Normalization of reverse transcription quantitative-PCR with housekeeping genes in rice.
Reverse transcription followed by real-time quantitative polymerase chain reaction (RT Q-PCR) is useful for the systematic measurement of plant physiological changes in gene expression. The validity of using 18S rRNA and three housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase, actin, and tubulin, was tested as a reference of RT Q-PCR. Under various growth stages of etiolated seedlings, different cultivars, and various times after UV-irradiation treatment, expression level of 18S rRNA correlated with total RNA suggesting the uniformity of RT Q-PCR efficiencies among samples. Relative expressions of housekeeping genes varied among samples and independently of experimental conditions, up to two-fold, signifying generally constant fraction of mRNA in total RNA. Results indicate 18S rRNA was the most reliable reference gene for RT Q-PCR of total RNA.
Kim
Bo-Ra
BR
School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea.
Nam
Hee-Young
HY
Kim
Soo-Un
SU
Kim
Su-Il
SI
Chang
Yung-Jin
YJ
D000199:Actins / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods / Q000592:standards*; D018506:Gene Expression Regulation, Plant / Q000235:genetics; D014644:Genetic Variation; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics* / Q000378:metabolism; D007723:Korea; D012275:Oryza / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D012337:RNA, Ribosomal, 18S / Q000235:genetics* / Q000378:metabolism; D012016:Reference Values; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D014404:Tubulin / Q000235:genetics* / Q000378:metabolism
D000199:Actins; D010940:Plant Proteins; D012337:RNA, Ribosomal, 18S; D014404:Tubulin; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)