An intron-based real-time PCR method for measuring vasopressin gene transcription.
The hypothalamus contains distinct neuronal populations that express distinguishing neuropeptides. The supraoptic nucleus contains magnocellular neurons that predominantly express either vasopressin or oxytocin. Transcriptional activators of vasopressin and other neuropeptides have been the subject of much research. Here we present a method of measuring neuropeptide transcription by tailoring one-step quantitative real-time PCR (qRT-PCR) for the analysis of processed and pre-mRNA (heteronuclear RNA). Using moderate and strong hyperosmotic stimuli to induce transcription, we report an increase in vasopressin transcription (pre-mRNA) of 141% and 406% over control levels in response to a 2% injection of 900 mOsm saline or a 1% body weight i.p. injection of 2 M NaCl, respectively. These results agree with a host of studies employing the more labor-intensive method of in situ hybridization histochemistry by which investigators also measured intron-containing heteronuclear RNAs. Furthermore, these results confirm that qRT-PCR with intron-specific primers can be used to rapidly analyze transcription, and suggest an important further benefit of a real-time PCR analysis, such as the ability of measuring transcription of multiple neuropeptides along with other genes from a single sample.
Ponzio
Todd A
TA
Laboratory of Neurochemistry, NINDS/NIH, Bethesda, MD 20892, USA.
Yue
Chunmei
C
Gainer
Harold
H
D000818:Animals; D017931:DNA Primers / Q000235:genetics; D007438:Introns / Q000235:genetics*; D008297:Male; D012322:RNA Precursors / Q000032:analysis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012462:Saline Solution, Hypertonic / Q000494:pharmacology; D013495:Supraoptic Nucleus / Q000187:drug effects / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics; D014667:Vasopressins / Q000096:biosynthesis* / Q000235:genetics*; D014882:Water-Electrolyte Balance / Q000187:drug effects / Q000235:genetics
D017931:DNA Primers; D012322:RNA Precursors; D012333:RNA, Messenger; D012462:Saline Solution, Hypertonic; D014667:Vasopressins