mircury LNA Universal RT microrna PCR

Transcription

1 Expression Analysis For biofluid samples, please use specific manual at exiqon.com/mirna-pcr mircury LNA Universal RT microrna PCR Instruction manual v6.1 #203301, April 2015

2 Table of contents Product Summary I. Reagent kits II. Primer Sets and Panels Storage Additional required materials Recommended accompanying products Product description Control Assays Before starting the Experiment Protocols A. Individual assays B. Human and Mouse&Rat microrna PCR Panels C. Focus microrna PCR Panels D. Pick-&-Mix microrna PCR Panels Tips to protocol Troubleshooting guide FAQs Related products

3 Product summary The mircury LNA Universal RT microrna PCR system is a microrna-specific, LNA -based system designed for sensitive and accurate detection of microrna by quantitative real-time PCR using SYBR Green. The method is based on universal reverse transcription (RT) followed by real-time PCR amplification with LNA enhanced primers (for more details please see page 12). The mircury LNA Universal RT microrna PCR portfolio is comprised of four types of reagent kits; including: Universal cdna synthesis kit II RNA Spike-in kit ExiLENT SYBR Green master mix kit microrna primer sets available in pre-defined Human, Mouse&Rat and Focus PCR panels and customized Pick-&-Mix PCR panels as well as individual primer sets and reference genes. All PCR panels are Ready-to-Use delivered with one 10 µl PCR reaction per well. Figure 1. Universal cdna synthesis kit II Description page 4 RNA Spike-in kit (optional) + microrna and reference gene primer sets Pick-&-Mix PCR panels (Ready-to-Use) Predefined PCR panels Human, Mouse&Rat, and Focus PCR panels (Ready-to-Use) Description page 6, protocol page 20 Description page 9, protocol page 43 + ExiLENT SYBR Green master mix 2,5 or 20ml Description page 7, protocol page Description page 5 3

4 I. Reagent kits Universal cdna synthesis kit II, 8-64 rxns (product # ) This kit contains all reagents required for first-strand cdna synthesis for 8-64 reactions 1). The UniSp6 RNA Spike-in template can be used by itself or in combination with the cel-mir-39-3p template provided in the RNA Spike-in kit: Contents 5 x Reaction buffer 2) 128 µl, 5 x concentrated Enzyme mix 64 µl, 10 x concentrated Nuclease free water 1 ml UniSp6, RNA Spike-in template 3) 12 fmol, dried down mircury LNA Universal RT microrna PCR, Starter Kit (product # ) This kit contains all reagents required to perform 20 cdna reactions of 10 µl volume and 100 PCR reactions. Kit includes primer set for the spike control UniSp6 (included), one candidate endogenous control primer set (mir-103a-3p) and two validated primer sets of your choice. Contents 5 x Reaction buffer 2) 128 µl, 5 x concentrated Enzyme mix 20 µl, 10 x concentrated UniSp6, RNA Spike-in template 12 fmol, dried down UniSp6 RNA Spike-in control primer set v2 4) 500 μl, 10x concentrated hsa-mir-103a-3p (also works for mmu+rno), lyophilized 200 rxn 2 primers free of choice from stocked primers 2x200 rxn ExiLENT SYBR Green master mix, 2x concentrated 500 µl Nuclease free water 1.25 ml 1) Number of reactions is based on a standard reaction volume of 10 µl to 80 µl. Reaction volume depends on the application and number of assays to profile. Please consult Figure 4 for details. 2) Includes universal reverse transcription primer. 3) Used exclusively for the UniSp6 RNA Spike-in control primer set included in the ExiLENT SYBR Green master mix kit. 4) Assay redesigned for improved performance, compared to the assay in product # , and Target sequence is unchanged. 4

5 ExiLENT SYBR Green master mix, 2.5 ml (product # ) This kit contains all reagents required for PCR amplification of micrornas. In addition, a positive control assay, the UniSp6 RNA Spike-in control primer set, is provided with this kit for amplification of the synthetic UniSp6 RNA spike-in provided in the Universal cdna synthesis kit II. The reagents are provided in amounts sufficient for 500 reactions of 10 µl. Contents ExiLENT SYBR Green master mix, 2x concentrated Nuclease free water UniSp6 RNA Spike-in control primer set v2 1) 2 x 1.25 ml, 2x concentrated 2 x 1.25 ml 500 µl, 10x concentrated ExiLENT SYBR Green master mix, 20 ml (product # ) This kit contains all reagents required for PCR amplification of micrornas. In addition, a positive control assay, the UniSp6 RNA Spike-in control primer set, is provided with this kit for amplification of the synthetic UniSp6 RNA spike-in provided in the Universal cdna synthesis kit II (the UniSp6 RNA Spike-in control primer set is provided 100x concentration and should be diluted to 10x concentration before using in the protocol for individual assays). The reagents are provided in amounts sufficient for 4000 reactions of 10 µl. Contents ExiLENT SYBR Green master mix, 2x concentrated Nuclease free water UniSp6 RNA Spike-in control primer set v2 1) 2 x 10 ml, 2x concentrated 1 x 20 ml 500 µl, 100x concentrated 1) Used exclusively for detection of the UniSp6 RNA spike-in provided with the Universal cdna synthesis kit II. Assay is redesigned for improved performance, compared to the assay in product # , and Target sequence is unchanged. 5

6 II. Primer Sets and Panels MicroRNA LNA PCR primer set (product # xxx and xxxxx) LNA PCR primer sets are designed for optimal performance with the Universal cdna Synthesis Kit II and the ExiLENT SYBR Green master mix, kit II. The performance of LNA primer sets will be affected if used in combination with less than optimal reagents. The primer sets are supplied in sufficient amounts for 200 reactions of 10 µl. Contents LNA PCR Primer mix (dried down) Reference gene primer set (product # varies) The Reference gene primer sets are designed for use with the microrna primers sets above as reference genes for normalization. The primer mix is supplied in sufficient amount for 200 reactions of 10 µl. Contents LNA PCR Primer mix (dried down) 6

