Bhatia P, Taylor W R, Greenberg A H, Wright J A
Manitoba Institute of Cell Biology, Winnipeg, Canada.
Anal Biochem. 1994 Jan;216(1):223-6. doi: 10.1006/abio.1994.1028.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been a gene of choice in Northern blot analyses as an internal RNA loading control. We have investigated the expression of GAPDH and 28S-ribosomal RNA (28S rRNA) genes in mouse 10T1/2 cells and in a variety of tumorigenic and highly malignant metastatic cell lines derived from the 10T1/2 cell line. We observed that GAPDH mRNA levels varied markedly among the tumorigenic and highly malignant cell lines and were elevated in these cell lines when compared to the normal mouse 10T1/2 cells. In contrast, the levels of 28S rRNA did not significantly vary among the tumorigenic and highly malignant cell lines and were approximately at the same level as that found in the normal parental mouse 10T1/2 cell line. These observations indicate that much caution should be taken when using GAPDH gene expression as an RNA loading control for Northern blots. Based upon these observations, we recommend the use of 28S rRNA gene expression as a preferred RNA loading control for Northern blot analysis in which total RNA is used.
甘油醛-3-磷酸脱氢酶（GAPDH）一直是Northern印迹分析中作为内部RNA上样对照的首选基因。我们研究了GAPDH和28S核糖体RNA（28S rRNA）基因在小鼠10T1/2细胞以及源自10T1/2细胞系的多种致瘤性和高恶性转移性细胞系中的表达情况。我们观察到，在致瘤性和高恶性细胞系中，GAPDH mRNA水平差异显著，与正常小鼠10T1/2细胞相比，这些细胞系中的GAPDH mRNA水平有所升高。相比之下，28S rRNA水平在致瘤性和高恶性细胞系中没有显著变化，与正常亲代小鼠10T1/2细胞系中的水平大致相同。这些观察结果表明，在将GAPDH基因表达用作Northern印迹的RNA上样对照时应格外谨慎。基于这些观察结果，我们建议使用28S rRNA基因表达作为在使用总RNA进行Northern印迹分析时首选的RNA上样对照。
