PCR-based cloning, sequencing, and exon mapping of lymphocyte-derived neuroendocrine peptides.
In this report a procedure for the analysis of mRNA expression in cells of limited availability by the reverse transcriptase-polymerase chain reaction (RT-PCR) method is described. The cells are lysed with Nonidet P-40, and the mRNA in the lysate is used directly as template for the cDNA synthesis reaction. Target cDNA is then amplified by PCR, and the products can be analyzed that same day by agarose gel electrophoresis. The oligonucleotide primers used for amplification are designed to include restriction sites to facilitate cloning for subsequent sequencing. We have demonstrated that luteinizing hormone-releasing hormone mRNA can be amplified from the hypothalamus and thymus of a 7-day rat pup, in which the starting cell number was limited. Furthermore, exon usage by target cDNA in different cell types can be easily determined by amplifying with exon-specific primers. Proopiomelanocortin (POMC) mRNA expressed in the pituitary utilizes all three exons, while a majority of POMC mRNA expressed in lymphocytes lacks exons 1 and 2. Thus, this provides an extremely rapid and sensitive means not only for analyzing mRNA expression but also for differential exon usage.
Department of Physiology and Biophysics, University of Alabama at Birmingham 35294.
C C
CC
Maier
J E
JE
Blalock
D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D005091:Exons; D005786:Gene Expression Regulation; D005796:Genes; D007987:Gonadotropin-Releasing Hormone / Q000096:biosynthesis / Q000235:genetics*; D007031:Hypothalamus / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism*; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D011333:Pro-Opiomelanocortin / Q000096:biosynthesis / Q000235:genetics*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D013950:Thymus Gland / Q000378:metabolism
D018076:DNA, Complementary; D012333:RNA, Messenger; D007987:Gonadotropin-Releasing Hormone; D011333:Pro-Opiomelanocortin