Qualitative Low-Level Internal Control for Nested RT-PCR

摘要

Analysis of genes expressed at low levels, or detection of RNAs from minute amounts of cells, is now possible by a combination of procedures: synthesis of a cDNA template (reverse transcription [RT]) followed by poly-merase chain reaction (PCR) amplification. This method relies on the preparation of undegraded RNA samples. In addition, these preparations must not be contaminated with inhibitors of reverse transcriptase or thermostable DNA polymerases. The most commonly used controls are (3-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and p-globin; and because expression of these "housekeeping" genes is high, partial degradation of the sample might not significantly impair the amount of product amplified To analyze very low abundance messenger RNAs, nested PCR can be performed; however, an internal control, corresponding to a gene expressed at low levels in a wide range of cell types, is then necessary. Herein we describe the identification of a low-level, internal nested RT-PCR control and its application in evaluating the presence of cancer cells in circulating blood.