Exogenous reference RNA for normalization of real-time quantitative PCR.
We have utilized an in vitro transcribed 3' mRNA fragment of the plant gene ribulose bisphosphate carboxylase (RuBisCO) as an exogenous standard for normalization of quantitative PCR data. Both K562 cells and primary erythroid CD34+ progenitor cells were treated with sodium butyrate and changes in gamma-globin mRNA levels were assayed using a previously published TaqMan probe and primer set, while RuBisCO levels were assayed by a SYBR Green detection assay. The data presented show that a correction to measured gamma-globin induction was necessary with both cell types. The correction for the CD34+ progenitor cells was a striking 95% increase, while that for the K562 cells was 44%. The use of an exogenous reference such as in vitro transcribed mRNA for the RuBisCO plant gene provides a robust and sample-independent method for the normalization of quantitative PCR data in bacterial and animal cells.
Smith
Reginald D
RD
Laboratory of Chemical Biology, NIDDK, NIH, Building 10, Room 9N318, 10 Center Drive, MSC 1822, Bethesda, MD 20892-1822, USA. smithrd@helix.nih.gov
Brown
Benjamin
B
Ikonomi
Pranvera
P
Schechter
Alan N
AN
D018952:Antigens, CD34 / Q000378:metabolism; D002478:Cells, Cultured; D020869:Gene Expression Profiling / Q000379:methods / Q000592:standards*; D005914:Globins / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D020014:K562 Cells / Q000378:metabolism; D012313:RNA / Q000032:analysis / Q000592:standards; D012333:RNA, Messenger / Q000032:analysis* / Q000592:standards*; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods / Q000592:standards*; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics* / Q000378:metabolism; D012680:Sensitivity and Specificity; D014481:United States
D018952:Antigens, CD34; D012333:RNA, Messenger; D012313:RNA; D005914:Globins; D012273:Ribulose-Bisphosphate Carboxylase