Establishment of Real-time TaqMan-Fluorescence Quantitative RT-PCR Assay for Detection and Quantification of mRNA Expression of RAc1 of Rice

The cDNA was cloned as the standard for real-time quantifying mRNA of RAc1 of rice and the TaqMan fluorescence quantitative PCR assay for detecting RAc1 gene expression was established as internal control gene for detecting other gene expression of rice.Total RNA extracted from leaf of rice was reverse transcribed to cDNA.The prospective amplicon was amplified and purified,then was ligated with pMD19-T Simple vector and transformed into bacterium JM-109.Plasmid DNA extracted from positive clones were verified by PCR amplification and sequenced.The concentration of purified DNA template was detected by analyzing absorbance in 260 nm and then the combined plasmid was diluted to series as standard for FQ-PCR.RAc1 was amplified by real-time fluorescence quantitative PCR from the plasmid DNA.The method of RAc1 mRNA real-time PCR was well established,which detected as low as 102 copies with the linear range from 102 to 107 copies.The standard curves showed high correlations(r=1.000) and PCR efficiency(E=98.2%).A series of standards for real-time PCR analysis had been constructed successfully,and real-time TaqMan-Fluorescence Quantitative RT-PCR was reliable to quantitatively evaluate mRNA of RAc1 of rice.Furthermore,RAc1 gene as the internal control gene for detecting other gene expression of rice.