mRNA Detection by In Situ RT-PCR

In situ polymerase chain reaction (PCR) is a recently developed technique whereby DNA (or cDNA) is enzymically amplified within cells or tissue sections, enabling the localization of specific low copy number sequences within a heterogeneous cell population. In situ reverse transcriptase PCR (RT-PCR) is an adaptation of the technique in which mRNA sequences are reverse transcribed to give a DNA copy (i.e., cDNA), which is subsequently amplified by PCR. The technique provides significantly enhanced sensitivity of detection over conventional mRNA in situ hybridization (ISH) as successful amplification can be achieved from a single mRNA target sequence. Amplification products are detected directly by incorporation of label during thermal cycling, or indirectly using a labeled probe and ISH (1 -9 ). Figure 1 shows the basic principal of the technique.