A method for analysis of gene expression patterns.
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.
Chalifour
L E
LE
Bloomfield Centre for Research in Aging, Sir Mortimer B. Davis-Jewish General Hospital, Montréal, Québec, Canada.
Fahmy
R
R
Holder
E L
EL
Hutchinson
E W
EW
Osterland
C K
CK
Schipper
H M
HM
Wang
E
E
D001483:Base Sequence; D015139:Blotting, Southern; D015342:DNA Probes; D018076:DNA, Complementary / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics*; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010957:Plasmids / Q000235:genetics; D012313:RNA / Q000032:analysis / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics
D015342:DNA Probes; D018076:DNA, Complementary; D008567:Membranes, Artificial; D012333:RNA, Messenger; D012313:RNA; D012194:RNA-Directed DNA Polymerase