I have used 18S primers as housekeeping genes for my yeast strains. These are showing 1.5-2.0 CT value on RT-PCR while genes under study are showing CT value in higher range from 23-30. I also checked the melting curves, which did not exhibit any DNA contamination. Are these low Ct values correct? I need some guidance in this respect.
In general ct value in most qpcr machine shoule be less than 30 whenever it become higher the results should be ignored since in the higher ct the reaction material will be consumed so the result are fulse positive
rRNAs are extremely highly abundant, so low Ct values are expected. Although I think that Ct values < 5-10 are odd, at least suspicious.
I wouldn't use rRNAs as reference genes. The reference genes should ideally have Ct values somwehere in the range where the Ct values of the target genes are. If you introduce some kind of bias in the determination of the Ct values with the background correction, then there is more hope that this bias is similar (and will cancel out, at least partially, in the delta-Cts) when the Ct values of the reference and the target gene are similar.
But generally Ct value of 18S fall in the range of 10-15 as it was referred by someone who was already using this as reference gene. Are these low ct values linked to the quality of RNA or cDNA?
It seems more to be a problem of the algorithm how the Ct values are determined. Can you show the amplification curves (once with and once without logarithmic Y scale; ideally also once with and once without background correction)?
Will you check the following article, it seems helpful:
"Validation of Reference Genes for Real-Time PCR of Reproductive System in the Black Tiger Shrimp"
Article Validation of Reference Genes for Real-Time PCR of Reproduct...
Best luck
I am also facing the same problem, the CT value is uniformly 5 using 18S RNA. Shall I proceed with the calculation. Or is the RNA conc. of 1000ng is to high? Shall I dilute my cDNA uniformly?
Please help.
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