|[Frontiers in Bioscience 1, c4-15, November 1, 1996]|
THE FOUNDATION OF SUCCESSFUL RT IN SITU PCR|
Director, MGN Medical Research Laboratories, Setauket, New York 11733, USA
Received: 07/25/96; Accepted: 10/02/96; On-line 11/01/96
The foundation of successful RT in situ PCR is the use of formalin fixed, paraffin embedded samples which have been digested for the optimal time in a protease. The optimal time, which is determined by testing a variety of protease digestion times, is defined by an intense signal in the nuclei of most cells irrespective of the primers used, and a loss of this signal with an overnight digestion in DNase. This permits the target specific direct incorporation of the labeled nucleotide into the amplified cDNA. A lack of signal with the negative control (DNase, no RT) and an intense nuclear signal in most cells with the positive control (no DNase) is prerequisite for success with RT in situ PCR. The localization of the signal (cytoplasmic for human mRNAs and restricted to certain cell types) is another important indicator of successful RT in situ PCR. The one step rTth system allows for the reproducible amplification and detection of low copy RNA targets within a few hours.