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.
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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DOI：
10.1267/ahc.09011
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发表时间：
2009-04-28
影响因子：
2.4
通讯作者：
Koji T
中科院分区：
文献类型：
--
作者：
Hishikawa Y;An S;Yamamoto-Fukuda T;Shibata Y;Koji T
原位聚合酶链反应（In situ polymerase chain reaction, In situ PCR）是为了更好地了解组织的生物学功能而发展起来的，它可以通过扩增靶基因来检测细胞内基因的少数拷贝。本研究优化了小鼠生殖器官石蜡包埋切片中X染色体连接磷酸甘油酸激酶-1 （pgk-1）基因检测的实验条件。考察了不同浓度的蛋白酶K （PK）和PCR循环数对其的影响。为了标记扩增的DNA，我们使用了地高辛- dutp （Dig）、Cy-3- dutp （Cy-3）或FluorX- dctp （FluorX）。卵巢PK最佳浓度为50µg/ml，睾丸PK最佳浓度为10µg/ml。在卵巢和睾丸中，Dig的最佳PCR周期为10个，FluorX和Cy-3的最佳PCR周期为25个。荧光和Cy-3对卵巢组织的信噪比优于Dig。在上述条件下，我们分别在颗粒细胞和生殖细胞的细胞核中检测到1-4个和1-2个pgk-1位点。结果表明，在优化的PCR循环数和PK浓度条件下，原位PCR可用于检测石蜡包埋切片中的特定基因。
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 µg/ml for the ovary and 10 µg/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.
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影响因子：
3.5
作者：
Pulimood, Anna B.;Peter, Shajan;Donoghue, Helen D.
通讯作者：
Donoghue, Helen D.
影响因子：
5.8
作者：
Sanno, N;Jin, L;Lloyd, RV
通讯作者：
Lloyd, RV
影响因子：
7.3
作者：
Chen, PCH;Pan, CC;Chiang, H
通讯作者：
Chiang, H
DOI：
10.1073/pnas.87.13.4971
发表时间：
1990-07-01
影响因子：
11.1
作者：
HAASE, AT;RETZEL, EF;STASKUS, KA
通讯作者：
STASKUS, KA
影响因子：
3.6
作者：
NAKAYAMA, H;KURODA, H;KITAMURA, Y
通讯作者：
KITAMURA, Y