Nested RT-PCR in a Single Closed Tube

There are several methods now in widespread use for detecting and characterizing specific RNA targets. These methods include in situ hybridization, Northern blotting, dot or slot blot, RNase protection assay, and reverse transcription coupled to polymerase chain reaction (RT-PCR). However, when the amount of RNA target is limited or restricted in its cellular or tissue distribution, the extreme sensitivity of the PCR allows the detection of minute quantities of RNA when coupled to an initial step that converts single-strand RNA to cDNA (1 –4 ). Nevertheless, when RT-PCR is applied for diagnostic purposes, the sensitivity usually afforded by this technique in routine detection tests is similar, or only slightly higher, to conventional enzyme-linked immunosorbent assay or hybridization techniques. This is frequently observed when dealing with poor-quality samples containing inhibitors of RT-PCR. The presence of different components of plant or animal origin, as well as specific RT-PCR conditions, may inhibit the reverse transcription and amplification by a number of mechanisms (5 ).