Real-Time PCR
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 or replicates required. Data analysis, including standard curve generation and copy number calculation, is performed automatically.
- An overview of RT-PCR
- ABI Sequence Detection Systems Chemistry Guide
- SYBR or TaqMan
- Getting started with RT-PCR
- TaqMan Gene Expression Assays for Validating Hits From Fluorescent Microarrays (ABI White Paper)
Our service includes providing instructional support that will assist you in operating the ABI-7900. Please contact Heather Hemric for support and questions. To sign up for usage of the ABI-7900 click here.
Custom Assays
1. Select the chemistry you will use for your assay - SYBRgreen or TaqMan. For an explanation of these two systems plus the advantages/disadvantages of each chemistry, please read this guide.
2. Design your assay. Please read the following documents about designing custom assays.
- How to design a SYBRgreen assay
- How to design a TaqMan assay
- For designing primers for use with these assays, use Primer Express. This program is available in the Microarray Facility Computer Lab, located at 2208 CIEMAS.
3. If you do not want to design your own assay, but cannot find the assay you want from the Assays-on-Demand, Applied Biosystems will design an assay for you.
4. Choose endogenous control for TaqMan assays only. You will need one endogenous control per sample per plate. No replicates are needed.
5. Fill out Setup File. This file explains how your assay plate should be set up. Download the setup file to your PC, fill out as directed below, then save as a .txt file. This will be required for submission of your assay.
- Setup file template
- Example of SYBRgreen setup file
- Example of TaqMan setup file
- Directions for filling out setup file
5. Fill out submission form and bring 96-well plate with samples ready for RT-PCR to the Microarray Facility, at 2208 CIEMAS.
Choosing Endogenous Controls
Using the comparative CT method, endogenous controls can normalize the expression levels of target genes by correcting differences in the amount of cDNA that is loaded into PCR reaction wells.
Two steps are important in the endogenous control selection process:
1. Verify that the endogenous control is consistently expressed in the sample set to be tested. Because the endogenous control normalizes differences in the amount of cDNA that is loaded into PCR reaction wells, endogenous control expression must be uniform across all samples in the study. Endogenous controls can be tested for uniform expression by comparing the gene expression of several samples. Samples should be representative of the target's gene expression and should span the target's expected range of expression. Specifically, it is important to demonstrate that while the target's expression levels may range widely, expression of the endogenous control remains constant. When conducting this test, it is crucial to load identical amounts of cDNA for each test sample. To ensure this, measure cDNA concentrations with a spectrophotometer. Gene expression of endogenous controls should vary only slightly. The TaqMan(R) Human Endogenous Control Plate (P/N 4309920) can evaluate 11 housekeeping genes for their potential as endogenous controls. These 11 endogenous controls are also available as individual reagents.
2. Ensure that the target(s) and endogenous control have identical PCR efficiency. The endogenous control and target assays must have identical PCR efficiency. TaqMan(R) Endogenous Controls (both controls and targets) are carefully designed for optimal performance in gene expression quantification. It is not necessary, therefore, to test PCR efficiency when using TaqMan(R) Endogenous Controls for normalizing TaqMan(R) Endogenous Control targets. However, when using TaqMan(R) Endogenous Controls to normalize targets that are not TaqMan(R) Endogenous Controls, ensure that PCR amplification efficiencies are identical. This can be done by serially diluting a cDNA sample and demonstrating that the CT difference between the target and endogenous control remains constant. A constant CT difference across a range of at least 3 logs (1000-fold) of initial template concentration will verify identical PCR efficiency.
Note for polyA RNA templates: The 18S rRNA endogenous control assay cannot accurately evaluate cDNA generated from polyA RNA samples because most of the rRNA has been removed from them.