How reliable is 18S RNA as reference - (Mar/01/2006 )
Recently I read a literature from Zhu LJ, Altmann SW. [Anal Biochem. 2005 Oct 1;345(1):102-9] about a promising method to normalize gene expressions using 18S RNA as reference.
Can anyone tell me how perfect this method is? What is the advantages and disadvantages in comparing to the popular endogenous method? And which aspects do I have to play caution to when applying this 18S method?
Millions thanks for my floods of questions
I have not done any realtime RT-PCR, but done a lot of regular RT-PCR using RNA from cell lines. Most of my RT-PCRs were verified by Western blotting. I use 18S RNA, GAPD and beta-actin as internal PCR control. 18S is very consistent across samples while GAPD and beta-actin are not, and may be affected by certain treatments. There is a paper published some time ago reporting that beta-actin and GAPD are not good control.
Please also check this thread http://www.protocol-online.org/archive/posts/3162.html
Beta actin is supposedly a much better control than GAPDH.
I really don't think 18S is that much better than Beta actin as an internal control; they're about the same.
The simple answer is that there is no simple answer. It is not adequate to chose an internal control based purely on heresay and literature. Every cell treatment and cell line will produce a different genetic profile and have different sensitivities. The only way to do it is to test a handful of genes and see which are unaffected.
We had initial chosen 18S RNA and GAPDH as our controls for drug-treated cells but found that our drugs decreased there expression. However, when we later tried beta-actin the was little effect so we now use that. Other classical genes include TBP.
Trial-and-error if you want to do it thoroughly.
you may want to look here