TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div class="page" title="Page 14">
<div class="layoutArea">
<div class="column">
<p>The EPIK™ miRNA Panel Assays protocol is a two‐step protocol consisting of:</p>
<p> </p>
<div class="page" title="Page 14">
<table>
<tbody>
<tr>
<td>
<div class="layoutArea">
<div class="column">
<p>Step 1.</p>
</div>
</div>
</td>
<td>
<div class="layoutArea">
<div class="column">
<p>Reverse transcription with miRNA-specific RT‐oligonucleotides and EPIK™ cDNA synthesis kit</p>
</div>
</div>
</td>
</tr>
<tr>
<td>
<div class="layoutArea">
<div class="column">
<p>Step 2.</p>
</div>
</div>
</td>
<td>
<div class="layoutArea">
<div class="column">
<p>Real-Time PCR using SensiSMART™ Master Mix and amplification primers</p>
</div>
</div>
</td>
</tr>
</tbody>
</table>
<p> </p>
<div class="page" title="Page 14">
<div class="layoutArea">
<div class="column">
<p>It is critical for the success of the experiment to follow the protocol carefully, from first‐strand cDNA synthesis to real‐time PCR amplification (approximately 2 hours). However, the procedure can be paused after the first‐strand cDNA synthesis and the undiluted cDNA may be stored at ‐20 °C for up to three days.</p>
</div>
</div>
</div>
</div>
</div>
</div>
</div>

[GUIDELINES]
<p><strong>KIT CONTENTS</strong></p>
<p> </p>
<div class="page" title="Page 4">
<table>
<tbody>
<tr>
<td>
<div class="layoutArea">
<div class="column">
<p>EPIK™ Cancer miRNA ASSAY COMPONENTS</p>
</div>
</div>
</td>
<td>
<div class="layoutArea">
<div class="column">
<p>KIT</p>
</div>
</div>
</td>
<td> </td>
</tr>
<tr>
<td>
<div class="layoutArea">
<div class="column">
<p>Cancer Assay Plates*</p>
</div>
</div>
</td>
<td>
<div class="layoutArea">
<div class="column">
<p>8 x 96-well plates**</p>
</div>
</div>
</td>
<td colspan="1" rowspan="3"> </td>
</tr>
<tr>
<td>
<div class="layoutArea">
<div class="column">
<p>RT Primer Pool - lyophilized</p>
</div>
</div>
</td>
<td>
<div class="layoutArea">
<div class="column">
<p>4 tubes (A, B, C and D)</p>
</div>
</div>
</td>
</tr>
<tr>
<td>
<div class="layoutArea">
<div class="column">
<p>RNA Spike - lyophilized</p>
</div>
</div>
</td>
<td>
<div class="layoutArea">
<div class="column">
<p>1 tube</p>
</div>
</div>
</td>
</tr>
<tr>
<td>
<div class="layoutArea">
<div class="column">
<p>EPIK 5x RT Buffer</p>
</div>
</div>
</td>
<td>
<div class="layoutArea">
<div class="column">
<p>1 x 32 μL</p>
</div>
</div>
</td>
<td colspan="1" rowspan="4"> </td>
</tr>
<tr>
<td>
<div class="layoutArea">
<div class="column">
<p>EPIK RT Enzyme</p>
</div>
</div>
</td>
<td>
<div class="layoutArea">
<div class="column">
<p>1 x 8 μL</p>
</div>
</div>
</td>
</tr>
<tr>
<td>
<div class="layoutArea">
<div class="column">
<p>2x SensiSMART™ SYBR Master Mix</p>
</div>
</div>
</td>
<td>
<div class="layoutArea">
<div class="column">
<p>4 x 1 mL</p>
</div>
</div>
</td>
</tr>
<tr>
<td>
<div class="layoutArea">
<div class="column">
<p>DEPC Water</p>
</div>
</div>
</td>
<td>
<div class="layoutArea">
<div class="column">
<p>3 x 1.8 mL</p>
</div>
</div>
</td>
</tr>
</tbody>
</table>
<div class="page" title="Page 4">
<div class="section">
<div class="layoutArea">
<div class="column">
<p>* For well definitions please see web site <a href="http://www.bioline.com/mirna" target="_blank">www.bioline.com/mirna</a><br /> ** Three 96-well configurations are available, depending on the real-time PCR machine used.</p>
<p> </p>
<p><strong>DESCRIPTION</strong></p>
<div class="page" title="Page 4">
<div class="section">
<div class="layoutArea">
<div class="column">
