in RTqPCR. if i have my target gene expression at ct 33 and beta actin at ct 17..i repeated and that's always the ratio..could my rna be degraded but i have good ct for beta actin how?
It doesn't mean your total RNA is degraded, it's most likely to be a los leves expression case. You are around hundreds of transcript copies, if you were getting a signal in every single one of your triplicates, then you would have to use some other stable and low expression level HK gene. The later, so you can get appropiate normalization data. I'd rather add 10 times more cDNA to the qPCR reaction, so the target gene average goes up by aproximately 3.32 cycles. In such a way, you could get suitable triplicate's average.
Either way, you'll have to change your house keeping gene. However, you want to be 100% sure about the integrity of your RNA samples, you can always run them un denaturing agarose electrophoresis and look for major and minor ribosomal RNA subunits.
Good luck
Hi Basma
It means (simply) the level of transcript abundance of beta actin is much higher comparing with your target gene. It happens a lot.
Regards
ct at 33? i agree with Adan: either loss of expression or large deviation between the target and reference gene amplification (difference in Tm of primers, melting temperature of amplicons).
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