Quantitative Polymerase Chain Reaction
The MB Core Facility maintains two Q-PCR instruments. Both are ABI Model 7500 Fast Q-PCR Systems located on the Hanover Campus in room 241 Remsen. These instruments are available for use 24/7 on a signup basis with a per run fee to cover the cost of maintenance and service. The Core facility staff offers advice and training for users of these instruments .
Quantitative PCR is an extremely sensitive technique for detecting gene expression. When compared to micro-arrays, quantitative PCR is much better for detecting poorly expressed RNA, finding subtle changes in gene expression, and working with small sample sizes. Quantitative RT-PCR has been used to detect genes expressed as single mRNA copies and can theoretically detect changes in gene expression as small as 5%. Quantitative RT-PCR can also be used to examine gene expression in single cells. Sensitivity is important since the sequencing of various genomes will reveal many genes that micro-arrays and other multi-gene expression techniques cannot detect because the gene is poorly expressed or expressed only in rare cell types.
No mRNA-based gene expression technique will always reflect the level of protein in the cell. In some situations, m-RNA levels can be closely correlated with protein expression. However, some highly transcribed genes may be under translational, rather than transcriptional control mechanisms and would not correlate with the amount of protein produced by the cell.
For a short disscussion of QPCR click here. You must first decide which indicator system you want to use. There are two choices: SYBR Green or another intercolating dye that measures the amount of double stranded DNA present in the reaction or ABI Taqman style FRET probes. Both of these methods offer advantages. For a discussion on the pros and cons of each method click here. If you are new to QPCR, it is advisable to read ABI User Bulletin #2 (PDF). This bulletin contains information on the various ways to quantitate gene expression using QPCR.
Primer and Probe design
The key to good data in Q-PCR is the design of your primers and probes. The PCR primers are key. Whether SYBR Green or FRET probes are used for a reporter, poor primers would lead to insufficient amplification and low signals from your reporter dye. The MB Core Facility has a PC copy of ABI's Primer Express software available for use in the Core Facility. In addition, we will be able to offer various bits of advice on primer design gleaned from our experience and that of other users.
Contact the core facility for more information.