7 Pre-defined microrna PCR Panels (product # varies) The Human, Mouse&Rat and Focus microrna PCR panels Ready-to-Use consist of either 96-well or 384-well PCR plates containing dried down LNA primer sets for one 10 µl realtime PCR reaction per well. The LNA primer sets are designed for optimal performance with the Universal cdna Synthesis Kit II and the ExiLENT SYBR Green master mix, kit. The performance of the LNA primer sets will be affected if they are used in combination with less than optimal reagents. Contents Human mirnome panel I and II, V4 384-well PCR plates supplied with LNA primer sets dried down, one 10 µl reaction per well: Panel I Panel II 372 LNA primer sets for the amplification of human micrornas 1) 380 LNA primer sets for the amplification of human micrornas 1) 3 inter-plate calibrators 3 inter-plate calibrators 3 primer sets for reference genes 2) 1 blank well 5 RNA Spike-in control primer sets 3) 1 blank well Contents Mouse&Rat mirnome panel I and II, V4 384-well PCR plates supplied with LNA primer sets dried down, one 10 µl reaction per well: Panel I Panel II 372 LNA primer sets for the amplification of mouse and rat micrornas 1) 380 LNA primer sets for the amplification of mouse and rat micrornas 1) 3 inter-plate calibrators 3 inter-plate calibrators 3 primer sets for reference genes 2) 1 blank well 5 RNA Spike-in control primer sets 3) 1 blank well 7

8 Contents Serum/Plasma Focus microrna PCR panel, V4 PCR plates compatible with various real-time PCR instruments are available and supplied with LNA primer sets dried down, one 10 µl reaction per well: 96-well (2 plates) 179 LNA primer sets for the amplification of human micrornas 1+2) 2x3 Inter-plate calibrators 384-well plate (2 panels per plate) 2x179 LNA primer sets for the amplification of human micrornas 1+2) 2x6 Inter-plate calibrators 5 RNA Spike-in control primer sets 3) 2x5 RNA Spike-in control primer sets 3) 2 blank wells - 1 in each plate 2x2 blank wells Contents Cancer Focus microrna PCR panel, V4 PCR plates compatible with various real-time PCR instruments are available and supplied with LNA primer sets dried down, one 10 µl reaction per well: 96-well (1 plates) 84 LNA primer sets for the amplification of human micrornas 1) 384-well plate (4 panels per plate) 4x84 LNA primer sets for the amplification of human micrornas 1) 3 Primer sets for potential reference genes 2) 4x3 Primer sets for potential reference genes 2) 3 Inter-plate calibrators 4x3 Inter-plate calibrators 5 RNA Spike-in control primer sets 3) 4x5 RNA Spike-in control primer sets 3) 1 blank well 1 blank well 1) Please go to to download plate layout files. 2) Human Panels and Cancer Focus Panel: Three snrnas (U6snRNA, SNORD38B, SNORD49A). Mouse&Rat Panels: Three snrnas (U6snRNA, RNU5G, RNU1A1). Serum/plasma Focus Panel: mir-103, mir-191, mir-423-5p, mir-16, mir-425, mir-93, mir-451 are regarded reference gene candidates. 3) The RNA Spike-in control primer sets targets the UniSp6 RNA spike-in supplied in the Universal cdna synthesis kit II and the 4 RNA spike-ins contained in the RNA Spike-in kit (UniSp2, UniSp4, UniSp5, and cel-mir-39-3p). 8

9 Pick-&-Mix microrna PCR Panel, (product # and ) The Pick-&-Mix microrna PCR Panels consist of either 96-well PCR plates or 384-well PCR plates containing custom selections of dried down microrna LNA PCR primer sets for one 10 µl real-time PCR reaction per well, ready-to-use. PCR plates compatible with various real-time PCR instruments are available. The LNA primer sets are designed for optimal performance with the Universal cdna Synthesis Kit II and the ExiLENT SYBR Green master mix kit. The performance of the LNA primer sets will be affected if they are used in combination with less than optimal reagents. Contents PCR plates supplied with customer defined LNA primer sets, reference gene primer sets, and RNA Spike-in control primer sets, dried down, one 10 µl reaction per well 1) : 96-well PCR plates 1) 384-well PCR plates 1) 10 primer sets in 8 replicates 22 primer sets in 16 replicates 22 primer sets in 4 replicates 46 primer sets in 8 replicates 92 primer sets in 1 replicate 94 primer sets in 4 replicates 96-well flexible layout 380 primer sets in 1 replicate 384-well flexible layout 1) Please go to to configure a Pick-&-Mix microrna PCR Panel. 9

10 Storage All microrna PCR Panels, LNA primer sets and Reference gene primer sets The PCR panels and primer sets are shipped dried down at room temperature. The primers can be stored between +4 C and -20 C. Under these conditions, all components are stable for at least 12 months. After resuspension, it is recommended to store LNA primer sets and Reference gene primer sets in aliquots at -20 C to avoid repeated freeze-thaw cycles. Universal cdna synthesis kit II and ExiLENT SYBR Green master mix These kits are shipped on dry ice in polystyrene containers and should be stored at -15 C to -25 C. Do not store in a frost-free freezer. Under these conditions, all components are stable until the expiry date on the package or vial. It is recommended that the RNA spike-in be stored in aliquots at -20 C after re-suspension to avoid repeated freeze-thaw cycles. Additional required materials Reagents not supplied ROX or other passive reference dye (required on some PCR cyclers) Materials and Equipment not supplied Nuclease-free PCR tubes or plates for use with individual assays Nuclease-free, aerosol barrier pipette tips Nuclease-free, low nucleic acid binding microcentrifuge tubes (e.g. Eppendorf DNA LoBind tubes product and original NUNC vials) Sealing foils for PCR plates Micro-centrifuge and plate centrifuge Heating block, thermal cycler or other incubators Real-time PCR instrument Optional but recommended product mircury LNA Universal RT microrna PCR, RNA Spike-in kit (product# ) 10

