Preparation of samples for polymerase chain reaction in situ

The purpose of this paper is to describe the key variables in sample and re agent preparation needed for successful polymerase chain reaction (PCR) in situ. Tissue or cell preparations should be fixed in a cross linking fixati ve, such as 10% buffered formalin, preferably from 15 to 48 hours. Tissues should be embedded in paraffin; cell preparations can be fixed when near co nfluence, then physically removed and processed. When possible three sample s (4 mu M tissue sections or 1-5000 cells) should be placed on silane coate d glass slides. Digestion in pepsin (2 mg/ml) for 30 min is adequate for DN A detection by PCR in situ hybridization whereas optimal protease digestion time is variable and related to formalin fixation time for reverse transcr iptase (RT) in situ PCR. RT in situ PCR requires an overnight digestion wit h DNase. The amplifying solution should contain 4.5 mM MgCl2, 0.05% bovine serum albumin, and, for RNA analysis, the reporter nucleotide. A false posi tive signal would be evident with incorporation of the reporter nucleotide for DNA targets due to DNA repair; this can be avoided with frozen, fixed t issues and the hot start maneuver. Otherwise, one needs to use a labeled pr obe and a hybridization step to detect amplified DNA targets in paraffin em bedded tissues.