Lab Technique - RT-PCR

RT-PCR stands for real-time polymerase chain reaction. Both PCR and RT-PCR are able to amplify target DNA sequences but the value-addedness of RT-PCR comes from the fact that it can also detect the amplified sequence in real-time. For PCR, agarose gels are required for the detection of the amplified sequence and it can only done after the amplification process. This becomes very time consuming. Because of the increased number of manual steps involved in the detection of PCR products, it is likely that the rate of error due to contamination or other human error is higher than RT-PCR.

(Image taken from http://bio-rad.gene-quantification.info/)

This is a real-time PCR system (CFX96™ Real-Time PCR Detection System from Bio-Rad) that my lab uses. It is able to perform several assays at any one time and is relatively user-friendly.

The procedures to run RT-PCR are easy as the vendor would have provided you with sufficient information to run your samples. For example, for the TaqMan probes that I was using, I was required to add in 10µL of TaqMan Universal PCR Master Mix, 1µL of Assay Mix, desired amount of sample and RNase-free water. The parameters for RT-PCR (not mentioned) and formula are all provided by the vendor. As long as the protocol is followed, every thing should be a-ok.

Lastly, RT-PCR can be used for quantification of gene expression, detection of pathogens and genotyping. There are of course, more applications out there than that that I've listed. It is really how creative anyone can get to exploit this useful machine.

Yvonee Chew 0703189A

P/S Where is Jacelyn?