11 Recommended accompanying products Exiqon recommends the Exiqon GenEx qpcr software for comprehensive and convenient data analysis. GenEx includes a wizard for import of Exiqon mircury Universal RT microrna PCR data and offers advanced methods to analyze real-time qpcr data in a few simple steps. The software includes tools for selection and validation of reference genes, data pre-processing and comprehensive statistical analyses. For more information and to download a free trial, please go to The following Exiqon GenEx products are available: Exiqon GenEx6 Industrial - Exiqon version of GenEx, qpcr analysis software, industrial license Exiqon GenEx6 Academic - Exiqon version of GenEx, qpcr analysis software, academic license Exiqon recommends the mircury RNA Isolation kits for purification of total RNA or small RNA fraction. RNA purified using the mircury RNA Isolation kits is fully compatible with the mircury LNA Universal RT microrna PCR System. The following kits are available: mircury RNA Isolation Kit Cell & Plant Provides a rapid method for purification of total RNA from cultured animal cells, small tissue samples, blood, bacteria, yeast, fungi and plants. mircury RNA Isolation Kit Tissue Specifically designed for purification of total RNA from tissue samples. mircury RNA Isolation Kit Biofluids Kit for purification of low abundant small RNAs from samples such as serum, plasma, urine and CSF. See Tip 2 11

12 Product description A unique system for microrna profiling mircury LNA Universal RT microrna PCR offers the best available combination of performance and ease-of-use on the microrna real-time PCR market because it unites two important features (Figure 2): 1. Universal RT One first-strand cdna synthesis reaction provides template for all microrna real-time PCR assays. This saves precious sample, reduces technical variation, requires use of less reagents, and saves time in the laboratory. 2. LNA PCR amplification Both PCR amplification primers (forward and reverse) are microrna specific and optimized with LNA. The result is: 1) exceptional sensitivity as well as extremely low background enabling accurate quantification of very low microrna levels and 2) highly specific assays that allow discrimination between closely related microrna sequences. mircury LNA Universal RT microrna PCR offers solutions both for high-throughput microrna expression profiling and for quantification of individual micrornas. Figure 2. Schematic outline of the mircury LNA Universal RT microrna PCR System. A poly-a tail is added to the mature microrna template (step 1A). cdna is synthesized using a poly-t primer with a 3 degenerate anchor and a 5 universal tag (step 1B). The cdna template is then amplified using microrna-specific and LNA -enhanced forward and reverse primers (step 2A). SYBR Green is used for detection (step 2B). Step 1: First-strand synthesis (RT) Mature microrna A) AAAAAAAAAAAAAAAAAAAA B) AAAAAAAAAAAAAAAAAAAA TTTTTTTTTTTTTTT 5 universal tag 3 degenerate anchor A) B) Step 2: Real-time PCR amplification mir-specific forward primer LNA LNA LNA TTTTTTTTTTTTTTT LNA LNA LNA mir-specific reverse primer 12

13 Control Assays There are 3 different types of control assays available in the mircury LNA Universal RT microrna PCR system: Reference assays and reference candidates Inter-plate calibrators RNA spike-in assays All of these control assays are available in the microrna PCR panels. One RNA spike-in template is provided with the Universal cdna Synthesis Kit II, while the assay that will detect this RNA spike-in is available in the ExiLENT SYBR Green master mix. Additionally 4 RNA spike-in templates are available as a spike-in kit. The assays for detection of these 4 templates as well as the reference assays are available as individual primer sets. Reference assays and reference candidates These assays detect small non-coding RNAs - either small nuclear RNA, small nucleolar RNA or microrna which frequently are found to be stably expressed across different cells or tissues. Reference assays may therefore be candidate assays for normalization in a profiling study with several samples. Though this is a good and recommended approach, great caution should be taken in the selection of reference genes. The danger of using endogenous reference genes lies in the assumption that a specific gene is expressed at the exact same level in all sample types. This is rarely true. The selection of reference genes should therefore be made with care, and should be specific to the sample set you are working with. The actual selection of reference genes to be used for normalization should always be based on a determination of the most stably expressed gene(s) which may be done using either GeNorm or NormFinder both tools that are integrated within Exiqon s GenEx data analysis software. When applicable, we recommend using microrna rather than small nuclear RNA or small nucleolar RNA for normalization. Firstly, small nuclear and nucleolar RNAs are longer RNA species than microrna and may purify differently from microrna. Moreover, small nuclear and nucleolar RNAs have entirely different functions as well as sub-cellular locations, and finally certain samples like blood plasma does not contain the small nuclear and nucleolar RNAs. Global mean normalization is a preferred alternative to using reference genes for normalization when working with panels and samples where many micrornas are screened per sample and where many microrna are called (detected) in all samples. Exiqon s GenEx will easily perform global mean normalization. To read more on normalization. See Tip 11 13

