Real-Time PCR
<p><strong><span style="color:Black"><a name="Background" style=";">Background</a><br /></span></strong><span style="color: Black">The recent introduction of fluorescence-based techniques to PCR has allowed the development of real-time PCR, also called qPCR or kinetic PCR, which is widely used in gene expression analysis either as a primary method or as a confirmation of other methods such as microarray analysis. Real-time PCR is also the most commonly used method to monitor the efficiency of gene silencing by RNA interference. Other applications include measurement of DNA copy number and transgene copy number, determination of viral titers, and single nucleotide polymorphism genotyping.<br /><br />Unlike conventional PCR that uses an end-point analysis of the amplicon, real-time PCR is based on the detection and quantitation of the PCR product as it is made, using either fluorogenic specific probes (such as Taqman probes) or double-stranded DNA binding fluorescent dyes such as SYBR Green.<br /><br />Early in the PCR amplification process (exponential phase), conditions are optimal and the amount of double-stranded DNA theoretically doubles at every cycle. Therefore, during this exponential phase of the reaction, the fluorescent signal increase is directly proportional to the amount of PCR product (<a href="/id0900b40a801fb4c4" style=";">Figure 1</a>)<br /><br />Real-time PCR data are analyzed after setting an arbitrary threshold that must intersect the signal curve <strong style="">in its exponential phase</strong>. The point at which the curve crosses the threshold is called <strong>T</strong><strong style="">hreshold Cycle (C<sub>T</sub>).</strong> The higher the starting copy number of the nucleic acid target is, the sooner a significant increase in fluorescence is detected, and the lower the C<sub>T</sub> value (<a href="/id0900b40a801fb4c3" style=";">Figure2</a>)<br /><br /><strong>STANDARD CURVE</strong><strong style="font-style: italic;"><br /></strong>Quantitative analysis of unknown samples commonly uses a calibration/standard curve representing C<sub>T</sub> values as a function of Log amount / copy number of starting material (<a href="/id0900b40a801fb4c5" style=";">Figure 3</a>)<br /></span></p>
<p><span style="color: Black">In �perfect� reactions, the number of amplicon copies doubles at each cycle, and the characteristics of the resulting standard curve are:<br /><br />- PCR Efficiency = 100%<br />- Correlation Coefficient = 1.000<br />- Slope = -3.300<br /><br />For accurate quantitation of unknown samples, the standard curve should be as close to a �perfect� curve as possible, like in the example presented in <a href="/id0900b40a801fb4c5" style=";">Figure 3</a>, and all test samples should be in the range of the standard curve.<br /><br /></span><strong><span style="color:Black">MELT CURVE<br /></span></strong><span style="color:Black">Applicable to assays using a fluorescent dye (SYBR Green) as detection method. A given piece of double-stranded DNA melts at a precise temperature which is primarily dependent on its base contents and length. As the temperature increases, the PCR product denatures and releases the associated SYBR Green, causing a sharp decline in fluorescence. The melt curve is obtained by plotting the negative first derivative values of the fluorescence intensity as a function of the temperature, which results in a peak.<br /><br />The melt curve is a good indication of how clean the PCR product amplified is; ideally a single, narrow peak (<a href="/id0900b40a801fb4c2" style=";">Figure 4</a>).<br /><br />The real-time PCR technology is widely and increasingly used, because it offers significant adv <!-- #993$ -->antages such as high sensitivity, wide quantification range, good reproducibility, and high throughput capabilities; it has become a critical tool for basic research and molecular diagnostics.<br /><br /><strong><a name="Guidelines" style=";"><span style="">Guidelines</span></a><br /></strong>The MBCF offers on a regular basis a 2 hour introductory class to qPCR. It is <strong style="">mandatory</strong> to take this class once if you wish to operate our machine. In addition to this tutorial, our staff can help you with different aspects of your qPCR project such as primer design, practical help, troubleshooting, etc... The MBCF is also available to run samples for you. In this case, you do not need to take the course. For more information, please contact us <a href="mailto:%28MBCF@mail.etsu.edu" style=";">(MBCF@mail.etsu.edu</a>) <br /><br />Here are some guidelines to get you started:<br /><br /><strong style=""><a name="Instrumentation_" style=";">Instrumentation</a><br /></strong>The MCBF is equipped with a Bio-Rad iCycler which combines a thermocycler unit (96-well plate format) and an optical module that reads fluorescence. This machine is very versatile and has PCR multiplexing capability since the optical module can collect data for up to 4 different fluorophores within the same well.<br /><br /><strong style=""><a name="Sample_RNA_" style=";">Sample RNA</a><br /></strong>In real-time RT-PCR experiments, using high quality total RNA is crucial.</span></p>
