FGSR Real-time PCR Services

The FGSR offers real-time PCR services to assist users in verifying microarray results. All FGSR services utilize Applied Biosystems' TaqMan Gene Expression Assays. Please note that both the FGSR and Applied Biosystems recommend running all reactions in quadruplicate in order to generate the most meaningful and trustworthy data. There are several ways to use TaqMan assays:

TaqMan Gene Expression Assays: More than 600,000 pre-designed assays for human, mouse, rat, Arabidopsis, and Drosophila genes. TaqMan Assays are provided in a single-tube, 20X format. To find the appropriate assay for your gene, visit the Applied Biosystems or PANTHER websites.
Custom TaqMan Gene Expression Assays: If the assay for your gene is not available as a standard TaqMan assay, or your study organism is not one of the five offered for TaqMan, you can create a custom assay.
TaqMan Low Density Array Gene Signature Panels: The Low Density Array format is a sealed 384-well plate preloaded with a set of TaqMan assays. Eight sample loading ports allow you to perform 48 assays on each sample, while minimizing handling and ensuring that an experiment is easily repeatable between technicians or laboratories. The Gene Signature Panels are preformatted with a battery of assays that target a single pathway or process.
TaqMan Low Density Array: The Low Density Array format is also completely customizable by an investigator who wants the advantages of a single plate to assess multiple assays.

Real-time PCR Software

Your data will be generated with and must be viewed in Applied Biosystems' SDS software (for details, see your project page). If you can't install this software, the FGSR has workstations available for our customers to use.

Real-time PCR Data

Your results will be calculated using the Comparative CT method of relative quantitation. This will yield a precise fold change value for each gene of interest in each sample. In order to understand your results, you should understand each of the following terms:

Threshold Cycle (Ct): For a given well, the threshold cycle (Ct) represents the PCR cycle at which the SDS software first detects a noticeable increase in reporter fluorescence above a baseline signal
ΔCt: The difference between the threshold cycle of a sample assay and the threshold cycle of the corresponding endogenous reference: ΔCt = Ct(target) - Ct(endogenous control).
ΔΔCt: For a given cDNA target, the difference between the average ΔCt value of a target sample and the average ΔCt for the corresponding calibrator sample: ΔΔCt(test sample) = AvgΔCt(test sample) - AvgΔCt(calibrator sample). This value is used to calculate expression fold value by the equation: Expression fold value = 2^-ΔΔCt
RQ: The relative quantity, compared to the baseline sample. This number is the fold change of your sample

When we export your results to an Excel file, we will include the following columns:

Position - Position of the well containing the associated sample (well numbering begins with 1 for well A-1 and increases from left-to-right, top-to-bottom)
Sample - The sample name assigned to the associated sample at the time it was run
Detector - The name of the detector assigned to the sample/well position
Task - The task applied to the detector assigned to the sample/well position (Target or Endogenous Control)
Ct - The calculated threshold cycle (Ct) for the associated sample
ΔCt - The calculated normalized Ct (ΔCt) value for the associated sample
Avg ΔCt - The average ΔCt for the replicate group of the associated sample
ΔCt SD - The calculated standard deviation for the ΔCt value of the replicate group of the associated sample
ΔΔCt - The calculated ΔΔCt value for the replicate group of the associated sample
RQ - The calculated relative quantity for the replicate group of the associated sample expressed as a multiple of the calibrator sample (fold change)
RQ Min - The minimum relative quantity for the replicate group of the associated sample calculated by the software using the confidence interval set in the Analysis Settings dialog box
RQ Max - The maximum relative quantity for the replicate group of the associated sample calculated by the software using the confidence interval set in the Analysis Settings dialog box
Outlier - A flag that indicates whether the software omitted the sample/well as an outlier

Instructions for importing data files are available in the SDS software by clicking "SDS Online Help" under the Help menu, and then choosing "Complete the Basic Software Skills Tutorial."