14 Inter-plate calibrators Three wells within the pre-defined Human, Mouse&Rat, and Focus PCR Panels contain the inter-plate calibrator assay (annotated as UniSp3 IPC in the plate layout files). Depending on the plate layout, the Pick-&-Mix Panels contain at least three inter-plate calibrators. Each of these wells, contain a pre-aliquoted primer pair and a DNA template and therefore the variation is very minimal from well-to-well and from plate-to-plate of these assays. The inter-plate calibrators are used for calibration between PCR plate runs which is very useful on some instruments that apply the cycle threshold method for Cq determination such as the ABI7900 PCR cycler. Since the inter-plate calibrators are independent of cdna quality in order to give a signal (but may be affected by PCR inhibitors in the sample) they may be used to quality control each plate run. Inter-plate calibration (IPC) can easily be performed in the data analysis software Exiqon s GenEx. Alternatively, IPC may be performed manually by using the IPC assay replicates as follows. For each plate, verify that the replicates have Cq standard deviation within 0.5. If this is not the case, eliminate the outlier if this can be identified. Calculate the average of the replicates for each plate, the overall average (average of IPC values from all plates). The calibration factor is calculated as the difference between plate average and overall average for each plate (calibration factor = IPCplate-IPCoverall). An example is shown in Table 1. Finally, calibrate each plate by subtracting the calibration factor from all Cq values in the plate. Table 1. Example of inter-plate calibration (IPC) Plate 1 Plate 2 Plate 3 let-7c IPC plate average IPC overall average Calibration factor let-7c calibrated

15 RNA spike-ins (synthetic control templates) The primary purpose of the RNA spike-ins and the matching primer pairs for detection of these is to provide controls for the quality of the RNA isolation, the cdna synthesis reaction and the PCR. RNA isolations may vary in yield, purity and integrity. Some sample types may contain compounds that inhibit the cdna synthesis or the PCR even though the RNA has been purified using the best standard procedures. This may result in different efficiencies of the reverse transcription or PCR between compared samples. One way to control for differences in efficiencies at each experimental level (isolation, cdna synthesis, and PCR) is by adding known RNA spike-ins to the sample prior to isolation and cdna synthesis. Use of the RNA spike-ins may also reveal if nucleases are present. After conducting the PCR but before initiating the data analysis, wells detecting RNA spike-ins are compared and outlier samples may be identified and considered for exclusion in the further data analysis. We have designed a flight of RNA spike-ins for this purpose. The UniSp6 RNA Spike-in template is provided with the Universal cdna synthesis kit II. Additionally four RNA spikein templates can be obtained with the separate RNA Spike-in kit. Here, a vial of three RNA spike-in templates, UniSp2, UniSp4 and UniSp5, mixed at different concentrations can be used during RNA isolation. The cel-mir-39-3p RNA template provided in a separate vial in the RNA Spike-in kit can be mixed with the UniSp6 template from the Universal cdna synthesis kit II to obtain two different template concentrations. This combination can be added during the cdna synthesis. Five wells in pre-defined PCR panel plates contain the matching primer sets. The selection of RNA Spike-in control primer set in the Pick-&-Mix PCR Panel can be customized to the specific need. A UniSp6 control primer set is also provided with the ExiLENT SYBR Green master mix kit, which is to be used with our non-plate based PCR primer set products.the RNA spike-ins are shipped dried down and must be re-suspended before use. If the RNA Spike-in kit with multiple RNA spike-ins is used, follow the protocol accompanying that product. If UniSp6 is to be used alone: 1. Re-suspend the UniSp6 RNA spike-in by adding 80 μl nuclease free water to the tube. 2. Mix by vortexing and spin down. Leave for min. on ice to fully dissolve RNA spike-in. Mix by vortexing and spin down. Store in aliquots at -20 C 3. Prior to the RT reaction, add 1 μl synthetic spike-in (10 8 copies/μl) per 20 ng sample RNA. 15

16 An alternative application of the UniSp6 RNA spike-in is as inter-plate calibrator. This is only relevant when using individual LNA primer sets and Reference gene primer sets in a multi-plate set-up. The microrna PCR panels already contain an inter-plate calibrator. Add 1 μl synthetic spike-in (10 8 copies/μl) to 20 ng of a complex RNA sample (e.g. total RNA from MS2, yeast, or a cell line; not provided with the kit). Proceed with first strand synthesis and subsequently real-time PCR as described in the protocols of the current instruction manual. At least one spike-in amplification reaction per PCR plate is used for inter-plate calibration. See Tip 9 Experimental design Before starting the experiment, it is essential to consider the experimental setup and consider the number of replicates needed for obtaining significant results replicates being technical as well as biological. The number of biological replicates required varies from experiment to experiment depending on the variation within and between the groups. We recommend that a No Template Control (NTC) is included in the study every time a new experiment is set up, to set the background level. The most optimal NTC is a mock up sample preparation including only carrier RNA as sample. The NTC should be run on all assays included in the study. We define the background of an assay as 5 Cq values below the NTC level. Furthermore we recommend including spike-ins found in the Spike-in kit to provide full quality control over all steps in the profiling (see figure 3). Finally it is necessary to include a number of candidate reference mirnas, which are expected to be constitutively expressed across the different experimental conditions, for data normalization. Figure 3. Experimental design Sold as separate Spike-in kit (203203) Comes with mirsign mir-21 Oncogene Assay kit Included in all panel products UniSp2 UniSp4 UniSp5 cel-mir-39-3p UniSp6 UniSp3 Sample n Sample n+1 Sample preparation evaluation cdna synthesis evaluation Inter plate Calibration NTC 16

