Coupled reverse transcription-polymerase chain reaction (RT-PCR) technique is comparative, quantitative, and rapid: uses in alcohol research involving low abundance mRNA species such as hypothalamic LHRH and GRF.
The measurement of alterations in low abundance mRNAs such as the hypothalamic hormones luteinizing hormone-releasing hormone (LHRH) and growth hormone-releasing hormone (GHRH or GRF) from individual hypothalamic tissues in rats has previously been difficult and usually required either isolation of poly(A) mRNA or the pooling of numerous animals to obtain a reasonable signal on Northern blots. Although more sensitive detection methods exist, such as the use of RNA probes or solution hybridization (RNase protection), we have found the most reliable, sensitive, rapid, and accurate method is the reverse transcription-polymerase chain reaction (RT-PCR) using histone H3.3 as an internal control for both steps of this procedure. H3.3 is a cell-cycle independent and constitutively expressed gene in all tissues. We have developed an RT-PCR assay for LHRH and GRF mRNA quantitation and comparative analysis for hypothalamic and extrahypothalamic brain tissues and present the use of RT-PCR for LHRH quantitation in ethanol (EtOH) studies.
Kelley
M R
MR
Department of Molecular and Cellular Biochemistry, Loyola University of Chicago, Stritch School of Medicine, Maywood.
Jurgens
J K
JK
Tentler
J
J
Emanuele
N V
NV
Blutt
S E
SE
Emanuele
M A
MA
D000818:Animals; D001483:Base Sequence; D007987:Gonadotropin-Releasing Hormone / Q000235:genetics*; D013007:Growth Hormone-Releasing Hormone / Q000235:genetics*; D007031:Hypothalamus / Q000737:chemistry*; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase; D051381:Rats; D017207:Rats, Sprague-Dawley
D012333:RNA, Messenger; D007987:Gonadotropin-Releasing Hormone; D013007:Growth Hormone-Releasing Hormone; D012194:RNA-Directed DNA Polymerase