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<p><span style="color:Black"> Good ratio OD<sub>260nm/280 nm</sub> (? 1.8)</span></p>
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<p><span style="color:Black"> No degradation (28S/18S rRNA ratio of 2:1 on an agarose gel)</span></p>
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<p><span style="color:Black"> No/low contamination with genomic DNA (DNase treat your RNA)</span></p>
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<p><span style="color:Black"><strong style=""><em style="">Suggested Total RNA isolation kits:<br /><br /></em></strong>- Qiagen RNeasy Mini Kit including an on-column DNase digestion using RNase-free DNase set<br /><br /><a href="http://www1.qiagen.com/default.aspx" style=";" target="_blank">http://www1.qiagen.com/default.aspx</a><br /><br /> - Ambion - RNAqueous Mini and Midi Kits � <a href="http://ambion.com/" style=";" target="_blank">http://ambion.com</a><br /> - <strong>For high throughput:</strong> Promega Automated SV96 Total RNA Isolation System � can be used on the Beckman Biomek 2000<br /><br />robot, present in the MBCF. This kit is designed to isolate total RNA from cultured cells but we have used successfully isolated<br /><br />RNA from animal tissue (rat brain)<br /><br /><strong>For small size samples or low abundance transcripts</strong>, an RNA <strong>linear amplification</strong> step can be performed prior qRT-PCR analysis. Several companies propose kits in which the entire transcript population present in a sample is amplified between 1,000 and 5,000 times. This amplification is linear so the differential gene expression is preserved. For more information, visit the following websites:<br /><br /><br /><a href="http://www.invitrogen.com/content.cfm?pageid=1" style=";" target="_blank">http://www.invitrogen.com/</a><br /><a href="http://www.epibio.com/main.asp" style=";" target="_blank">http://www.epibio.com/main.asp</a><br /><br /><strong style="">Reverse Transcription<br /><br /></strong>Most of the time, first-strand cDNA synthesis is done using oligo(dT) and/or random hexamers, but gene specific primers can also be used<br /><br />Note:<br /><strong style="">We keep in stock IDT Ready-Made Oligo(dT) and Random Hexamers<br /></strong>Many commercial RT kits are available - The Invitrogen Superscript II kit is <span style="font-weight: 700;">in</span> <strong style="">stock in the MBCF<br /><br />Primer design - <em>The most important determinant for accurate/specific results<br /><br /></em></strong><em><strong>Predesigned primer sets for <span style="text-decoration:underline">many</span> genes are available.</strong> </em><strong style=""><em> </em>Qiagen <span style="text-decoration:underline">www.qiagen.com/GeneGlobe</span> and Invitrogen <span style="text-decoration:underline">www.invitrogen.com</span> </strong> (TaqMan primer/probes) are just two companies that offer validated primers.<br /><strong style=""><br /></strong>We can design qPCR primers for you but here are some recommendations if you prefer doing the design yourself.</span></p>
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<p><span style="color:Black">The primers should span a DNA region with no secondary structures. DNA folding programs are available online to use for free. Suggested link: <span style=""><span style="text-decoration:underline;"><a href="http://www.bioinfo.rpi.edu/applications/mfold/" style=";" target="_blank">http://www.bioinfo.rpi.edu/applications/mfold/</a></span><a href="http://www.bioinfo.rpi.edu/applications/mfold/" style="text-decoration: none;" target="_blank"> </a></span> Make sure to include folding/annealing temperature (55?C-68?C) and salt concentrations of the assay (commonly 50 mM Na+; 3 mM Mg2+)</span></p>
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<p><span style="color:Black">Ideal amplicon size: 50 to 200-250 bp</span></p>
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<p><span style="color:Black">If working on cDNA: primers should span at least one intron. Suggested link: <span style="text-decoration:underline;"><span style=""><a href="http://www.ensembl.org/" style=";" target="_blank">http://www.ensembl.org/</a></span></span></span></p>
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<p><span style="color:Black">Database including information regarding exon/intron junctions, transcript variants, protein information, etc...</span></p>
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<p><span style="color:Black">Ensure that the designed primer pair is specific for the gene of interest � We strongly recommend doing a BLAST search (<a href="http://www.ncbi.nlm.nih.gov/blast/" style=";" target="_blank">http://www.ncbi.nlm.nih.gov/blast/</a>) on these primers<br /><br /><strong style=""><em>Primer design softwares</em></strong></span></p>
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<p><span style="color:Black">Free webtools at <a href="http://www.idtdna.com/" target="_blank" style=";">http://www.idtdna.com</a>; Primer Quest, mFold, OligoAnalyzer 3.0</span></p>