17 Before starting the Experiment Important note RNA work requires specific handling and precautions to prevent RNase contamination of the reagents and degradation of the RNA sample. Find information on how to handle RNA in the tips section starting on page 52. The tips section also provides simple guidelines for good laboratory practice to ensure optimal performance of PCR experiments. Before setting up a real-time PCR experiment, there are a number of practical experimental design parameters that should be considered: RNA input - The mircury LNA Universal RT microrna PCR protocol is optimized for use of 20 ng total RNA per 20µL cdna synthesis reaction. The exact amount of total RNA needed depends on whether the downstream application is individual assays or panels. Furthermore, the amount of total RNA to be used may also vary depending on the microrna expression levels in the cells or tissue to be analyzed. For highly expressed micrornas it is possible to use down to 10 pg total RNA as starting material. For weakly expressed micrornas it may be possible to use up to 200 ng of total RNA; however, in samples with high amounts of PCR inhibitors (e.g. FFPE tissue samples), this may not be feasible. Finally, inhibitors may be present in RNA preparations from certain samples e.g. serum and plasma. Prior to conducting a larger microrna profiling study, it is recommended to optimize the amount of input RNA to the RT reaction in order to avoid conducting a larger study where inhibition occurs sporadically throughout the data set. Information on how to extract and handle RNA can be found in the tips section. In short, total RNA should be prepared using a method that preserves small RNA species. DNase treatment may be necessary. When using commercially available kits, please ensure that the total RNA preparation is guaranteed to contain micrornas. 17

18 Note: Blood serum and plasma are particular sample types that require special RNA purification procedures and the amount of RNA present in the samples can usually not be accurately determined. Due to the low levels of micrornas and potentially high levels of inhibitors in samples derived from serum and plasma, specific recommendations for how to set up experiments using these types of sample can be found in the mircury LNA Universal RT microrna PCR, Instruction Manual Biofluid samples. Normalization when running individual assays or when configuring a Pick-&-Mix microrna PCR panel it is important to consider how the data will be normalized. For tips and recommendations on choosing the correct reference genes and how to test and validate reference genes, please see section on reference assays (page13) and the Tip 11 (page 58). See Tip 11 Excess volumes required for pipetting Liquid handling with pipettes or pipetting robots require excess volumes of reagents due to loss during pipetting. The loss depends on the available pipetting system but losses in the range from 10% -25% are not uncommon. All protocols in the current instruction manual reflect the required reaction volumes and pipetting volumes should be adjusted according to accommodate the pipetting loss of the available pipetting system. ROX ROX is a passive reference dye used by some PCR cycles to obtain a robust read over the entire array of wells in a 96- or 384-well PCR plate. The requirement for ROX is instrument dependent and we recommend to follow the instrument manufactures guidelines on this (see also Tip 8). See Tip 8 ABI instruments the default settings on ABI real-time PCR cyclers are not suitable for running mircury LNA Universal RT microrna PCR. Settings need to be changed from automatic to manual background and threshold settings to obtain valid PCR data (see also Tip 10). Furthermore, if the dataset is to be analyzed using the GenEx software, it is important that the experiment is set up as an AQ experiment, not RQ. To ensure correct settings, download the instrument settings file at See Tip 10 18

19 RNA spike-ins consider how the RNA spike-ins should be applied in the planned study. Please consult the section on RNA spike-ins on page 15. Protocols for the first-strand cdna synthesis and real-time amplication follows on the next pages: A. Individual assays, please go to page 20 B. Human and Mouse&Rat microrna PCR Panels, please go to page 29 C. Focus microrna PCR Panels, please go to page 36 D. Pick-&-Mix microrna PCR Panels, please go to page 43 Figure 4 gives an overview and helps identifying which protocol to follow as well as the recommended cdna reaction volume needed for a given sample and assay type. Figure 4. Protocol overview Sample type Cells or tissue Serum plasma or other biofluids Panels or primer sets: mirnome panel I mirnome panel I+II Pick-&-Mix/Focus Individual assays (<96) mirnome panel I mirnome panel I+II Pick-&-Mix/Focus Individual assays (<96) Universal cdna reactions per kit Universal cdna reaction volume 20 µl 40 µl 10 µl 20 µl 10 µl 40 µl 80 µl 10 µl 20 µl 10 µl Dilution of cdna in ExiLENT Master Mix 100x 80x 50x 40x Use standard printed manual or download from Use biofluids manual from 19

20 Protocol A - Individual assays This protocol is used for conducting the first-strand cdna synthesis and real-time PCR, using the individual assays for human, mouse and rat (product numbers xxx). If working with serum plasma samples or other biofluids, please refer to the specific mircury LNA Universal RT microrna PCR Instruction Manual for biofluid samples at Before using the LNA PCR primer mix or the Reference gene primer mix for the first time, the primers must be re-suspended: Re-suspend the primer mix by adding 220 μl nuclease free water to the tube. Mix by vortexing and spin down. Leave on ice for minutes. The UniSp6 RNA spike-in control primer mix v2 is supplied with the master mix and is already dissolved. Ensure primer concentration is 10x before proceeding with the protocol (see page 5 for details). Additional required materials: 96- or 384-well plate real-time PCR cycler Thermocycler for first-strand cdna synthesis 96/384-well plates or tube strips compatible with available real-time PCR cycler Micro centrifuge Swing bucket centrifuge for 96-/384-well plates Protocol - Individual assays 20

21 Checklist: Have you considered excess volumes required for using liquid handling robotics see page 18 Did you consider how to use the RNA spike-ins please see page 15 ROX: The ExiLENT SYBR Green master mix, does not include the ROX passive reference dye. Please follow instrument manufactures recommendations ABI instruments: The use of manual background and threshold settings is necessary for obtaining correct PCR data. Make sure to have the optimal settings by downloading the instrument settings file at Furthermore, if the data is to be analyzed using GenEx, the experiment must be set up as an AQ experiment, not RQ Have you optimized the input amount to the RT reaction in order to avoid inhibition? Protocol - Individual assays 21