<p>Mature microRNAs (miRNAs) are endogenously biosynthesized across many species of eukaryotes. These single-stranded RNAs (~ 22 nucleotides long) are known to play important regulatory roles in animals and plants by targeting mRNA transcripts for cleavage or translational repression. To date, thousands of unique miRNAs have been identified (www.mirbase.org). Their expression levels vary greatly among species, tissues and in disorders.</p>
<p> </p>
<p>Detecting miRNAs remains a significant challenge, mainly due to the short lengths of the nucleotide sequences. Various methods for miRNA measurement are currently available and quantitative real-time PCR remains the method of choice for both convenience and reliability. Stem-loop structure-based assays have been successfully used for the quantification of replicating viruses and mature miRNAs, however these methods rely on sequence‐dependent probes or chemically modified primers for optimal specificity and are time‐consuming, labour‐intensive and suffer from sample-to-sample variability. There are many distinct advantages of EPIK™ miRNA Panel Assays over these miRNA‐detection methods, including:</p>
<ul>
<li>Highly specific; targeting only mature miRNA and not precursors and can discriminate between highly similar miRNAs.</li>
<li>Ultra-sensitive; can detect mature miRNA from as little as 10 pg of total RNA</li>
<li>Wide dynamic range of quantitation; all assays can detect mature miRNAs with greater than six logs of dynamic range (1 million fold changes).</li>
<li>Fast reaction time; the simple two-step protocol takes less than 2 hours from RNA to result.</li>
</ul>
</div>
</div>
</div>
</div>
<p>The workflow of EPIK™ miRNA Panel Assays consists of 2 independent stages, where users have the choice of pausing between each stage (Fig 1).</p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/gr4cz36.png" data-src="https://s3.amazonaws.com/pr-journal/gr4cz36.png" data-ofn="Screen Shot 2016-11-23 at 4.04.34 PM.png" /></p>
<p><strong>Fig. 1 EPIK™ miRNA Panel Assay powered by MiRXES™ technology.</strong> The EPIK™ miRNA Panel Assay comprises of a set of three specific primers. The conformationallyrestricted RT primer allows efficient hybridization only to the mature form of the target miRNA (Stage 1). The miRNA-specific forward and nested reverse real-time PCR primers confer further specificity and enable robust amplification of the target cDNA (Stage 2).</p>
<p> </p>
<p>All EPIK™ miRNA Panel Assays have been validated using both synthetic miRNA templates and total human RNA. Typically the assays detect as few as 100 copies of template per RT reaction with excellent assay efficiency and linearity (Fig. 2). These stem-loop structure-based assays are designed using MiRXES™ proprietary thermodynamics-based algorithms, enabling these assays to demonstrate superior sensitivities and specificities.</p>
<p> </p>
<p>For the amplification and detection stage with SensiSMART™ qPCR mixes, a commonly available DNA-binding dye (SYBR Green) is used, rather than a probe-based system. This leads to remarkable sensitivities as well as extremely low background, enabling the accurate detection of very low miRNA levels. In addition, these assays, allow the clear discrimination between miRNA sequences with high similarity.</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/gr5cz36.png" data-src="https://s3.amazonaws.com/pr-journal/gr5cz36.png" data-ofn="Screen Shot 2016-11-23 at 4.09.05 PM.png" /></p>
<p><strong>Fig. 2 Performance of EPIK™ miRNA Panel Assay for human miRNA.</strong> A synthetic miRNA was reverse-transcribed and amplified by SensiSMART™. The results illustrate the sensitivity and efficiency (99.97%) of the assay, allowing the detection of miRNAs at varying expression levels (10<sup>8</sup> to 100 copies), including low expressers.</p>
<p> </p>
<p>Please read this manual carefully to familiarize yourself with the EPIK™ miRNA Panel Assay protocol before starting (also available on <a href="http://www.bioline.com/mirna" target="_blank">www.bioline.com/mirna</a>).</p>