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<p><span style="color:Black">GeneRunner</span></p>
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<p><span style="color:Black">Vector NTI � license site on ETSU Campus</span></p>
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<p><span style="color:Black">Visual OMP4 (DNA Software Inc.) � purchased by the MBCF; for primer pair and probe design; integrated DNA folding module; �single-plexing� and multiplexing � contact us for details</span></p>
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<p><span style="color:Black"><strong style="font-style: italic;">Primers</strong></span></p>
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<p><span style="color:Black">No formation of primer dimers</span></p>
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<p><span style="color:Black">No runs of 4 or more same nucleotides, especially G</span></p>
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<p><span style="color:Black">No more than 2 G or C in the final 5 nucleotides (primer 3� end) <span style=""> </span></span></p>
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<p><span style="color:Black">Match Tms as close as possible (? 0.5?C if possible)</span></p>
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<p><span style="color:Black">GC content of 20-80% - 50-60% is recommended</span></p>
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<p><a name="controlgenes" style=";"><span style="color:Black"><strong style="">Endogenous Control Genes for Normalization</strong></span></a></p>
<p><span style="color:Black">To minimize variations in the amount of input material (DNA, cDNA) used in the qPCR reaction, the results for your target gene should be normalized to those obtained for an endogenous control gene. The "perfect" control does NOT exist but should be validated for your system/model. Ideally, this control gene should be constitutively expressed in all cells, preferably expressed at similar levels as the gene(s) of interest and, most importantly, its expression should <span style="text-decoration:underline;">NOT</span> change in your application.</span></p>
<p><span style="color:Black"><em><strong>Examples of commonly used controls:</strong></em> GAPDH, GUSB, b2-Microglobulin, TATA Binding Protein (TBP), Transferrin Receptor (TfR)</span></p>
<p><span style="color:Black"> </span></p>
<p><span style="color:Black">The MBCF has, available to users, primer sets for human GAPDH and b2-Microglobulin, and for rat GAPDH, assays for which the qPCR conditions have already been optimized (SYBR Green assays).</span></p>
<p><span style="color:Black"><strong style="">Standard Curve</strong></span></p>
<p><span style="color:Black">The standard curve can be dilution series of:</span></p>
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<p><span style="color:Black"> DNA/cDNA � Arbitrary/<span style="text-decoration:underline;">Relative</span> number</span></p>
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<p><span style="color:Black"> Cloned PCR product � Copy number = <span style="text-decoration:underline;">Absolute</span> number</span></p>
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<p><span style="color:Black"> If amplicon size < 100 bp, a purified synthetic single-stranded oligonucleotide of the same sequence as the PCR product can be used � Copy number = <span style="text-decoration:underline;">Absolute</span> number</span></p>
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<li><span style="color:Black">T7-transcribed RNA template - accurate quantitation?</span></li>
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<p><span style="color:Black"><strong style="font-style: italic;">Important:</strong> For an accurate quantitation of unknowns samples, make sure all test points are in the range of the standard curve</span></p>
<p><span style="color:Black">How do I calculate copy numbers from a DNA concentration? <a href="/id0900b40a8030fda5" style=";">click here</a></span></p>
<p><span style="color:Black"><strong>Quantitation Strategies</strong></span></p>
<p><span style="color:Black">Different quantitation strategies are currently used:</span></p>
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<p><span style="color:Black"><strong><em>Absolute Quantitation</em></strong></span></p>
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<p><span style="color:Black"><strong> -</strong> Standards must be <strong>accurately quantitated</strong></span></p>
<p><span style="color:Black"> - <span style="">Requires </span><strong>multiplexing</strong>; amplification of the internal control and of the gene(s) of interest is performed in a single tube</span></p>
<p><span style="color:Black"> - Final result is usually reported relative to a <strong>defined unit</strong> (copies per ng of total RNA, per genome, per cell or mg of tissue - Used for example for <strong>viral load determination</strong> and <strong>inter-lab comparisons</strong></span></p>
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<p><span style="color:Black"><strong><em>Relative Quantitation</em></strong></span></p>