22 Workflow for individual primer sets (per sample) Phase I: Prepare RNA sample See page 52 for recommendations Phase II: cdna synthesis See protocol page 23. Phase III: real-time PCR amplification See protocol page Prepare adequate amount of pre-mixed primer + PCR Master mix and distribute into wells - Add cdna to all primer sets to be analyzed Protocol - Individual assays LNA TM primer sets Relative expression (log2) mir-21 let-7a Normal Tumor Total Tumor Tumor stroma Phase IV: Data analysis See data analysis guide online - Export data for further analysis - Data pre-processing, normalization and statistical analysis 22

23 Protocol The mircury LNA Universal RT microrna PCR protocol is a two-part protocol consisting of: 1. First-strand cdna synthesis (Step 1-5) 2. Real-time PCR amplification (Step 6-11) Important: Keep reagents and reactions on ice (or at 4 C) at all times. First strand synthesis Step 1 Dilute template RNA Adjust each of the template RNA samples to a concentration of 5 ng/μl using nuclease free water. Step 2 Prepare reagents Gently thaw the 5x Reaction buffer and nuclease-free water, and immediately place on ice. Mix by vortexing. Re-suspend the RNA spikeins according to the appropriate RNA Spike-ins protocol (see page 15), leave on ice for minutes. Immediately before use, remove the Enzyme mix from the freezer, mix by flicking the tubes and place on ice. Spin down all reagents. Protocol - Individual assays 23

24 Step 3 Combine reagents according to Table 2 Note: remember to calculate necessary excess volume for pipetting and robotic dead volume. If performing first-strand cdna synthesis on multiple RNA samples, it is recommended to prepare an RT working solution of the 5x Reaction buffer, water, Enzyme mix and RNA spike ins (in the proportion indicated in the first four lines of Table 2). The following procedure is recommended: 1. Prepare the required amount of RT working solution and place it on ice. 2. Dispense RT working solution into nuclease free tubes. 3. Dispense template RNA in each tube. Table 2 Reverse transcription reaction setup Reagent Volume (µl), RT reaction 5x Reaction buffer 2 Nuclease-free water 4.5 Enzyme mix 1 Synthetic RNA spike ins, optional 0.5 replace with H 2 O if omitted Template total RNA (5 ng/µl) 2 Total volume 10 Step 4 Mix and spin reagents Mix the reaction by very gentle vortexing or pipetting to ensure that all reagents are thoroughly mixed. After mixing, spin down. Protocol - Individual assays Step 5 Incubate and heat inactivate 1) Incubate for 60 min at 42 C. Heat-inactivate the reverse transcriptase for 5 min at 95 C. Immediately cool to 4 C. Store at 4 C or freeze. 1) The protocol can be interrupted at this stage. The undiluted cdna may be kept at -20 C for up to 5 weeks (optional store at 4 C for up to 4 days). It is recommended that synthesized cdna is stored in low-nucleic acid binding tubes or plates. 24

25 qpcr protocol Step 6 Prepare reagents for real-time PCR Place cdna (from Step 5), nuclease free water and PCR Master mix on ice and thaw for min. Protect the PCR Master mix vials from light. Immediately before use, mix the PCR Master mix by pipetting up and down. The rest of the reagents are mixed by vortexing and spun down. Step 7 Dilute cdna template 80x in nuclease free water 2) Immediately before use, dilute only the amount of cdna template needed for the planned real-time PCR reactions 80x in nuclease free water (e.g. add 395 μl nuclease free water to each 5 μl of reaction). It is important that low-nucleic acid binding tubes or plates are used. It is not recommended to store the 1:80 dilution of cdna. Recommendation: Include a passive reference dye in the cdna dilution if advised by instrument manufacturer. Please note that the PCR Master mix does not include ROX. The amount of ROX required is instrument dependent and it is important to refer to the manufacturer s recommendations when deciding how much ROX to use, see Tip 8. See Tip 8 Protocol - Individual assays 2) Adjust volumes to accommodate your in-house liquid handling system volume loss when pipetting 25

26 Step 8 Combine PCR Master mix, PCR primer mix and cdna according to Table 3. Mix thoroughly Note: remember to calculate necessary excess volume for pipetting and robotic dead volume. When multiple real-time PCR reactions are performed with the same microrna primer set, it is recommended to prepare a primer master mix working-solution of the PCR primers and the PCR Master mix (in the proportion indicated in Table 3). The following procedure is recommended: 1. Prepare the required amount of primer:master mix working-solution (see Table 3) and place it on ice. It is recommended to include excess of all reagents in the master mix to compensate for pipetting excess material. 2. Place the relevant volume of primer:master mix working-solution in PCR tubes/wells (see Table 3) and spin tubes/plate briefly in a centrifuge (1500g for 1 minute), to remove air bubbles. 3. Add cdna template to each tube/well. Table 3 Real-time PCR reaction, pr. 10 µl reaction 3) Reagent Volume (µl), 96/384-well plate, tubes or strips PCR Master mix 5 PCR primer mix 4) 1 Diluted cdna template 4 Total volume 10 Step 9 Mix and spin reagents Mix the reaction by gentle pipetting to ensure that all reagents are mixed thoroughly. After mixing cap tubes or strips or seal the plate with optical sealing as recommended by the manufacturer. Spin down in a centrifuge (1500g for 1 minute). The experiment can be paused at this point. Store the reactions protected from light at 4 C for up to 24 hours. Protocol - Individual assays 3) If using a 96-well cycler with a minimum recommended volume of 20 µl (as some ABI instruments), then use 10 µl reaction volume and set the instrument settings at 20 µl. 4) The PCR primer mix must be dissolved prior to real-time PCR set-up, see page

