Quantitation of apolipoprotein epsilon gene expression by competitive polymerase chain reaction in a patient with familial apolipoprotein E deficiency.
A simple method of obtaining semiquantitative and reliable data on apolipoprotein (apo) sigma gene expression is described. We detected apo sigma specific sequences by reverse transcription (rT)-PCR. For quantitative measurement, an apo sigma DNA standard was produced allowing the development of a competitive PCR-method. The efficiency of RNA extraction and cDNA synthesis was controlled by quantitation of a housekeeping gene (glyceraldehyde-3-phosphatedehydrogenase, G3PDH) in separate reactions. To imitate a defined induction of apo sigma gene expression, serial twofold dilutions of total RNA were reversely transcribed and the respective cDNAs used to perform a competitive apo sigma and G3PDH PCR. The change in apo sigma cDNA and G3PDH cDNA was 1.7-2.3-fold with an expected value of 2.0-fold. Standard deviations in three independently performed experiments were within a range of < 15% of the mean, indicating low intra-assay variation and high reproducibility. To illustrate this method, apo sigma gene expression was measured in a patient with complete lack of functional active apo E in comparison to healthy controls. The method presented here might be valuable in assessment of apo sigma gene expression in human disease.
Department of Internal Medicine I, Ruprecht Karls-University, Heidelberg, Germany. dobmeyer@em.unifrankfurt.de
J M
JM
Dobmeyer
M
M
Rexin
T S
TS
Dobmeyer
S A
SA
Klein
R
R
Rossol
G
G
Feussner
D000293:Adolescent; D000328:Adult; D001057:Apolipoproteins E / Q000172:deficiency* / Q000235:genetics*; D001161:Arteriosclerosis / Q000235:genetics; D002478:Cells, Cultured; D005260:Female; D015870:Gene Expression; D006801:Humans; D008264:Macrophages / Q000378:metabolism; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis; D015203:Reproducibility of Results; D014158:Transcription, Genetic
D001057:Apolipoproteins E; D012333:RNA, Messenger