We have a reverse transcriptase PCR-thermo cycler in our lab. I want to study 4 different gene expressions and have the primers. Can I do it? Can anyone suggest any protocol?
Hello, the first thing you have to do is to make sure that you have all the primers that you need, not only the primers for the genes that you are aiming to check the expression, but also a house-keeping gene (which is expressed as a constant levels which can be used as a baseline for comparisons of levels of expression).
In fact in the research group where I work we researched different protocols for the analysis of gene expression (in our particular case in bacterial biofilms) and you can check these publications
PLoS One. 2012;7(5):e37480. doi: 10.1371/journal.pone.0037480. Epub 2012 May 21.
Optimizing a qPCR gene expression quantification assay for S. epidermidis biofilms: a comparison between commercial kits and a customized protocol.
França A, Freitas AI, Henriques AF, Cerca N.
Hope this helps
It should be pretty straightforward, you just need to construct cDNA from good quality RNA (for two-step rt-PCR) and then proceed like normal PCR for your genes alongwith houskeeping genes.
If you are planning for multiplexing, the expected product sizes should be different for each gene and have same annealing temperature (which has to be standardized).
hope this helps
regards
nav
If the product size of your primers have enough difference, and tm and other amplification conditions are same then you can use in one PCR tube, its really interesting experience
Thank you guys for you suggestions. I just wonder if i do multiplexing in one PCR tube then how to run gel for the products? I mean, all gene products are in same tube then How can i compare with housekeeping gene? can anyone give any picture?
@ Pia: Thank you very much for your detail discussion. however, how would i know the 4 genes will amplify in different sizes before PCR? Yes I would do semi-quantitative PCR.
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