27 ABI instrument user? Apply manual baseline and threshold settings! Go to to download settings file Step 10 Real-time PCR amplification Perform real-time PCR amplification followed by melting curve analysis according to Table 4. Table 4 Real-time PCR cycle conditions Process step Settings, LC480 instrument 5) Settings, other instruments 3) Polymerase Activation/Denaturation 95 C, 10 min 95 C, 10 min Amplification 45 amplification cycles at 95 C, 10 s 60 C, 1 min, ramp-rate 1.6 C/s 6) Optical read 40 amplification cycles at 95 C, 10 s 60 C, 1 min, ramp-rate 1.6 C/s 6) Optical read Melting curve analysis 7) Yes Yes Step 11 Analyze data Perform initial data analysis using the software supplied with the realtime PCR instrument to obtain raw Cq values (Cp or Ct, depending on PCR instrument). If you are using an ABI instrument, please note that it is not recommended to use auto Ct settings. Furthermore, if the data is to be analyzed using Exiqon GenEx, the experiment must be set up as an AQ experiment, not RQ. For a guide on how to set manual baseline and threshold, refer to Tip 10, page 56 in the tips section. If you are using a Roche LC480 instrument, we recommend analysis using the 2nd derivative method. For tips on normalization, please see Tip 11, page 58. We recommend performing normalization and further data analysis with the Exiqon GenEx qpcr analysis software ( com/mirna-pcr-analysis). Please refer to our data analysis guide for recommendations. Protocol - Individual assays See Tip 11 27

28 5) Five additional amplification cycles are required when using the LC480 instrument to allow collection of assay data with Cp-values up to 40. 6) The ramp-rate of cooling from 95 C to 60 C should be set to 1.6 C/s. This is equivalent to 100% under standard cycling conditions on the ABI 7500, 7900 and Viia7 instruments. If the ramp rate of cooling is too rapid, performance may be compromised. 7) Melting curve analysis of the PCR product(s) is recommended to verify specificity and identity of the amplification reaction. Melting curve analysis is an analysis step built into the software of instruments. Please follow the instructions provided by the supplier. Note: The Tm of a PCR product depends on buffer composition, salt concentration and the PCR instrument. Protocol - Individual assays 28

29 Protocol B - Human and Mouse&Rat microrna PCR Panels This protocol is used for conducting the first-strand cdna synthesis and real-time PCR using the following products: Human mirnome PCR Panel (product numbers to ) Mouse&Rat mirnome PCR Panel (product numbers to ) If working with serum plasma samples or other biofluids, please refer to the specifi c mircury LNA Universal RT microrna PCR Instruction Manual for biofluid samples at Additional required materials: 384-well plate real-time PCR cycler Thermocycler for first-strand cdna synthesis Micro centrifuge Tube for mixing water and master mix (10 ml) Sealing foils for PCR plates Swing bucket centrifuge for 96/384-well plates Recommended: Liquid handling robot for pipetting Checklist: Have you considered excess volumes required for using liquid handling robotics see page 18 Did you consider how to use the RNA spike-ins please see page 15 ROX: The ExiLENT SYBR Green master mix, does not include the ROX passive reference dye. Please follow instrument manufactures recommendations ABI instruments: The use of manual background and threshold settings is necessary for obtaining correct PCR data. Make sure to have the optimal settings by downloading the instrument settings file at Furthermore, if the data is to be analyzed using GenEx, the experiment must be set up as an AQ experiment, not RQ. Have you optimized the input amount to the RT reaction in order to avoid inhibition? Protocol - Human, Mouse&Rat Panels 29

30 Workflow for Human and Mouse&Rat microrna PCR Panels (per sample) Phase I: Prepare RNA sample See page 52 for recommendations Phase II: cdna synthesis See protocol page 31. Phase III: real-time PCR amplification See protocol page Mix cdnas with PCR Master mix - Add cdna:pcr Master mix to PCR plates Protocol - Human, Mouse&Rat Panels Relative expression (log2) mir-21 let-7a Normal Tumor Total Tumor Tumor stroma Phase IV: Data analysis See data analysis guide online - Export data for further analysis - Data pre-processing, normalization and statistical analysis 30

31 Protocol The mircury LNA Universal RT microrna PCR protocol is a two-part protocol consisting of: 1. First-strand cdna synthesis (Step 1-5) 2. Real-time PCR amplification (Step 6-9) Important: Keep reagents and reactions on ice (or at 4 C) at all times. First strand synthesis: Step 1 Dilute template RNA Adjust each of the template RNA samples to a concentration of 5 ng/μl using nuclease free water. Step 2 Prepare reagents Gently thaw the 5x Reaction buffer and nuclease-free water, and immediately place on ice. Mix by vortexing. Re-suspend the RNA spikein(s) according to the appropriate RNA Spike-in protocol (see page 15), leave on ice for minutes. Immediately before use, remove the Enzyme mix from the freezer, mix by flicking the tubes and place on ice. Spin down all reagents. Protocol - Human, Mouse&Rat Panels 31

32 Step 3 Combine reagents according to Table 5 Note: remember to calculate necessary excess volume for pipetting and robotic dead volume. If performing first-strand cdna synthesis on multiple RNA samples, it is recommended to prepare an RT working solution of the 5x Reaction buffer, water, Enzyme mix and RNA spike ins (in the proportions indicated in the first four lines of Table 5). The following procedure is recommended: 1. Prepare the required amount of RT working solution and place it on ice. 2. Dispense RT working solution into nuclease free tubes. 3. Dispense template RNA in each tube. Table 5 Reverse transcription reaction setup Reagent Panel I Volume (µl) 5x Reaction buffer 4 8 Nuclease-free water 9 18 Enzyme mix 2 4 Synthetic RNA spike ins, optional replace with H2O if omitted 1 2 Template total RNA (5ng/µL) 4 8 Total volume Panel I+II Volume (µl) Step 4 Mix and spin reagents Mix the reaction by very gentle vortexing or pipetting to ensure that all reagents are thoroughly mixed. After mixing, spin down. Protocol - Human, Mouse&Rat Panels Step 5 Incubate and heat inactivate 1) Incubate for 60 min at 42 C. Heat-inactivate the reverse transcriptase for 5 min at 95 C. Immediately cool to 4 C. Store at 4 C or freeze. 1) The protocol can be interrupted at this stage. The undiluted cdna may be kept at -20 C for up to 5 weeks (optional store at 4 C for up to 4 days). It is recommended that synthesized cdna is stored in low-nucleic acid binding tubes or plates. 32