<p> </p>
<p><strong>STORAGE</strong></p>
<div class="page" title="Page 6">
<div class="section">
<div class="layoutArea">
<div class="column">
<p style="display: inline !important;">When stored under the recommended conditions and handled correctly, full activity of reagents is retained until the expiry date indicated on the outer box label.</p>
<p> </p>
<p>Avoid subjecting any plate or reagent to repeated freezing and thawing. Reagents should be stored according to their label, with plates and the reagent box being stored at -20 °C and the RNA Spike stored at -80 °C after reconstitution.</p>
<p> </p>
<p><strong>SAFETY INFORMATION</strong></p>
<p>When working with chemicals, always wear suitable personal protective equipment (PPE), including lab coat, gloves and safety glasses. <br /> <br />For detailed information, please consult the material data safety sheets (MSDSs) available on our website at <a href="http://www.bioline.com/mirna" target="_blank">www.bioline.com/mirna</a>.</p>
<p> </p>
<p><strong>PRODUCT SPECIFICATIONS</strong></p>
<div class="page" title="Page 7">
<div class="section">
<div class="layoutArea">
<div class="column">
<p>EPIK™ miRNA Panel Assays are powered by MiRXES™ technology. The conformationally restricted RT primers are designed so that there is specific hybridization to the mature miRNA target. Following a reverse transcription stage, a robust amplification of the newly synthesized cDNA is accomplished using miRNA-specific forward and reverse real-time PCR primers to confer further specificity and sensitivity.</p>
<p> </p>
<p>The EPIK™ miRNA Panel Assay protocol is optimized for use of up to 100 ng human total RNA per cDNA synthesis reaction (20 μL). The exact amount of human total RNA needed depends on the tissue state and can vary, depending on the type of cell, tissue or biofluid of interest and on the expression levels of the target miRNAs. As low as 10 pg of total RNA is sufficient for accurate quantification of highly expressed targets whereas up to 100 ng may be required for low expression miRNAs.</p>
<p> </p>
<p>The qPCR primers for EPIK™ miRNA Panel Assays are supplied lyophilized in 96-well white plates, which are compatible with the machines listed below (see<a href="http:// www.bioline.com/mirna" target="_blank"> www.bioline.com/mirna</a>).</p>
<p> </p>
<p>Plates with white wells provide improved signal-to-noise ratio in qPCR experiments and give the highest chance of detection of low-copy number miRNA. The white plates supplied can be used in most machines without re-calibration and as a direct substitute for clear plates.</p>
<p> </p>
<p>It is essential that the right plate type and the right reagent type are chosen for the machine you intend to use.</p>
</div>
</div>
</div>
</div>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/gr6cz36.png" data-src="https://s3.amazonaws.com/pr-journal/gr6cz36.png" data-ofn="Screen Shot 2016-11-23 at 4.12.16 PM.png" /></p>
<p><strong>EQUIPMENT AND REAGENTS TO BE SUPPLIED BY THE USER</strong></p>
<div class="page" title="Page 9">
<div class="section">
<div class="layoutArea">
<div class="column">
<ul>
<li>The following additional items are required:</li>
<li>Nuclease-free disposable plasticware</li>
<li>Plate seals suitable for qPCR</li>
<li>Microcentrifuge for 1.5 mL tubes</li>
<li>Plate centrifuge suitable for 96-well plates</li>
<li>Cooling block or ice bucket suitable for 96-well plates</li>
<li>Heating block or thermocycler capable of isothermal heating at 42 °C and 70 °C</li>
<li>Vortex</li>
</ul>
<p> </p>
<p><strong>REAL-TIME PCR MACHINE AND ROX LEVEL</strong></p>
<div class="page" title="Page 9">
<div class="section">
<div class="layoutArea">
