Preparation of samples for polymerase chain reaction in situ.
The purpose of this paper is to describe the key variables in sample and reagent preparation needed for successful polymerase chain reaction (PCR) in situ. Tissue or cell preparations should be fixed in a cross linking fixative, such as 10% buffered formalin, preferably from 15 to 48 hours. Tissues should be embedded in paraffin; cell preparations can be fixed when near confluence, then physically removed and processed. When possible three samples (4 microM tissue sections or 1-5000 cells) should be placed on silane coated glass slides. Digestion in pepsin (2 mg/ml) for 30 min is adequate for DNA detection by PCR in situ hybridization whereas optimal protease digestion time is variable and related to formalin fixation time for reverse transcriptase (RT) in situ PCR. RT in situ PCR requires an overnight digestion with DNase. The amplifying solution should contain 4.5 mM MgCl2, 0.05% bovine serum albumin, and, for RNA analysis, the reporter nucleotide. A false positive signal would be evident with incorporation of the reporter nucleotide for DNA targets due to DNA repair; this can be avoided with frozen, fixed tissues and the hot start maneuver. Otherwise, one needs to use a labeled probe and a hybridization step to detect amplified DNA targets in paraffin embedded tissues.
Nuovo
G J
GJ
MGN Medical Research Laboratories, Setauket, NY 11733, USA. gnuovomd@mem.po.com
D001921:Brain / Q000166:cytology / Q000648:ultrastructure; D001923:Brain Chemistry; D004247:DNA / Q000032:analysis*; D018076:DNA, Complementary / Q000032:analysis; D005557:Formaldehyde; D016591:Histocytological Preparation Techniques; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D007202:Indicators and Reagents; D016133:Polymerase Chain Reaction / Q000379:methods*; D016610:Tissue Embedding; D016707:Tissue Fixation
D018076:DNA, Complementary; D007202:Indicators and Reagents; D005557:Formaldehyde; D004247:DNA