5] Use of quantitative polymerase chain reaction to study retinoid receptor expression

Abstract

Publisher Summary This chapter describes different methods employed for the expression analysis of various retinoid receptors. Different one-tube reverse transcription–polymerase chain reaction (RT–PCR) that assembles the reverse transcription reaction and the cyclic amplification reaction in a single tube are described. One of the protocols described exploits the reverse transcriptase activity of thermostable polymerases. In the presence of Mn 2+ ions the enzyme isolated from Thermus thermophilus (Tth polymerase) uses RNA as a template for DNA polymerization. In the second step of the one-tube reaction, Mn 2+ ions are sequestered by EGTA, a chelator with a high affinity for Mn 2+ as compared to Mg 2+ ; the latter ion and PCR primers are added to the diluted reaction mix and amplification of the cDNA produced can be performed in the same tube. Depending on the expression level of the receptors in the sample analyzed and on the amount of RNA used for the reaction, the cycle number of the first round of amplification can be adjusted and different amounts of the first round of amplification can be used as a template for the second round to calibrate the sensitivity of the system.