaction. RNA poor quality or no mirna include in total RNA. Check the concentration, integrity, and purity of the template RNA before starting the protocol. Protect reaction from RNase contamination or re purified RNA of sample. RNA concentration is too high and too low. Adjust the RNA concentration according to the instruction manual Check that the filter in the real time PCR cycler was set to either SYBR Green or FAM/FITC. The amount of template may not be enough or the template may be degraded. Use the highest concentration of diluted template samples possible to set up PCR. PCR annealing temperature too high. Decrease annealing temperature. Not enough PCR cycles. For good sensitivity, one should generally set up more than 35 PCR cycles, but more than 45 cycles may result in too much background signals. Insufficient starting template. Increase the amount of template cdna. RNA samples may contain PCR inhibitors. Further purification or an alternative RNA extraction method may be necessary. Check if there are PCR products to exclude the possibility of instrument detector malfunction. 17

V. Appendix A. Related Products NucleoSpin mirna Kit (50) (Cat.No:200001) Parallel isolation of small and large RNA from human / animal tissue and cultured cells, from plant tissue, No phenol chloroform during the procedure NucleoSpin mirna Plasma Kit(50) (Cat.No:200021) Isolation of small RNA from plasma or serum for cancer research (e.g., reliable monitoring of clinical parameters) NanoFectin Transfection Reagent (Cat.No: EMB750A 1) Deliver more DNA and sirnas/mirnas to cells than the leading lipid based transfection reagents Lenti mirs (Cat.No: PMIRHxxA 1) Over express microrna precursors using lentivectors. mirzips (Cat.No: MZIPxxA 1) Permanent microrna knockdown using RNAi lentivectors with anti sense micrornas.. B. Technical Support For more information about miracle products and to download manuals in PDF format, please visit our web site: http://www.genetimes.com/miracle For additional information or technical assistance, please call or email us at: Phone: 400 820 5565 (Toll Free) (86)21 33676611 Fax: (86)21 33676258 E mail: Technical Support: tech@genetimes.com.cn Ordering Information: order@genetimes.com.cn 18

VI. Licensing and Warranty Statement Limited Use License Use of the miracle related Kit (i.e., the Product ) is subject to the following terms and conditions. If the terms and conditions are not acceptable, return all components of the Product to Genetimes Technology within 7 calendar days. Purchase and use of any part of the Product constitutes acceptance of the above terms. Purchase of the product does not grant any rights or license for use other than those explicitly listed in this Licensing and Warranty Statement. Use of the Product for any use other than described expressly herein may be covered by patents or subject to rights other than those mentioned. Genetimes Technolgoy disclaims any and all responsibility for injury or damage which may be caused by the failure of the buyer or any other person to use the Product in accordance with the terms and conditions outlined herein. Genetimes Technology has pending patent applications related to the Product. Limited Warranty Genetimes Technology warrants that the Product meets the specifications described in the accompanying Product Datasheet. If it is proven to the satisfaction of Genetimes Technology that the Product fails to meet these specifications, Genetimes Technology will replace the Product. In the event a replacement cannot be provided, Genetimes Technology will provide the purchaser with a refund. This limited warranty shall not extend to anyone other than the original purchaser of the Product. Notice of nonconforming products must be made to Genetimes Technology within 30 days of receipt of the Product. Genetimes liability is expressly limited to replacement of Product or a refund limited to the actual purchase price. Genetimes liability does not extend to any damages arising from use or improper use of the Product, or losses associated with the use of additional materials or reagents. This limited warranty is the sole and exclusive warranty. Genetimes Technology does not provide any other warranties of any kind, expressed or implied, including the merchantability or fitness of the Product for a particular purpose. Genetimes Technology is committed to providing our customers with high quality products. If you should have any questions or concerns about any miracle related products, please contact us at 400 820 5565 2012, Genetimes Technology, Inc. 19

http://www.genetimes.com/miracle For additional information or technical assistance, please call or email us at: Phone: 400 820 5565 (Toll Free) (86)21 33676611 Fax: (86)21 33676258 E mail: Technical Support: tech@genetimes.com.cn Ordering Information: order@genetimes.com.cn