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Quantification of human H1 histamine receptor mRNA from peripheral blood
C J Rosebraugh1, R P Yasuda, S U Yasuda
1Department of Medicine, Georgetown University Medical Center, Washington, DC, USA.
Study Objective:
To develop a reverse transcription (RT)-polymerase chain reaction (PCR) technique to detect and quantify human histamine1 (H1) receptor mRNA in peripheral blood.
Methods:
Primer pairs were based on the human H1 receptor nucleotide sequence. A competitive reference standard (CRS) was developed that used the same primers as wild-type mRNA but contained a 92-bp deletion. RT-PCR was performed with 5 microg of total RNA obtained from venous blood of six subjects that was added to known concentrations of CRS RNA. Linear regression comparing wild-type with CRS product was used to quantify wild-type mRNA.
Measurements And Main Results:
Three subjects had detectable H1 mRNA, with a range of 31-435 pg. In three subjects PCR product was not detected, although the presence of RNA was confirmed. Redesigned primer pairs showed mRNA to H1 receptor in two of the remaining subjects, but it was undetectable in the third.
Conclusion:
RT-PCR can be used to detect and quantify human H1 receptor mRNA from peripheral blood.