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<p><span style="color:Black"><em><strong> </strong> -</em> Results usually reported as a <strong>ratio Gene of Interest/Endogenous Reference</strong></span></p>
<p><span style="color:Black"><strong> - Best used for gene expression studies</strong> </span></p>
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<p><span style="color:Black"><strong><em>Comparative Quantitation</em></strong></span></p>
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<p><span style="color:Black"><em><strong> </strong> -</em> Requires a <strong>thorough preliminary assay optimization</strong></span></p>
<p><span style="color:Black"><strong> - Samples all prepared identically</strong></span></p>
<p><span style="color:Black"><strong> - No standard curve; Calibrator sample</strong> used as 1X standard in every run</span></p>
<p><span style="color:Black"> - Suitable for <strong>high throughput</strong> applications</span></p>
<p><span style="color:Black"> - <strong>Mathematical determination</strong> of fold increase / decrease in gene of interest expression across samples using<strong>: </strong></span></p>
<p><span style="color:Black"><strong> *</strong> No normalization</span></p>
<p><span style="color:Black"> <strong> *</strong> Normalization using at least one reference gene and with efficiency correction (REST method) or without real-time</span></p>
<p><span style="color:Black"> efficiency correction (DDCt method)</span></p>
<p><span style="color:Black"> </span></p>
<p><span style="color: Black" lang="EN-GB">For more information about quantitation methods, visit <span style="color:Black"><a href="http://www.gene-quantification.info/" style="color: rgb(0, 0, 102); text-decoration: underline;" target="_blank">http://www.gene-quantification.info/</a></span></span></p>
<p><span style="color:Black"><strong style=""><a name="qPCR-setup" style=";">qPCR setup</a></strong></span></p>
<p><span style="color:Black"><strong style="font-style: italic;">MANUAL:</strong> takes about 2 hours to setup a full plate</span></p>
<p><span style="color:Black"><em><strong style="">AUTOMATED:</strong></em> using the Eppendorf 5070($20/plate), plus tips</span></p>
<p><span style="color:Black">Side-by-side comparisons of manual vs. automated plate setups have shown very comparable results in term of C<sub>T</sub> values and reproducibility between replicates (<a href="http://qcom.etsu.edu/mbcf/pcr_files/Real-time%20PCR/qPCR%20fig5.htm" style=";">Figure 5</a>, <a href="http://qcom.etsu.edu/mbcf/pcr_files/Real-time%20PCR/qPCR%20fig6.htm" style=";">Figure 6</a>)</span></p>
<p><span style="color:Black">How do I set-up my SYBR Green assay? <a href="/id0900b40a80224f1c" style=";">click here</a></span></p>
<p><span style="color:Black">Note: Prepare a sufficient volume of PCR mixture for the number of wells needed, including 4+ extra wells if the Eppendorf 5070 is used to setup the plate; minimal extra volume required, regardless of the number of wells used</span></p>
<p><span style="color:Black"><strong style="">Suggested run</strong></span></p>
<p><span style="color:Black">Run samples in triplicate</span></p>
<p><span style="color:Black">3 blank wells (No template)</span></p>
<p><span style="color:Black"><strong> </strong>3 negative control wells (RT minus) � at least once when using a new set of primers</span></p>
<p><span style="color:Black">4-5 point standard curve (each in triplicate)</span></p>
<p><span style="color:Black"><strong> </strong>1-27 samples in triplicate</span></p>
<p><span style="color:Black"><strong> </strong>If you need to run multiple plates for the same primer set, make sure to put <span style="text-decoration:underline;"><strong>at least one common sample on all plates</strong></span> for accurate comparison</span></p>
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<p><span style="color:Black">After the first run using a new set of primers, we recommend to check the amplicon:<span style=""> </span></span></p>
<p><span style="color:Black"><span style=""> </span> - <strong>size</strong> on an agarose gel</span></p>
<p><span style="color:Black"><span style=""> </span> - <strong>sequence</strong></span></p>
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<p><span style="color:Black"><strong style="">iQ/Cfx software</strong> - After running your assay on the either of the real-time PCR machines, data analysis must be done in your laboratory. Software is available at no charge for download on your machine</span></p>
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<p><span style="color:Black"><strong style="">Fees and requirements</strong></span></p>
<p><a name="PrimerDesign" style=";"><span style="color:Black"><strong>Primer Design by the MBCF</strong></span></a></p>
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<p><span style="color:Black"><strong> </strong>Design primers-probes</span></p>
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<p><span style="color:Black"> <span style=""> </span> - $75 - includes 1 free re-design</span></p>
<p><span style="color: Black"> - $100 non ETSU</span></p>
