Hi!
I am trying to standardise certain cytokines' m-RNA expression in glial cells, but the amplification happens at ~35-40 cycles. Considering that the cytokines are lowly expressed in physiologically normal cells, a low copy number is obvious, but i have read that reactions beyond ~35 cycles are not much acceptable. Any inputs? I have changed the primers and taqman probes twice, the result being the same. Can ~40 cycles be considered permissible?
Just FYI: I have been successfully running singleplex and duplex reactions in the same samples with different not-so-low expressed genes, with ~100+_1 % efficiency, comparable with the reference gene. so:
The C-DNA is working well
Reaction mixture/ buffer/machine are working fine. Same samples in duplex give a proper signal for 18 s gene.
there does appear amplification, albeit late; and taqman probes will ensure specificity, so can be speculated that the amplification is actually happening, only the copy no. might be low.
I have tried different primer/probe concentrations, different temperature range... not to much avail. Increasing concentration too, doesn't sound good, for even upon increasing the concentration of cDNA from 1 ng/ul to 10 ng/ul, the Cq shift is not more than ~2-3 cycles.
Any suggestion is very much welcome.
Thanks .
Dear Poojashri Mishra
U can increase the cDNA concentration up to 50 ng.
Are your primers validated ?
If not then amplify your template with primers only (without probe) with normal PCR and see the amplification on gel and check the correct size. If u see good amplification then the problem is your Probe. If you dont see any amplification the the problem is your primers.
Thanks Abhishek Vats,
My concentration is 40 ng already, i use 4 ul of the cDNA in a 20 ul reaction. increasing more will cause a shift in 18 s expression, which happens at~ 10 cycles already.
The amplification seems to happen alright, it is jsut that it is tad too late. My question is, forgive my naivity at it; could the late amplification be attiributed to wrong set primers, or should i consider the cDNA amount as culprit and go ahead toggling the same...
Also, does MgCl2 concentartion have to play a role in it?
Thanks
Dear Poojashri
First of all I want to suggest u that you should use more then one endogenous gene because 18 S is always showing this problem of low Ct value. One thing I also want to clear you that 18 S is not a very good housekeeping gene because its copy number is different in different individuals and different cell lines. beta actin or gapdh both show higher Ct then 18S so u can use higher concentration of cDNA with beta actin or gapdh.
Your second concern is right that different primer set will show different Ct value.
And if your probes are not bind to your template properly then it will amplify but not show any Ct. As i recommend earlier you should do normal PCR with only primers, Or do the RT-PCR with SYBR green chemistry.
I think u are using master mix that include everything so forget about MgCl2 concentration
Abhishek's points are important.
Do not use 18S RNA as a reference gene!
As a control, use endotoxin and IL-8 or IL-6 expression as an end point.
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