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Transcriptional activity of the vasopressin gene measured by onestep quantitative RT‐PCR.
Author(s) -
Ponzio Todd A,
Yue Chunmei,
Gainer Harold
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1391-a
Subject(s) - vasopressin , microbiology and biotechnology , messenger rna , biology , gene expression , in situ hybridization , gene , ribonuclease , northern blot , real time polymerase chain reaction , hypertonic saline , rna , endocrinology , genetics
Traditional methods for detecting transcriptional activity of neuropeptide genes include in situ hybridization histochemistry and classical transcriptional run‐on assays for examining pre‐mRNA, and northern blots, ribonuclease protection assays and gene chip arrays for mRNA. These methods are labor intensive, costly, can require much tissue and are often too insensitive to detect small changes in low abundant transcripts. Here we describe a method for measuring changes in vasopressin (VP) pre‐mRNA using one‐step quantitative reverse‐transcription PCR (qRT‐PCR), with intron‐specific primers. Supraoptic nuclei were collected 30 minutes following an intraperitoneal injection of iso‐ or hypertonic saline. Rats injected with 900 mOsm saline expressed an increase in VP pre‐mRNA (173% above 290 mOsm isotonic controls, n=12). Injections of 2M NaCl, the strongest stimulus tested, resulted in pre‐mRNA levels 204% above control levels, n=6. These results agree with earlier studies and suggest qRT‐PCR can be used to measure transcription of VP and other neuropeptides in both quantitative and qualitative studies.