Chalifour L E, Fahmy R, Holder E L, Hutchinson E W, Osterland C K, Schipper H M, Wang E
Bloomfield Centre for Research in Aging, Sir Mortimer B. Davis-Jewish General Hospital, Montréal, Québec, Canada.
Anal Biochem. 1994 Feb 1;216(2):299-304. doi: 10.1006/abio.1994.1045.
mRNA can be copied into cDNA with the use of reverse transcriptase so that the relative abundance of individual mRNAs is reflected in the cDNA product. With further manipulation a replica of the mRNA expression pattern can be duplicated into a radioactive double-stranded DNA probe. DNA from a series of genes inserted into plasmids can be fixed to a membrane using a slot blot manifold and probed with the RNA-derived DNA probe. The intensity of the hybridization signal for a given gene is a result of its relative abundance in the RNA-derived DNA probe. Quantitation can be achieved through the use of housekeeping genes as baseline monitors. Inclusion of vector sequences can negate any spurious hybridization to vector rather than insert sequences. We have successfully used this method to obtain gene expression patterns for RNA isolated from diverse sources including rodent tissues, various cell lines, and Drosophila and Caenorhabditis elegans samples. Northern blots have verified the results obtained. The pattern of expression of many genes can be determined from as little as 10 micrograms of total RNA, making this method ideally suited for studies in which RNA is rare or in short supply.
利用逆转录酶可将信使核糖核酸（mRNA）转录成互补脱氧核糖核酸（cDNA），从而使cDNA产物反映出各个mRNA的相对丰度。经过进一步操作，mRNA表达模式的复制品可被复制成放射性双链DNA探针。一系列插入质粒的基因的DNA可使用狭缝印迹装置固定在膜上，并用RNA衍生的DNA探针进行检测。给定基因的杂交信号强度是其在RNA衍生的DNA探针中相对丰度的结果。通过使用管家基因作为基线监测指标可实现定量分析。包含载体序列可消除与载体而非插入序列的任何虚假杂交。我们已成功使用此方法获得从多种来源分离的RNA的基因表达模式，这些来源包括啮齿动物组织、各种细胞系以及果蝇和秀丽隐杆线虫样本。Northern印迹法已验证了所获得的结果。仅从10微克总RNA就可确定许多基因的表达模式，这使得该方法非常适合用于RNA稀少或供应短缺的研究。