<div class="column">
<p>Please ensure that you have the correct plate type and SensiSMART™ ROX level for the machine you intend to run the assays on. Different qPCR machines from different manufacturers have specific requirements for the method of normalization employed, as well as for the plate size and shape. Using the wrong plate may damage your instrument (see <a href="http://www.bioline.com/mirna" target="_blank">www.bioline.com/mirna</a>).</p>
<p> </p>
<p><strong>ROX</strong></p>
<div class="page" title="Page 9">
<div class="section">
<div class="layoutArea">
<div class="column">
<p>EPIK™ miRNA Panel Assays have been optimized for use in SYBR<sup>®</sup> Green- based real-time PCR on the real-time PCR instruments listed below, each of these instruments having the capacity to analyze the real-time PCR data with the passive reference signal either on or off.</p>
<p> </p>
<div class="page" title="Page 9">
<div class="section">
<div class="layoutArea">
<div class="column">
<ul>
<li>The EPIK™ Hi-ROX miRNA Panel Assays can be used on the ABI StepOne Plus.</li>
<li>The EPIK™ Lo-ROX miRNA Panel Assays can be used on: ABI 7500, 7500 Fast QuantStudio<sup>®</sup> 3&amp;5 Real-Time PCR System; QuantStudio™ 6 Real-Time PCR system; QuantStudio™ 7 Real-Time PCR system; QuantStudio™ 12K Flex Real-Time PCR system and ViiA7™. The EPIK™ Lo-ROX miRNA Panel Assays can also be used on the BioRad<sup>®</sup> CFX96 and Roche LightCycler<sup>®</sup> 480 that do not require the use of ROX.</li>
</ul>
<p> </p>
<p><strong>96-well plates</strong></p>
<div class="page" title="Page 10">
<div class="layoutArea">
<div class="column">
<p>The type of qPCR plate depends on which qPCR machine is to be used to run the experiment. The plates supplied will only fit machines fitted with a 96-well block. For advice on other plate types (384-well or 48-well) please contact Bioline Technical Support. We do not provide these assays for Rotor-Gene qPCR machines at the present time.</p>
<p> </p>
<div class="page" title="Page 10">
<div class="layoutArea">
<div class="column">
<p>Due to the low volume of the qPCR reaction, we recommend the use of one of the following machines in combination with the following plate types:</p>
<p> </p>
<div class="page" title="Page 10">
<table style="width: 579px;">
<tbody>
<tr>
<td style="width: 144px;">
<div class="layoutArea">
<div class="column">
<p>Plate type 1<br /> (0.1 mL, “low profile”)†</p>
</div>
</div>
</td>
<td style="width: 427px;">
<div class="layoutArea">
<div class="layoutArea">
<div class="layoutArea">
<div class="column">
<p>ABI StepOne Plus; ABI ViiA7 FAST; ABI 7500 FAST; QuantStudio<sup>®</sup><br /> 3 Real-Time PCR System FAST; QuantStudio® 3&amp;5 Real-Time<br /> PCR System FAST; QuantStudio™ 6 Real-Time PCR system FAST; QuantStudio™ 7 Real-Time PCR system FAST; QuantStudio™ 12K Flex Real-Time PCR system FAST</p>
</div>
</div>
</div>
</div>
</td>
</tr>
<tr>
<td style="width: 144px;">
<div class="layoutArea">
<div class="column">
<p>Plate type 2 (0.2 mL)††</p>
</div>
</div>
</td>
<td style="width: 427px;">
<div class="layoutArea">
<div class="layoutArea">
<div class="layoutArea">
<div class="layoutArea">
<div class="column">
<p>ABI ViiA7; ABI 7500; QuantStudio<sup>®</sup> 3 Real-Time PCR System; QuantStudio<sup>®</sup> 3&amp;5 Real-Time PCR System; QuantStudio™ 6 Real-Time PCR system; QuantStudio™ 7 Real-Time PCR system; QuantStudio™ 12K Flex Real-Time PCR system</p>
</div>
</div>
</div>
</div>
</div>
</td>
</tr>
<tr>
<td style="width: 144px;">
<div class="layoutArea">
<div class="column">
<p>Plate type 3 (0.1 mL)</p>
</div>
</div>
</td>
<td style="width: 427px;">
<div class="layoutArea">
<div class="column">
<p>BioRad CFX96; Roche LC480 (96-well block only)</p>
</div>
</div>
</td>
</tr>
</tbody>
</table>
