Quantitative RT-PCR for Neuroendocrine Studies

Determination of mRNA levels of specific genes is becoming increasingly important as a measure of gene expression. With the recent advent of RT-PCR, the sensitivity for mRNA determination has been increased dramatically, and this technique is becoming widely used in neuroendocrine studies which involve small tissue samples and/or isolated nuclei. Nevertheless, the exact procedure for reliable quantification of RT-PCR has been widely debated. This minireview attempts to assimilate the available literature on the RT-PCR technique and discuss the various approaches commonly used to obtain quantitative results using the technique. An example from our laboratory of the use of RT-PCR for the measurement of several gene products in the same sample using exogenous internal standards is also provided. Particular attention is paid to the choice of endogenous vs. exogenous internal standards, the length of the transcript of the standard and its relationship to the target sequence being amplified, the amplification pattern of the target gene and internal standard, the reproducibility of the method, and the overall usefulness and suitability of RT-PCR for neuroendocrine studies.