DNA Microarray Core Facility

Real-Time PCR

Real-Time PCR

RT-PCR (ABI 7900 HT)

Overview

Real-Time PCR (RT-PCR) (reverse transcription-polymerase chain reaction) is the most sensitive technique for mRNA detection and quantitation currently available. Compared to Northern blot analysis and RNase protection assay, RT-PCR can be used to quantify mRNA levels from much smaller samples and, ultimately, quantitation of RNA from a single cell.
The benefit of RT-PCR is the ability to obtain quantitative results from the sensitive PCR assay. Since Real-Time PCR quantitates reaction products for each sample in every PCR cycle, the result is an amazingly broad dynamic range, with no user intervention required. Data analysis, including standard curve generation and copy number calculation, is performed automatically by the SDS software connected to our ABI 7900 HT RT-PCR machine.

Instrument

Use of the ABI 7900 HT is available for Real-Time assays or plate reads. This is a charged instrument service, please refer to pricing. The RT-PCR machine is located in 2196AB CIEMAS Building. First time users please email Heather Hemric to schedule a tutorial before using the machine. Online Signup is required to schedule use of this instrument.

High-Throughput Service

Full RT-PCR service with plates prepared with liquid handler is available. Please check our Liquid Handling Service page for details.

Pricing

Contact microarraylab@duke.edu or call (919)668-1583 for pricing

Supplies Needed

  1. Compatible PCR plate (Applied Biosystems P/N 4306737)
  2. Optical Adhesive Covers (AppliedBiosystems P/N 4313663)
  3. Commercially available RT-PCR reagents
    -cDNA synthesis
    -RT-PCR Master Mix (TaqMan or SYBR Green)
    -Sequence-specific primers for TaqMan reactions

Available Heat Blocks

  • 96-well
  • 384-well
  • TLDA microfluidic card block

Software

SDS 2.3 is available to RT-PCR customers free of charge

Workflow

  1. Read our RT-PCR Information and Instructions
  2. Design Primers
  3. Gather supplies and reagents
  4. Reserve time to use the 7900HT
  5. Perform the RT reaction to generate cDNA
  6. Make master mixes and load plate
  7. Run plate on 7900HT
  8. Retrieve data
  9. Analyze data

FAQ

link to FAQ page
If you have any further questions, please contact Heather Hemric at hhemric@duke.edu.