How 18s gene can be amplified when I use oligo dt for cDNA synthesis - (Mar/07/2013 )
Hi,
I have done few real time PCR works recently, and I used the 18s gene for endogeneous control.
Its Ct value was approximately 11 for every cDNAs used as template.
Here, I have a question.
Since 18s gene doesn't have poly A tail, it can't be amplified with oligo dT.
and cDNAs I used were synthesized using oligo dT, I wonder how 18s gene can be amplified with its Ct value approximately 11.
Thank you, all.
I did the same thing some time ago and obtain amplification. I was also wondering why. I found this paper which may answer your question
Polyadenylation of ribosomal RNA in human cells
apart from polyadenylation, there are other explanations I think. For example, OligodT primers could bind to polyA region within rRNA, In addition, the amplification could be from DNA. You have to rule out that possibility.
One question is why got 18S transcribed.
More interesting question would be, if it is in such case a valid reference gene.
I always thought that although 18S rRNA does not have a polyA tail, the whole transcript that it is transcribed from (majority of rRNA) does have a polyA tail. Can anyone clarify? It is still used as a qPCR control because it gives a constant sample of all actively transcribed RNA in the cell, not just the 18S subunit.
It is a DNA contamination.
18s is not a good reference gene, because:
1) It has multiple copies in genome. 300 to 400 copies, thus they are much more abundant than target mRNA transcripts, which are usually rare. Thus:
2) You have to dilute sample for 18s as 1:6000 while you dilute sample for other gene 1:20 to bing its CT value between 20-30. Thus:
3) 18s is diluted much more than that of other genes, so it has less inhibitors because of more dilution.
4) It has pseudogenes.
https://www.roche-ap...notes/lc_15.pdf
Check it yourself: goto Primer-Blast website and past both forward and reverse primers in that website and click "Get Primers" buttom.
https://www.ncbi.nlm...s/primer-blast/
You will see that it is not possible to design a pair of primers for 18s that do not bind to more than one place.
5) Ribosomal RNA is resistant to degradation much more than mRNA.
Thus use Ribosomal Proteins instead of 18s. (edited)
http://openwetware.o...R_normalisation
and my suggestion based on my experience:
If you can not find a gene (except 18s )with stable CT value in order to use it as a reference gene, it is your fault.
Because 100% I am sure that you have not done RT, RNA normalization and sampling very well.
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Babak
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