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About Real Time PCR

The Molecular Genomics Core (MGC) offers both custom and pre-optimized assays for an assortment of applications. We offer support in all aspects of the project from assay design to data analysis. It’s our attention to detail from sample QC to statistical analysis that sets our service apart from all others.  Real Time PCR is the gold standard for validating gene expression data. Typically a list of genes with associated NCBI accession numbers are submitted for design of either TaqMan or SYBR assays.  Whenever possible, assays will be designed such that a primer or TaqMan probe transverses an exon-exon junction in order to reduce the contribution of genomic DNA.  The resulting probe/primers will be compared to the public database to ensure specificity.  Samples should be submitted in the form of total RNA either frozen or as an ethanol precipitate.  Once received, sample quantity will be determined via the NanoDrop which will be used to normalize RNA input for the reaction.  Two hundred nanograms of sample will then be used to assess the integrity of the sample via the Agilent BioAnalyzer.  Upon completion of QC, real-time analysis with be carried out on our ABI 7500 and a detail report generated.  Each report consists of sample concentration, sample quality (Electropherogram and Gel-like image), relative ratios, p-values, and bar graphs.

Real-Time PCR Services

  • Gene expression profiling
  • siRNA knockdown validation
  • Allelic discrimination / SNP genotyping
  • Transgenic mouse genotyping

Service Features & Options

  • Sample Quantitation via NanoDrop® ND-1000
  • Qualitative Sample analysis via Agilent BioAnalyzer®
  • Assay (primer/probe) design & optimization
  • SYBR/TaqMan Chemistry Options
  • Relative/Absolute Quantitation Options
  • Easy to understand data report with statistical analysis

Useful Links

To schedule an appointment or to discuss experimental design, send an email to Debbie Prusak or Dr. Tom Wood.

Gemonics Real Time Service Information

Q: How much total RNA do I need to supply?
A: We recommend submitting 1-5ug of Total RNA for our standard service. Amount of RNA required for Real Time Arrays is directly proportional to the number of assay plates being analyzed.

Q: What protocols do you recommend for RNA isolation?
A: Although there are a number of good alternatives we’ve experienced the greatest success with Ambion's line of products.

Q: How long will it take to get my results back?
A: Samples are handled on a first come first serve basis. It is a good idea to contact the MGC in the early stages of planning an experiment so that we can anticipate the samples arriving to us for processing. Once the samples are in the MGC, data will be available in 1-2 weeks.

Q: Are you able to assist with data analysis?
A: Yes, you will receive a detailed custom report with validity scores, fold changes and graphical presentation.

Q: Do I need to do any type of replications (biological or technical)?
A: In order to have data that is statistically relevant, you must submit a minimum of 3 biological replicates per treatment.

Q: Are you able to custom design assays for my project?
A: Yes, provide us with an NCBI accession number and we will design a custom assay.

Q: Are you able to assist with the Experimental design of My project?
A: All investigators who wish to pursue a project through the core facility are strongly encouraged to meet with the Genomics Core Staff to ensure proper experimental design and a clear understanding of sample requirements for processing through the facility.

To schedule an appointment or to discuss experimental design, send an email.

Terms Used in Quantitation Analysis

Active reference - Active reference means the signal is generated as the result of PCR amplification. The active reference has its own set of primers and probe.

Endogenous and exogenous controls are examples of active references.

Amplicon   A short segment of DNA generated by the PCR process.

Amplification plot The plot of fluorescence signal versus cycle number.

Baseline The initial cycles of PCR, in which there is little change in fluorescence signal.

Calibrator A sample used as the basis for comparative results.

CT (threshold cycle) The fractional cycle number at which the fluorescence passes the fixed threshold

Endogenous control – This is an RNA or DNA that is present in each experimental sample as isolated. By using an endogenous control as an active reference, you can normalize quantitation of a messenger RNA (mRNA) target for differences in the amount of total RNA added to each reaction.

Exogenous control – This is a characterized RNA or DNA spiked into each sample at a known concentration. An exogenous active reference is usually an in vitro construct that can be used as an internal positive control (IPC) to distinguish true target negatives from PCR inhibition. An exogenous reference can also be used to normalize for differences in efficiency of sample extraction or complementary DNA (cDNA) synthesis by reverse transcriptase. Whether or not an active reference is used, it is important to use a passive reference containing the dye ROX in order to normalize for non-PCR-related fluctuations in fluorescence signal.

Normalized amount of target - A unitless number that can be used to compare the relative amount of target in different samples.

NTC (no template control) - A sample that does not contain template. It is used to verify amplification quality.

Nucleic acid target (also called “target template”) - DNA or RNA sequence that you wish to amplify

Passive reference - A dye that provides an internal reference to which the reporter dye signal can be normalized during data analysis. Normalization is necessary to correct for forestallment fluctuations caused by changes in concentration or volume.

Reference A passive or active signal used to normalize experimental results.

Rn (normalized reporter) - The fluorescence emission intensity of the reporter dye divided by the fluorescence emission intensity of the passive reference dye

Rn+ The Rn value of a reaction containing all components, including the template

Rn- The Rn value of an un-reacted sample. The Rn- value can be obtained from:

  • The early cycles of a real-time PCR run (those cycles prior to a detectable increase in fluorescence), OR
  • A reaction that does not contain any template

ΔRn (delta Rn) - The magnitude of the signal generated by the given set of PCR conditions. The Δ Rn value is determined by the following formula: (Rn+) – (Rn-)

Standard A sample of known concentration used to construct a standard curve. By running standards of varying concentrations, you create a standard curve from which you can extrapolate the quantity of an unknown sample.

Threshold The average standard deviation of Rn for the early PCR cycles, multiplied by an adjustable factor. The threshold should be set in the region associated with an exponential growth of PCR product.

Unknown A sample containing an unknown quantity of template. This is the sample whose quantity you want to determine.

To schedule an appointment or to discuss experimental design, send an email, or call (409) 772-6349.