<p><span style="color: Black"> - multiplex designs: 2=$200 3=$300</span></p>
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<p><span style="color:Black"> You <strong><span style="text-decoration:underline;">must</span></strong> provide the Genbank Accession number of your gene of interest</span></p>
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<p><span style="color:Black"> We strongly recommend you to <strong><span style="text-decoration:underline;">sequence</span></strong> the amplicon you obtain to make sure this is the expected product</span></p>
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<p><span style="color:Black"><strong>Reaction setup</strong></span></p>
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<p><span style="color:Black">We recommend you setting up your qPCR using the benchtop UV-cabinet located in the MBCF, i.e. standard, unknown and primer dilutions, preparation of the PCR mixture, plate loading, etc...</span></p>
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<p><span style="color:Black">The MBCF provides the SYBR Green</span></p>
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<p><span style="color:Black">You must bring your own supplies (tips, tubes, gloves)</span></p>
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<p><span style="color:Black">Unless the plate is sealed prior to bringing to MBCF, you must use a dUTPase system (see us for details if needed)</span></p>
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<p><span style="color:Black"><strong style="">Equipment use fee</strong></span></p>
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<p><span style="color: Black" lang="FR">iCycler: $25 / run ETSU; $50 / run non ETSU</span></p>
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<p><span style="color:Black">Eppendorf 5070 robot: $20 / full plate</span></p>
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<p><span style="color:Black"><strong style=""><a name="Disposables" style=";">Disposables</a></strong></span></p>
<p><span style="color:Black">The MBCF sells disposable for use in qPCR, such as:</span></p>
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<p style="margin-left: 0.25in; margin-top: 4px; margin-bottom: 4px;" class="MsoNormal"><span style="color:Black"> Individual plates $4.39</span></p>
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<p style="margin-left: 0.25in; margin-top: 4px; margin-bottom: 4px;" class="MsoNormal"><span style="color:Black"> Individual optically clear tapes $1.20</span></p>
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<p style="margin-left: 0.25in; margin-top: 4px; margin-bottom: 4px;" class="MsoNormal"><span style="color:Black"> Pipet tips for the Eppendorf 5070</span></p>
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<p style="margin-left: 0.25in; margin-top: 4px; margin-bottom: 4px;" class="MsoNormal"><span style="color:Black"><strong> </strong>qPCR Supermix (Invitrogen, Bio-Rad, Qiagen, etc.)</span></p>
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<p><span style="color:Black"><span style="font-weight: 700">To use the iCycler</span></span></p>
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<p><span style="color:Black"> Must take the introductory course to qPCR offered by the MBCF at least once. You can re-take the course as a refresher as needed.</span></p>
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<p><span style="color:Black"> Must schedule time with MBCF</span></p>
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<p><span style="color:Black"> Must have an MBCF account</span></p>
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<p><span style="color:Black"> Do NOT analyze your data on the MBCF machine, software (Windows) is free to all users - Collect Data on Zip disk or burn to CD-R</span></p>
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<p><span style="color:Black"><span style="font-weight: 700">How can the MBCF aid you with your qPCR project?</span></span></p>
<p><span style="color:Black">We can help you with different aspects of your qPCR project depending on your needs. We can:</span></p>
<p><span style="color:Black"> - design your primers for a small <a href="#PrimerDesign" style=";">fee</a></span></p>
<p><span style="color:Black"> - help you get started with the design of your assay</span></p>
<p><span style="color:Black"> - sell you <a href="http://www.etsu.edu/com/mbcf/Services/RealTimePCR.aspx#Disposables" style=";">disposables</a> for qPCR</span></p>
<p><span style="color:Black"> - provide <a href="#controlgenes" style=";">endogenous control primers</a> we have designed & tested</span></p>
<p><span style="color:Black"> - show you how to use the iCycler</span></p>
<p><span style="color:Black"> - help you setup the plates using the Eppendorf 5070 robot - for a small fee</span></p>
<p><span style="color:Black"> - troubleshoot your qPCR assays and help you optimize your experimental conditions</span></p>
<p><span style="color:Black"> - and, if you are running short on time and technical expertise, we can set up and run your assays for you. Ask us for details!</span></p>
Real-Time PCR
Real-Time PCR
Real-Time PCR