<div class="page" title="Page 10">
<div class="layoutArea">
<div class="column">
<p>† This plate may also fit the following machines, though this operation is performed entirely at the users’ own risk, and results may be variable. Bioline does not accept any responsibility for damage caused by using the incorrect plate type: ABI 7900HT FAST. Please contact Bioline Technical Support for more advice.<br /> † † This plate may also fit the following machines, though this operation is performed entirely at the users’ own risk, and results may be variable. Bioline does not accept any responsibility for damage caused by using the incorrect plate type: ABI 7900HT; ABI 7300; ABI 7700; ABI 7000; BioRad iCycler IQ; BioRad IQ4; BioRad IQ5; BioRad Opticon; BioRad Chromo; Eppendorf Mastercycler ep realplex; Stratagene Mx3000; Stratagene MX3005; Stratagene MX4000; Techne Quantica. Please contact Bioline Technical Support for more advice.</p>
<p> </p>
<div class="page" title="Page 11">
<div class="section">
<div class="layoutArea">
<div class="column">
<p>Each plate is supplied with dried down PCR primers specific for the following</p>
<p>reactions:</p>
<ul>
<li>
<p>88 miRNA-specific detection primer pairs (arrayed in columns 1 to 11, rows A to H)</p>
</li>
<li>
<p>2 RNA Spike control primers pairs (in duplicate) for the detection of the RNA</p>
<p>Spike (column 12, rows A to D)</p>
</li>
<li>
<p>2 primer pairs (in duplicate) detecting artificial control DNA for plate-to-plate calibration (column 12, rows E to H)</p>
</li>
</ul>
<div class="page" title="Page 11">
<div class="section">
<div class="layoutArea">
<div class="column">
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/gr7cz36.png" data-src="https://s3.amazonaws.com/pr-journal/gr7cz36.png" data-ofn="Screen Shot 2016-11-23 at 4.13.41 PM.png" /></p>
<p><strong>Fig. 4 EPIK™ miRNA Panel Plate layout.</strong> Wells A1 to H11 (red) each contain specific miRNA qPCR primers. Wells A12 and B12 contain replicate RNA Spike Control 1 (orange). Wells C12 and D12 contain replicate RNA Spike 2 (orange). Wells E12 and F12 contain replicate Inter-plate Calibrator 1 (green). Wells G12 and H12 contain replicate Inter-plate Calibrator 2 (green).</p>
<p> </p>
<p>The EPIK™ Cancer miRNA Assay comprises eight plates. The miRNAs most likely to be found in cancer samples (as shown in the published literature) are arrayed over plates 1 to 4, to give a total of 352 separate miRNA-specific reactions. The 4 plates are supplied in duplicate so that the user can either:</p>
<ul>
<li>
<p>Perform a preliminary analysis of the experimental variation in one sample</p>
</li>
<li>
<p>Or perform an initial screen against two conditions as a singlicate experiment</p>
<p>(e.g. comparison of cancerous versus non-cancerous)</p>
</li>
</ul>
<p> </p>
<p><strong>IMPORTANT NOTES</strong></p>
<div class="page" title="Page 12">
<div class="layoutArea">
<div class="column">
<p>Handle RNA carefully to avoid contamination by RNases, often found on labware, fingerprints and dust. For optimal RNA stability, keep RNA frozen at -20 °C for short-term or -80 °C for long-term storage.</p>
<p> </p>
<p>It is important to work quickly when purifying RNA (see hints and tips on working with RNA at <a href="http://www.bioline.com/uk/rna-hints-and-tips" target="_blank">www.bioline.com/uk/rna-hints-and-tips</a>).</p>
<p> </p>
<p><strong>Handling and storing starting material</strong></p>
<div class="page" title="Page 12">
<div class="layoutArea">
<div class="column">
<p>We recommend using purified miRNA or total RNA with this panel rather than attempting direct detection of miRNA in partly purified sample types.</p>
<p> </p>
<p>It is recommended that the ISOLATE II miRNA Kits are used for the preparation of the samples, as this allows rapid, unbiased, phenol-free isolation of miRNA.</p>
