Ballagi-Pordány A, Ballagi-Pordány A, Funa K
Ludwig Institute for Cancer Research, Biomedical Center, Uppsala, Sweden.
Anal Biochem. 1991 Jul;196(1):89-94. doi: 10.1016/0003-2697(91)90122-a.
A method for quantitative determination of specific cellular mRNA is described. The mRNA in a dilution series of total RNA was reverse transcribed by an oligo-dT primer and the cDNA was amplified by the polymerase chain reaction (PCR) using sets of specific primers. A 32P- or biotin-labeled specific probe was hybridized to the PCR products immobilized on nitrocellulose membrane. The intensity of the hybridization signals was evaluated for quantification of the PCR products. A standard curve was produced by the known amount of the in vitro transcribed cRNA, which contained the same sequence as the mRNA. The series of standard cRNA dilutions were reverse transcribed, amplified and hybridized in the same manner. The amount of the specific RNA was deduced by fitting to the standard curve. Two tissue specimens of intestinal tumors, evaluated on the basis of hybridization signals by three different methods, were shown to contain similar amounts of beta-actin mRNA. Furthermore, a Chinese hamster ovary (CHO) cell line transfected with platelet-derived growth factor (PDGF) beta-receptor cDNA was found to contain similar amounts of beta-actin mRNA as the untransfected CHO cell line. However, the transfected CHO cell line contained over 10(11) copies of the PDGF beta-receptor mRNA per microgram of total RNA, while the untransfected one showed no detectable RNA, indicating that the latter contained less than 10(6) copies per microgram of total RNA in this assay.
描述了一种定量测定特定细胞mRNA的方法。总RNA稀释系列中的mRNA用寡聚dT引物进行逆转录，然后使用特定引物组通过聚合酶链反应（PCR）扩增cDNA。将32P或生物素标记的特异性探针与固定在硝酸纤维素膜上的PCR产物杂交。评估杂交信号的强度以定量PCR产物。由已知量的体外转录cRNA产生标准曲线，该cRNA含有与mRNA相同的序列。一系列标准cRNA稀释液以相同方式进行逆转录、扩增和杂交。通过拟合标准曲线推断特定RNA的量。通过三种不同方法基于杂交信号评估的两个肠道肿瘤组织标本显示含有相似量的β-肌动蛋白mRNA。此外，发现用血小板衍生生长因子（PDGF）β受体cDNA转染的中国仓鼠卵巢（CHO）细胞系含有与未转染的CHO细胞系相似量的β-肌动蛋白mRNA。然而，转染的CHO细胞系每微克总RNA含有超过10（11）个PDGFβ受体mRNA拷贝，而未转染的细胞系未检测到RNA，表明在该测定中后者每微克总RNA含有少于10（6）个拷贝。
