Quantitative analysis of beta-actin, beta-2-microglobulin and porphobilinogen deaminase mRNA and their comparison as control transcripts for RT-PCR.
Quantitation of target mRNAs using the reverse-transcription polymerase chain reaction found a widespread field of application in diverse biomedical diagnostic assays. However, the problem of varying sample quality has to be solved by correcting target molecule amounts through detection of an endogenous control template. The choice of an appropriate reference gene is still object of debate as pseudogene co-amplification and expression level variations may limit the usefulness of some currently used reference reactions. We compared quantitative expression levels of the commonly used endogenous reference genes beta-actin (beta-actin), beta-2-microglobulin (beta2-MG) and porphobilinogen deaminase (PBDG) using the TaqMan chemistry. With these assays we investigated the respective expression patterns in K562 cells and leucocytes of normal individuals as well as of malignoma patients. In K562 cells 1544+246 beta-actin, 65+30 beta2-MG and 22+/-8 PBDG copies/cell were detected. In normal leucocytes 491+/-97 beta-actin, 40+/-17 beta2-MG and <1 PBDG copies/cell were quantified. Leucocytes of various malignancies exhibited 84+/-51 beta-actin, 106+/-8 beta2-MG and <1 PBDG copies/cell. We conclude that beta2-MG is the most suitable reference gene tested as its variation between different sample origins and within distinct cell types was acceptable low.
Lupberger
J
J
Department of Medicine, Division of Hematology/Oncology, Charité Virchow-Klinikum, Humboldt University Berlin, FRG.
Kreuzer
K A
KA
Baskaynak
G
G
Peters
U R
UR
le Coutre
P
P
Schmidt
C A
CA
D000199:Actins / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D011163:Hydroxymethylbilane Synthase / Q000235:genetics*; D007962:Leukocytes / Q000502:physiology; D009369:Neoplasms / Q000097:blood / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D012016:Reference Values; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D001613:beta 2-Microglobulin / Q000235:genetics*
D000199:Actins; D012333:RNA, Messenger; D001613:beta 2-Microglobulin; D011163:Hydroxymethylbilane Synthase