Which gene is the best internal control for RT-PCR - (May/25/2004 )
I want to know which gene is the best internal control for RT-PCR. I usually use b-actin as a control, however, it sometimes seems to express differently in cells after certain treatment. I am not sure if the changes are really due to regulated expression or just technical issues.
Beta-actin is not proper control especially when cell culture condition is different (2D, monolayer vs. 3D, suspension). Beta-actin will be much lower in 3D culture.
5S rRNA is the best control we use to correct this problem.
To be a good internal control, a gene should have a constant basal level of expression which is consistent, non-regulated and independent of the cell-cycle. b-actin expression has been found by a number of studies to be influenced by some factors such as by matrigel, thus is not a ideal internal control. Instead, 18S rRNA shows less variance in expression across a variety of treatment conditions than b-actin and GAPDH. However because expression of 18S rRNA is very abundant, PCR conditions such as the ratio of control to target gene primers need to be optimized, otherwise amplification of 18S rRNA can exhaust reaction reagents quickly.
how about beta-tubulin?
in our lab, histone H4 is generally used as internal control. it has uniform expression across cell lines aas far as i know
the referees in our latest two or three publications objected using GAPDH as internal control in pregnant rat uterus tissue. Their reason was that the change in the hormonal environment during pregnancy alter the GAPDH expression. What is your opinion about this? What internal control would be the alternative?