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.
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- Rasmussen R, Morrison T, Herrmann M, Wittwer C (1998) Quantitative PCR by continuous fluorescence monitoring of a double strand DNA specific binding dye. Biochemica 2: 8–11.Google Scholar
- Vandesompele J, de Preter K, Pattyn F, Poppe B, Van Roy N, de Paepe A, Speleman F (2002b) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3: 1–11.Google Scholar