EFFECT OF UNDILUTED cDNA on QPCR

[ashu330327]

Dear Sir
I have synthesised cDNA using 5microgram RNA with fermentas kit and directly used this qpcr reaction mixture(10 microlitres). High cp value is obtained.
So my question is that is it necessary to dilute the cDNA or used it directly.

Thanks

[mchlbrmn]

If you run a standard dilution series of a very positive sample, you will see if there is inhibition. The spacing between 1/10 dilutions should be 3.3 cycles. If the spacing is less, this means that inhibition could be delaying the concentrated sample.
I couldn't tell whether you used 10ul cDNA in an unkown volume PCR reaction, or used the entire 5ug cDNA reaction in a 10 ul PCR volume. Either way, it sounds to me like there very well could be inhibition of the PCR. I use at most 10% volume of cDNA in a PCR reaction, and usually to quantify I dilute the cDNA 1/10.

[ashu330327]

Dear Sir
Thanks for reply. According to your experience , there could be inhibition of pcr. I have used 5microgram cDNA directly in total 10 MICROLITRES reaction.
Thanks Sir!!!

[mchlbrmn]

Yes, I forgot to say that directly; there could be inhibition. That is a lot of cDNA in there. Contamination in the RNA can be one source, but with a large volume of RT reaction used, components of the reaction, such as the dNTPs may contribute. I once added extra dNTPs to a PCR reaction, and was surprised to see less product. the nucleotides can interact with the Mg++ and leaving less free for the polymerase, I believe.

[ashu330327]

Thanks Sir!!!