Quantification of estrogen receptor messenger RNA by quantitative polymerase chain reaction using internal standard fragment.
A simple and reliable polymerase chain reaction-based method for quantifying human and rat estrogen receptor (ER)-mRNA is described. This method involves co-amplification of cDNA transcribed from sample RNA with an internal standard to reduce tube to tube amplification variations. Human and rat internal standards were synthesized by insertion of a DNA fragment near the midportion of human and rat target ER cDNA molecules. After co-amplification, both products of target ER cDNA and internal standard were separated on agarose gel by electrophoresis and transferred onto a membrane filter. The radioactivity hybridized with 32P-labeled ER cDNA was counted. The ER mRNA content was calculated by linear regression analysis after obtaining a logarithm of ratios between the sample and the internal standard radioactivity. The coefficient of variation of this assay was 18.8%. An absolute amount of ER mRNA in less than 10(4) cultured cells could be measured.
Kagami
I
I
Department of Obstetrics and Gynecology, Gunma University School of Medicine, Japan.
Mizunuma
H
H
Miyamoto
S
S
Ibuki
Y
Y
Uchida
T
T
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D016889:Endometrial Neoplasms / Q000378:metabolism; D004717:Endometrium / Q000378:metabolism; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D006801:Humans; D008297:Male; D016254:Mutagenesis, Insertional; D009919:Orchiectomy; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D017208:Rats, Wistar; D011960:Receptors, Estrogen / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012015:Reference Standards; D015203:Reproducibility of Results; D014158:Transcription, Genetic / Q000187:drug effects; D014599:Uterus / Q000187:drug effects / Q000378:metabolism*
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D011994:Recombinant Proteins; D004958:Estradiol