<p> </p>
<p>The EPIK™ miRNA Panel Assay protocol is optimized for use of up to 100 ng total RNA per cDNA synthesis reaction (20 μL). Although the ratio between total RNA and specific miRNA is not fixed, measurement of total RNA provides a convenient way of estimating miRNA loading and an approximate methods for normalizing between experiments. If the ISOLATE II miRNA (BIO-52083) is used for example, the large RNA fraction concentration (as ng/μL), can be calculated from the final pure eluate and used as an estimate of total miRNA from the ISOLATE II miRNA column used to extract the miRNA fraction. This will only be correct if the elution volume of the large RNA fraction and miRNA fraction are identical.</p>
<p> </p>
<p><strong>Controls and calibrators</strong></p>
<div class="page" title="Page 13">
<div class="section">
<div class="layoutArea">
<div class="column">
<p>The RT-qPCR miRNA detection procedure outlined in this manual includes a synthetic RNA control (RNA Spike) which provides an accurate and convenient internal control for the experiment (for the suitability of such controls, see Redshaw <em>et al</em> (2013) Biotechniques <strong>54</strong>: 155-164 and Ho <em>et al</em> (2013) PLoS One <strong>8</strong>: e72463.</p>
<p> </p>
<p>The RNA Spike once reconstituted from its lyophilized state can be used in two modes:</p>
<ul>
<li>as an RNA spike to be used during the sample isolation, i.e. added to the lysis buffer of the RNA isolation kit: after reconstitution in 30 μL of nuclease-free water, 5 μL should be used per sample isolation and this can be used to detect differences in RNA purification.</li>
<li>an RT control for pre-purified miRNA, i.e. added during the reverse transcription stage: once reconstituted in 30 μL of nuclease-free water, addition of the RNA Spike in the reverse transcription stage (1 μL per RT reaction) will allow normalization between RT reactions, in the same way as reference genes are used in qPCR measurements of mRNA.</li>
</ul>
<p>Users should not attempt to use RNA Spike in both modes in the same experiment.</p>
<p> </p>
<p>If the user wishes to use their own calibration or plate-to-plate calibrator, then column 12 can be used to aliquot user-supplied primers. Control primers sets supplied in the 96-well plate target artificial sequences with no known homology to natural or artificial sequences deposited in miRBase (release 21, June 2014). As optimisation is required to ensure that user controls function properly, we recommend using the controls supplied.</p>
<p> </p>
<p><strong>Analysis</strong></p>
<div class="page" title="Page 13">
<div class="section">
<div class="layoutArea">
<div class="column">
<p>For convenience an Excel spreadsheet can be downloaded from the product page at <a href="http://www.bioline.com/mirna" target="_blank">www.bioline.com/mirna</a>, which can be used to analyse plate to plate variation as well as for analysis and presentation of the data.</p>
<p> </p>
<p><strong>WORKFLOW</strong></p>
<div class="page" title="Page 14">
<div class="layoutArea">
<div class="column">
<p>When working with the EPIK™ Cancer miRNA Panel Assay, it can be difficult for a single user with a single qPCR machine to run all the plates in one day. We suggest that in order for all the panels to be treated the same, storage should occur just after cDNA synthesis. All the cDNA reactions must be treated identically, so if it is not possible to run all the plates within one day, all the cDNA reactions must be frozen at ‐20 °C once completed. This will ensure that all cDNA reactions are subjected to the same number of freeze- thaw cycles.</p>
<p> </p>
