A PCR method for the quantitative assessment of mRNA for laminin A, B1, and B2 chains.
Laminin, a basement membrane glycoprotein, is involved in the development of normal kidney and its dysregulation contributes to glomerulosclerosis in renal disease. Studies designed to assess the regulation of this molecule at the level of transcription have been hindered by the relatively low abundance of the mRNA, making standard techniques such as Northern hybridization and RNase protection difficult and inaccurate. In this report, we have utilized the polymerase chain reaction (PCR) to quantitate differences in laminin mRNA expression during normal development of the mouse kidney. We have constructed a synthetic template to be used as an internal standard for mRNA quantitative of laminin chains A, B1 and B2, and beta-actin. This DNA template can be used to generate complementary RNA which can be reverse transcribed and amplified simultaneously with 0.5 microgram of total cellular mRNA allowing for accurate and absolute quantitation of laminin mRNA by PCR.
Horikoshi
S
S
Laboratory of Developmental Biology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland.
Fukuda
K
K
Ray
P E
PE
Sawada
M
M
Bruggeman
L A
LA
Klotman
P E
PE
D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D015152:Blotting, Northern; D007797:Laminin / Q000737:chemistry / Q000235:genetics*; D051379:Mice; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000378:metabolism*
D007797:Laminin; D015335:Molecular Probes; D012333:RNA, Messenger