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Acta Histochem Cytochem. 2009 Apr 28;42(2):15-21. doi: 10.1267/ahc.09011. Epub 2009 Apr 23.

Improvement of in situ PCR by optimization of PCR cycle number and proteinase k concentration: localization of x chromosome-linked phosphoglycerate kinase-1 gene in mouse reproductive organs.

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1
Department of Histology and Cell Biology, Nagasaki University Graduate School of Biomedical Sciences.

Abstract

In situ polymerase chain reaction (in situ PCR), which can detect a few copies of genes within a cell by amplifying the target gene, was developed to better understand the biological functions of tissues. In this study, we optimized the protocol conditions for the detection of X chromosome-linked phosphoglycerate kinase-1 (pgk-1) gene in paraffin-embedded sections of mouse reproductive organs. The effects of various concentrations of proteinase K (PK) and PCR cycle numbers were examined. To label the amplified DNA, we used digoxigenin-dUTP (Dig), Cy-3-dUTP (Cy-3), or FluorX-dCTP (FluorX). The optimal concentration of PK was 50 microg/ml for the ovary and 10 microg/ml for the testis. Ten PCR cycles were optimal for Dig and 25 cycles were optimal for FluorX and Cy-3 in the ovary and testis. The signal-to-noise ratio of FluorX and Cy-3 for ovarian tissue was better than that of Dig. Using the above conditions, we detected 1-4 and 1-2 spots of pgk-1 in the nuclei of granulosa and germ cells, respectively. Our results indicate that in situ PCR is useful for detecting a specific gene in paraffin-embedded sections under optimized conditions of both PCR cycle number and PK concentration.

KEYWORDS:

in situ PCR; ovary; pgk-1; proteinase K; testis

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