Buck K J, Harris R A, Sikela J M
Dept. of Pharmacology, University of Colorado Health Sciences Center, Denver 80262.
Biotechniques. 1991 Nov;11(5):636-41.
We have developed a sensitive, PCR-based method for quantitating changes in mRNA levels of members of gene families. In this approach, total mRNA is converted to cDNA and then PCR is carried out on family members simultaneously, using primers derived from regions conserved among family members. This is followed by gel electrophoresis and blotting of the product to filters. The level of expression of individual family members is determined by separate hybridizations using probes unique for each member and derived from sequences between the PCR primers. In this manner the same aliquot of mRNA, the same reverse transcriptase reaction, PCR, gel electrophoresis, and denaturation and blotting are used for analysis of each family member. Thus, experimental variation is minimized, and changes in mRNA levels of family members relative to one another can be monitored with precision. In addition, if a family member is known not to change as a result of the treatment employed, this mRNA can be used to normalize the data from other members and thereby allow individual variations to be quantitated. We have applied this approach to members of the GABAA receptor subunit gene family and studied effects of chronic ethanol treatment on mRNAs corresponding to several GABAA receptor subunits.
我们开发了一种基于聚合酶链反应（PCR）的灵敏方法，用于定量基因家族成员mRNA水平的变化。在这种方法中，总mRNA被转化为cDNA，然后使用源自家族成员间保守区域的引物，对家族成员同时进行PCR。随后进行凝胶电泳，并将产物转移至滤膜上。通过使用针对每个成员独特且源自PCR引物之间序列的探针进行单独杂交，来确定各个家族成员的表达水平。通过这种方式，相同的mRNA等分试样、相同的逆转录反应、PCR、凝胶电泳以及变性和印迹，都用于分析每个家族成员。因此，实验变异被最小化，并且可以精确监测家族成员mRNA水平相对于彼此的变化。此外，如果已知某个家族成员不会因所采用的处理而发生变化，那么该mRNA可用于对其他成员的数据进行标准化，从而能够对个体差异进行定量。我们已将这种方法应用于γ-氨基丁酸A型（GABAA）受体亚基基因家族的成员，并研究了慢性乙醇处理对对应于几种GABAA受体亚基的mRNA的影响。
