A total extract dot blot hybridization procedure for mRNA quantitation in small samples of tissues or cultured cells.
A simple method for the estimation of specific mRNA concentrations in small tissue samples (as little as 1 mg) or cultured cells (lower limit 10(5) cells) is described. Guanidine hydrochloride extracts of whole cells or tissues are applied directly onto nitrocellulose and hybridized with the appropriate nick-translated probe. Loading according to DNA content allows expression of the result as concentration per cell. Hybridizing with a ribosomal RNA probe allows expression of results relative to rRNA and estimation of the RNA/DNA ratio in the sample. We describe the application of this procedure to the measurement of ceruloplasmin mRNA in tissues and cultured hepatocytes.
Grimes
A
A
Murdoch Institute for Research Into Birth Defects, Royal Childrens Hospital, Parkville Victoria, Australia.
McArdle
H J
HJ
Mercer
J F
JF
D000818:Animals; D002478:Cells, Cultured; D003101:Collodion; D004247:DNA / Q000032:analysis; D015151:Immunoblotting; D007668:Kidney / Q000032:analysis; D008099:Liver / Q000032:analysis; D008297:Male; D008832:Microchemistry; D009693:Nucleic Acid Hybridization; D012313:RNA / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis*; D012335:RNA, Ribosomal / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains
D012333:RNA, Messenger; D012335:RNA, Ribosomal; D012313:RNA; D003101:Collodion; D004247:DNA