I am trying to check gene expression through real time but somehow not getting Ct values, in that case what can be the possibilities to troubleshoot this. Can we check whether the cDNA template used in the run is synthesized properly or not??
Mansi..
Have you checked your RNA concentration and its integrity on gel before synthesizing cDNA? if RNA is alright then you can use a cDNA synthesis kit other than the one already used. Use proper concentration of RNA for cDNA synthesis. run a normal pcr with your real time primers and check if u get amplification.
I think you first check the concentration of cDNA and make it constant i.e.100 ng/microL. Then you should check the amplification of house keeping gene with this cDNA to confirm that whether it is working or not. After this, you should perform the amplification of desired gene with this cDNA and checked on gel for single band. After this, you should do the real time PCR analysis. I hope this will work.
Check your cDNA by normal PCR for your target gene and something else that you know will be expressed. Use primers across exon-exon boundaries to ensure only cDNA can be detected. (Even if you DNase treat your RNA, it's still possible some gDNA could make it into your cDNA - you don't want to pick that up as a false positive).
If you can't pick up your target gene by normal PCR, but can detected other genes, then your target probably isn't expressed.
Hi Gaurav
Yes I have checked RNA concentration, and it is quite fine, with A260/280 in range.So do you mean to say that I should use cDNA template for normal pcr and check on gel before running real time??
Yes Mansi you should use this cDNA in normal PCR to amplify your target gene and as varun said you can also amplify your housekeeping gene and check it in gel. One more thing, were your primers working previously in normal PCR during standardization?
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