33 qpcr protocol: Step 6 Prepare reagents for real-time PCR Place cdna (from Step 5), nuclease free water and PCR Master mix on ice and thaw for min. Protect the PCR Master mix vials from light. Immediately before use, mix the PCR Master mix by pipetting up and down. The rest of the reagents are mixed by vortexing and spun down. Step 7 Combine PCR Master mix, water and cdna and add to PCR plates 2) Mix thoroughly The following procedure is recommended to avoid low concentrations of cdna from adhering to tube surface: 1. Before removing the plate seal, briefly spin down the plate(s) in a plate centrifuge. 2. Combine 2x PCR Master mix and water. Panel I: 2000 μl 2x master mix and 1980 μl water, Panel I+II: 4000 μl 2x master mix and 3960 μl water. 3. Mix gently and spin down. 4. Add 20 µl cdna (panel I) or 40 µl cdna (panel I+II) and mix. 5. Add 10 µl PCR Master mix: cdna mix to each well 3). 6. Seal the plate with optical sealing as recommended by the instrument manufacturer. 7. Spin plate briefly in a plate centrifuge (1500g for 1 minute), to to collect the sample. The experiment can be paused at this point. Store the reactions protected from light at 4 C for up to 24 hours. Recommendation: Include a passive reference dye in the cdna dilution if advised by instrument manufacturer. Please note that the PCR Master mix does not include ROX. The amount of ROX required is instrument dependent and it is important to refer to the manufacturer s recommendations when deciding how much ROX to use, see Tip 8. See Tip 8 Protocol - Human, Mouse&Rat Panels 33

34 ABI instrument user? Apply manual baseline and threshold settings! Go to to download settings file Step 8 Real-time PCR amplification Perform real-time PCR amplification followed by melting curve analysis according to Table 6. Table 6 Real-time PCR cycle conditions Process step Settings, LC480 instrument 4) Settings, other instruments Polymerase Activation/Denaturation 95 C, 10 min 95 C, 10 min Amplification 45 amplification cycles at 95 C, 10 s 60 C, 1 min, ramp-rate 1.6 C/s 5) Optical read 40 amplification cycles at 95 C, 10 s 60 C, 1 min, ramp-rate 1.6 C/s 5) Optical read Melting curve analysis 6) Yes Yes Protocol - Human, Mouse&Rat Panels Step 9 Analyze data Perform initial data analysis using the software supplied with the realtime PCR instrument to obtain raw Cq values (Cp or Ct, depending on PCR instrument). If you are using an ABI instrument, please note that it is not recommended to use auto Ct settings. For a guide on how to set manual baseline and threshold, refer to Tip 10, page 56 in the tips section. Furthermore, if the data is to be analyzed using Exiqon GenEx, the experiment must be set up as an AQ experiment, not RQ. Alternatively, use ABI settings files available from If you are using a Roche LC480 instrument, we recommend analysis using the 2nd derivative method. For tips on normalization, please see Tip 11, page 58. We recommend performing normalization and further data analysis with the Exiqon GenEx qpcr analysis software ( Please refer to our data analysis guide for recommendations. See Tip 11 34

35 2) Adjust volumes to accommodate your in-house liquid handling system and the inaccuracy these have when pipetting. 3) Corresponding to 0.05ng total RNA starting material pr. PCR reaction. 4) Five additional amplification cycles are required when using the LC480 instrument to allow collection of assay data with Cp-values up to 40. 5) The ramp-rate of cooling from 95 C to 60 C should be set to 1.6 C/s. This is equivalent to 100% under standard cycling conditions on the ABI 7500, 7900 and Viia7 instruments. If the ramp rate of cooling is too rapid, performance may be compromised. 6) Melting curve analysis of the PCR product(s) is recommended to verify specificity and identity of the amplification reaction. Melting curve analysis is an analysis step built into the software of instruments. Please follow the instructions provided by the supplier. Note: The Tm of a PCR product depends on buffer composition, salt concentration and the PCR instrument. Protocol - Human, Mouse&Rat Panels 35

36 Protocol C - Focus microrna PCR Panels This protocol is used for conducting the first-strand cdna synthesis and real-time PCR, using the Cancer Focus microrna PCR panels or any of the Toxicology Focus microrna PCR Panels, 96-well plates and 384-well plates. If working with serum plasma samples or other biofluids, please refer to the specific mircury LNA Universal RT microrna PCR Instruction Manual for biofluid samples at Additional required materials: 96- or 384-well plate real-time PCR cycler Thermocycler for first-strand cdna synthesis Micro centrifuge Tube for mixing water and master mix (10 ml) Sealing foils for PCR plates Swing bucket centrifuge for 96/384-well plates Recommended: Liquid handling robot for pipetting Protocol - Focus Panels Checklist: Have you considered excess volumes required for using liquid handling robotics see page 18 Did you consider how to use the RNA spike-ins please see page 15 ROX: The ExiLENT SYBR Green master mix does not include the ROX passive reference dye. Please follow instrument manufactures recommendations ABI instruments: The use of manual background and threshold settings is necessary for obtaining correct PCR data. Make sure to have the optimal settings by downloading the instrument settings file at Furthermore, if the data is to be analyzed using GenEx, the experiment must be set up as an AQ experiment, not RQ Have you optimized the input amount to the RT reaction in order to avoid inhibition? 36