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

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.

Fig. 1

PCR of pgk-1 gene in mouse ovary and testis. PCR products of pgk-1 (170 bp) were found in mouse ovary and testis.

Fig. 2

Effects of various concentrations of PK in mouse ovary using FluorX. Upper panels (A, B, C) were carried out Taq polymerase and lower panels (D, E, F) were performed omitting Taq polymerase. PK was evaluated at varying concentrations from 5 µg/ml (A, D), 20 µg/ml (B, E), and 50 µg/ml (C, F). One to 4 green spots were detected in the nuclei of granulosa cells (C). Red arrow; 4 spots. Yellow arrows; 3 spots. White arrows; 2 spots. Bar=20 µm.

Fig. 3

Effects of different PCR cycles in mouse ovary using FluorX. Five cycles (A), 15 cycles (B), 25 cycles (C), and 30 cycles (D) of PCR were performed using Taq polymerase. One to 4 green spots were detected in the nuclei of granulosa cells (C). As a negative control, 25 cycles of PCR were performed omitting Taq polymerase (E). Red arrows; 4 spots. Yellow arrows; 3 spots. White arrows; 2 spots. Bar=20 µm.

Fig. 4

Detection of pgk-1 in mouse ovary using Dig. One to 2 red spots were detected in the nuclei of granulosa cells using image analysis system (A). PCR cycles were 10 times, and PK concentration was 50 µg/ml. Taq polymerase was omitted as a negative control (B). Bar=20 µm.

Fig. 5

Detection of pgk-1 in mouse testis using Cy-3. One to 2 red spots of Pgk-1 gene were detected in the nuclei of germ cells with 10 µg/ml PK concentration and 25 cycles (A). Yellow arrow; 2 spots. White arrows; 1 spot. Taq polymerase was omitted as a negative control (B). Bar=20 µm.

Fig. 6

Pgk-1-positive spots in mouse granulosa cells. The number of pgk-1-positive spots after employing 15 cycles and 25 cycles (A). The percentage of pgk-1-positive granulosa cells after using 25 cycles for in situ PCR (B). Data represent mean±SD. *P<0.0001.