@article{oai:nagasaki-u.repo.nii.ac.jp:00015776,
 author = {Hishikawa, Yoshitaka and An, Shucai and Yamamoto-Fukuda, Tomomi and Shibata, Yasuaki and Koji, Takehiko},
 issue = {2},
 journal = {Acta histochemica et cytochemica},
 month = {Apr},
 note = {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., Acta Histochemica et Cytochemica, 42(2), pp.15-21; 2009},
 pages = {15--21},
 title = {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.},
 volume = {42},
 year = {2009}
}