Evaluation of eleven reference genes for Reverse Transcriptase Quantitative PCR of rubber tree under water deficit
Reverse Transcriptase Quantitative PCR (RTqPCR) is a powerful technique in order to detect low abundance of mRNA in the plant cell. The measurement of transcript abundance is relative to the control of expression such as housekeeping genes. Therefore, the reliability of RT-qPCR depends essentially to the choice of these internal controls also called reference genes. That is the reason why a prior validation of reference genes is suggested for every set of cDNA samples used in a new RT-qPCR experiment. This study aimed to analyze the stability of eleven selected housekeeping genes in three Hevea brasiliensis tissues (leaf, bark and root) under15 days of moderate water deficit. Total RNA was isolated from 18 samples consisting of control and stressed-plants collected at day-0 (D0), day-5 (D5) and day-15 (D15).The quality of cDNA synthesized was examined by PCR using HbActin primer. The eleventh primers encoding Hevea housekeeping genes (HbActin, HbelF1Aa, HbUBC4, HbUBC2b, HbYLS8, HbRH2b, HbRH8, HbUBC2a, HbαTub, Hb40S and HbUBI) were validated using PCR amplification. The Crossing-point (Cp) values were measured using a second derivative method after RT-qPCR analysis revealing a significantly higher Cp mean values for 11 housekeeping genes at D5 compared to D0 and D15 sampling points. This study suggests that a simple coefficient of variation (CV) method can be used to rank Hevea reference genes based on its stable expression. Five housekeeping genes (HbRH2b, HbRH8, HbUBC4, HbαTUB and HbActin) can be used for RT-qPCR analysis in Hevea brasiliensis under moderate water deficit. The HbRH2b gene was the most stable among others.