犬甘油醛-3-磷酸脱氢酶互补DNA：聚合酶链反应扩增、克隆、部分序列分析以及在核糖核酸酶保护分析中用作上样对照

Canine glyceraldehyde-3-phosphate dehydrogenase complementary DNA: polymerase chain reaction amplification, cloning, partial sequence analysis, and use as loading control in ribonuclease protection assays.

作者信息

Gröne A, Weckmann M T, Capen C C, Rosol T J

机构信息

Department of Veterinary Biosciences, College of Veterinary Medicine, Ohio State University, Columbus 43210, USA.

出版信息

Am J Vet Res. 1996 Mar;57(3):254-7.

Abstract

OBJECTIVE

To use canine glyceraldehyde-3-phosphate dehydrogenase complementary DNA (GAPDH cDNA) as a template in ribonuclease (RNase) protection assays to measure canine GAPDH mRNA expression.

DESIGN AND PROCEDURE

Primers designed from the human GAPDH gene were used to amplify a 191-base pair canine GAPDH cDNA by reverse-transcription polymerase chain reaction. The cDNA was sequenced, and used as a template for RNase protection assay.

SAMPLE POPULATION

Total RNA was isolated from a canine squamous carcinoma cell line.

RESULTS

Canine GAPDH cDNA had a high degree of homology to human, rat, and mouse GAPDH. In vitro transcription of canine GAPDH cDNA was used to produce complementary RNA that detected canine GAPDH mRNA by RNase protection assay.

CONCLUSION

Canine GAPDH cDNA is a useful loading control to be used in RNase protection assays measuring mRNA expression in canine cells or tissues.

摘要

目的

在核糖核酸酶（RNase）保护分析中使用犬甘油醛-3-磷酸脱氢酶互补DNA（GAPDH cDNA）作为模板，以测量犬GAPDH mRNA的表达。

设计与步骤

从人GAPDH基因设计的引物用于通过逆转录聚合酶链反应扩增191个碱基对的犬GAPDH cDNA。对该cDNA进行测序，并用作RNase保护分析的模板。

样本群体

从犬鳞状癌细胞系中分离总RNA。

结果

犬GAPDH cDNA与人、大鼠和小鼠的GAPDH具有高度同源性。犬GAPDH cDNA的体外转录用于产生互补RNA，通过RNase保护分析检测犬GAPDH mRNA。

结论

犬GAPDH cDNA是一种有用的上样对照，可用于在测量犬细胞或组织中mRNA表达的RNase保护分析中。