mRNA expression too low? - (Mar/30/2007 )
Does anyone has an experience with siRNA/real-timePCR. I need some advices.
I check the effect of siRNA using two-step real-time PCR but the mRNA expression of my gene is very low = 30 cycle but GAPDH = 18 cycle. And maybe I have difficulty in seeing the effect of the siRNA (I have checked 5 different siRNAs).
Cells were transfected with 10nM - 100nM and 48h later,total RNA was isolated and converted to cDNA (FastLane (qiagen) directly from cultured cells without RNA purification for use in two-step RT-PCR (QuantiTect SyberGreen PCR) ). I have noticed that if I try to knockdown genes, which expression is higher (cycle 25-28) than this one I do not have any problems.
What should I do? Should I take a more template to real-time (now, I use 1ul cDNA (from the RT reaction)per well, final PCR volume= 25ul )?
I wil be grateful for help.
Do you see expression of your gene of interest without knockdown? What kind of Cts do you get? What's your 1ul cDNA concentration? You can bump this up as high as you can if you don't have to conserve your RNA. I'd even try a range concentrations of RNA conversion if you are restricted to 1ul per rxn.
I wouldn't rule out bad primer/probes.
Thank you for answer:)
The expression of my gene without knockdown is very low (ct = 30 cycle when ct for GAPDH = 18).
And I use a primer to this gene form qiagen and SuperArray. I am sure that primers work well. But I have problems with siRNAs. Recently, I have checked 5 different pre-design siRNAs from companies, and I have not got a good results.
I would like to know if I have a very low expression of my gene, it is possible to knockdown this kind of gene (to achieve silencing). All the time, as controls I use positive control for GAPDH and fluorescently labeled control siRNA to monitor of siRNA delivery efficiency, which is estimated 80-90%.
Now, I do not know if I have problem with siRNAs or low expression of my gene!
Maybe if I will take more cDNA I will see differences. But I can not take a lot of template because I have read that GAPDH in real-time sholud be expressed after 15-18 cycle. Is it true???