<p>When a qPCR machine is available, remove a cDNA reaction from storage and prepare the qPCR as detailed in the protocol. Thaw the qPCR master mix and cDNA as late as possible before running the qPCR. Once mixed the cDNA:qPCR master mix should be loaded into the PCR plate and run on the instrument immediately. Storage of the ready to run plate at this point, even on ice, may result in non-specific primer-dimer formation.</p>
<p> </p>
<p>When the first run is nearly finished, remove the next cDNA reaction tube and repeat the process with the next plate.</p>
<p> </p>
<div class="page" title="Page 15">
<div class="section">
<div class="layoutArea">
<div class="column">
<p>The remaining tubes and plates can be deployed in a similar way. The user should allow sufficient time so that all the real-time data can be collected in as short a time as possible, as we recommend that the cDNA is stored at -20 °C for no more than three days. It should be possible for a single user with a single qPCR machine to run all the samples within 72 hours.</p>
<p> </p>
<p>In order to minimize the effect of -20 °C storage, we we recommend processing the plates of the same panel as close as possible in time, running successively panels A, B, C and D.</p>
<p> </p>
<p><strong>Using a single qPCR Machine</strong></p>
<div class="page" title="Page 21">
<div class="section">
<div class="layoutArea">
<div class="column">
<ol>
<li>The same qPCR machine type should be used to perform all of the qPCR cycling and analysis. While some plate types will fit in qPCR machines from different manufacturers, you are at risk of damaging your qPCR machine by using the wrong plate type. In addition, data from different makes of qPCR machines are processed using different methods. Using multiple manufacturers’ qPCR machines will make your analysis more complicated.</li>
<li>Perform the cDNA synthesis.</li>
<li>Store all the cDNA reactions at ‐20 °C once complete.</li>
<li>When the qPCR machine is available, remove the first cDNA reaction tube and thaw gently on ice. This storage step ensures that all the samples have been treated in a similar way, in that all the samples are subjected to one cycle of freeze/thaw.</li>
<li>Remove the first cDNA reaction tube from ice and follow the protocol steps. Make sure that the plate is sealed with a qPCR-compatible plate seal.</li>
<li>Place the plate in the qPCR machine and run the qPCR program.</li>
<li>Once prepared, plates should NOT be frozen at -20 °C. Plates should be processed immediately after preparation.</li>
<li>Start preparation of the next plate so that it is ready to be placed in the qPCR machine immediately after all the data from the first plate has been collected. We recommend starting 15-20 min before the end of the run.</li>
<li>Repeat steps 1 to 8 with the remaining plates. There are 8 plates in total for the Cancer panel, 4 plates for both the Biofluid and Stem Cell panels.</li>
</ol>
<p> </p>
<div class="page" title="Page 21">
<div class="section">
<div class="layoutArea">
<div class="column">
<p>The user should allow sufficient time so that all the real-time data can be collected in as short a time as possible. We recommend that the cDNA is stored at -20 °C for no more than three days. It should be possible for a single user with a single qPCR machine to run all the samples within 72 hours.</p>
<p> </p>
<p><strong>Using multipe qPCR machines</strong></p>
<div class="page" title="Page 22">
<div class="layoutArea">
<div class="column">
<p>If the user has multiple identical qPCR machines, all the reactions (RT and qPCR) should be performed in parallel. Take steps to ensure that all the RT reactions and qPCR reactions are treated identically. You should make sure that the same number of freeze/thaw steps is applied across replicates.</p>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.