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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

RT-QPCR vs Traditional PCR
Essentials of Real Time PCR
User Bulletin #2

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