what is the recomended conc. to prepar cDNA to run rt-pcr for brain rat tissue.. i used 50ng/ul some primer gave ct value btw 20-22 but other above 30.. ?
Typically, 1 ug RNA in a 20 uL RT reaction yields plenty of cDNA to use 1 uL of in the Q-PCR reaction. Differences in Ct values are likely differences in copy number (high expressing gene, like GAPDH vs a less expressed gene, such as MYCN).
If you are using the same primer and that is giving very discordant results, I would check the purity and integrity of the RNA input. Using nano-drop, A260/280 and A260/230 ratios should be very close to 2. An A260/280 ratio of 1.8 indicates ~50% of your A260 is coming from protein, not RNA. Lower values are worse.
If your RNA has been chewed up and spat out (certainly not unheard of...) you shouldn't see nice rRNA bands by agarose/formaldehyde gel, but a smear instead.
good or bad of the CTs depends on the expression level of the genes.
As Matthew said before, the ct value is differed for reference (GAPDH, Ct values around 15-20) and target (MYCN) genes.
After cDNA synthesis, the best way to check whether the cDNA work well or not, is validating all cDNA by one stable reference gene. If your reference gene's CTs are not deviating much across experiments then it should be fine.
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