1 reply to this topic

[eleceyes]

I am trying out Real Time PCR using TaqMan system (GAPDH).  I used cDNA made (Ambion Retroscript kit) from 2 ug HeLa RNA (RNA was extracted using Qiagen RNaesy kit) to generate a standard curve.

The R-square value of the standard curve is >0.99 and PCR efficiency is almost 90% but my Ct value for the undiluted HeLa cDNA is quite high (e.g. 30 cycles).  I read somewhere saying sample masses that give high Ct values (34-40 cycles) will give rise to poor precision and less power to detect low-fold changes.

Can I go ahead trying my real samples? If not, what step(s) should I take in order to solve this problem (high Ct)?

Thank YOU!

[jadefalcon]

as long as the copy number of your sample lies between the highest and the lowest standard, and the standard is ok (r2), i'd see no problem. high ct values mean there's not many copys of your target gene present in the sample, so that may pose a problem, but then i'd try to use higher concentated samples for standard determination...

mike