Co-labeling Using In Situ PCR

Abstract

S U M M A R YIn situ amplification permits the histological localization of low-copy DNA and RNA targets. However, in many instances it would be useful to know the specific phenotype of the target-containing cell or to ascertain the distribution of a different nucleic acid sequence in the same tissue section. This review describes a methodology that allows co-in situ localization of two nucleic acid targets or a DNA/RNA sequence and a protein in paraffin-embedded, formalin-fixed tissue. The key variable for detection of low-copy RNA targets by RT in situ PCR is optimal protease digestion to permit cDNA target-specific incorporation of the reporter nucleotide. This is achieved via inactivation of nonspecific DNA synthesis by overnight DNase digestion. The key variable for immunohistochemical localization of proteins is to determine the effect of protease digestion on the antigen-based signal intensity. Background for DNA targets by in situ hybridization or, for targets present in 1-10 copies per cell, PCR ISH is dependent primarily on probe concentration and the stringency of the post-hybridization wash. Radioactive 3 H-labeled nucleotides permit an excellent distinction with colorimetric signals for co-localization, although two distinct chromogens can in many instances allow successful localization of two different targets.

RT in situ PCR detection of MART-1 and TRP-2 mRNA in formalin-fixed, paraffin-embedded tissues of melanoma and nevi

“…13 Available proteinases include proteinase K, pepsin, pronase and trypsin. 36,37 To avoid overdigestion, we used trypsin, which is more manageable than stronger proteases such as proteinase K. The mRNA signal detected by RT in situ PCR is mainly in the cell cytoplasm.…”

“…Nonspecific amplification of residual DNA will create a 'repair' artifact. 13 A third consideration is the design of the primer. Because we used a specific oligonucleotide primer pair for both reverse transcription and PCR MART-1 and TRP-2 in melanoma and nevi E Itakura et al amplification, optimal annealing temperatures for reverse transcription and cDNA amplification were within a close range.…”

“…13,14 The oligonucleotide primer sequences (Invitrogen, Carlsbad, CA, USA) were derived from a previous paper 15 as shown in Table 1. For RT-PCR of MART-1 mRNA, the forward primer, 5 0 -CACGGCCACTCTTACAC CAC-3 0 , and the reverse primer, 5 0 -GGAGCATTGG GAACCACAGG-3 0 , yielded a product of 254 bp.…”

Impaired cervical homeostasis upon selective ablation of RXRα in epithelial cells

“…Direct in situ PCR was performed as previously described with some modifications (Martinez, 1998;Nuovo, 2001). Briefly, dried dewaxed sections on sylanecoated slides were incubated with 0.5 lg/ml Proteinase K. After washing with ultrapure water, 50 ll of the PCR master mix solution containing digoxigenin-11-(2 0 -deoxy-uridine-5 0 )-triphosphate (Roche), were added.…”

Primary Effusion Lymphoma: Cytopathologic Diagnosis Using In Situ Molecular Genetic Analysis for Human Herpesvirus 8

“…A review of this methodology has recently been described (11). In brief, cell block and paraffinembedded tissue samples were digested with pepsin (2 mg/mL, 37°C) for 20 to 60 minutes.…”