Quantitative determination of gene expression in human lymphocytes assessed by reverse transcription-polymerase chain reaction coupled to high-performance liquid chromatography.
Gene expression in human lymphocytes was assessed using reverse transcription and polymerase chain reaction amplification followed by ion-pair reversed-phase chromatography analysis. Competitive PCR was used to quantitate the desired cDNAs with a polivalent competitor adaptable to multiple novel mRNAs estimations with minor changes. Accuracy was 11.27+/- 11.87% (n = 7), as determined using standards. The coefficients of variation of the assessment of human OK12b were 7% (n = 6), 7.68 attmol/microg of total RNA, and 21% (n = 6), 0.93 attmol/microg of total RNA. Sample-to-sample variation in the reverse transcription and in the quantity and quality of RNA was attenuated by normalising results to beta-actin mRNA expression. The correlation between the OK12b/beta-actin ratio and competitive assessments of OK12b was 0.984, n = 6. The correlation between HPLC results and an independent method based on radionuclide uptake by the product, detected by electrophoretic separation, was 0.848, n = 10.
Andí
I
I
Departamento de Investigación Neuroquímica, Hospital Psiquiátrico de Zamudio, Vizcaya, Spain. iandia@hzam.osakidetza.net
Zumárrag
M
M
Dávil
R
R
Miller
J C
JC
Friefhoff
A J
AJ
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D017931:DNA Primers; D020869:Gene Expression Profiling; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D012333:RNA, Messenger