how to optimize rt-pcr for gene expression analysis - (Oct/13/2008 )
i am trying to optimize conditions for rt-pcr in an attempt to do some gene expression analysis. im pretty new to this, so any pointers would be great. fyi, i am using abi 7700 and abi's taqman gene expression master mix.
in short, i have tried various amounts of cdna for my protocol - from 250 to 2000 ng and i am still getting pretty high Ct numbers. i was suggested to try even more cdna (4000 ng) and see what happens. problem with this is that i i will need to make tons of cdna for my experiment (cant just do one tube). however, i was wondering if instead i can increase the amount of my primer/probe that i use. i buy the primer/probe from abi at 20x stock and i use 2.5 ul (total reaction volume is 50 ul) as suggested by abi's protocol.
i know my Ct numbers should be better than what im getting and im not quite sure hot to optimize my conditions.
It seemed to have some problems with your samples (RNA), how was your RNA quality such as integrity. You could increase primer/probe concentration, and then compare Ct to that with low concentration.
hmm...regarding rna quality, do you mean my 260/280 ratios or my concentration? the ratio was a bit low (1.6ish)...but my conentrations are ok..
First make sure your RNA quality is OK and RNA is not degraded by RNase. You can run an agarose gel to see whether you can see two bright 18s and 28s bands. If the bands are not good, you should take measures to prevent RNase contamination.
If your 260/280 ratios are under 1.8, your RNA is not pure, try clean up your RNA.
How about the Ct for housekeeping genes such as Gapdh, beta-actin, do they also have low Ct values?
Are you sure your RT reaction is OK? How much RNA did you use for RT? Before doing real-time PCR, do a regular RT-PCR to amplify house keeping genes to see if your RT is successful.
So troubleshoot from RNA->RT->qPCR.