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18S rRNA as internal control for mRNA-cDNA? - internal control (May/02/2007 )


This may be a really stupid question...

I have used an oligodT primer (mRNA) for my cDNA synthesis. Can I use 18S rRNA and rpl19 (rRNA) as internal controls?

You see I have performed q-RT-PCR with these internal controls and I have gotten pretty high Ct values (14-15)..??
Don't really understand how this is possible..


if you are using 18S as reference gene you should use random hexamer primers during reverse transcription since there are no polyAs in the 18S RNA. oligodT primers are useful if you only want to reverse transcribe the mRNA, so both your GOI and reference gene should be available as mRNA in your sample. this can give you higher yields of your target cDNA and therefore lower Ct values.

interestingly, there are also mRNAs without polyA tails e.g. histone mRNA

However, i cannot explain why you got those strange Ct values. glare.gif

-Ned Land-