Setting Up Reverse Transcription Quantitative-PCR Experiments

Quantitative real-time PCR (qRT-PCR), in conjunction with reverse transcriptase, has been used for the systematic measurement of plant physiological changes in gene expression. In the present paper, we describe a qRT-PCR protocol that illustrates the essential technical steps required to generate quantitative data that are reliable and reproducible. To demonstrate the methods used, we evaluated the expression stability of five [actin (ACT ), actin1 (ACT1 ), β-glyceraldehyde-3-phosphate dehydrogenase (GAPDH ), cyclophilin (CYC ), and elongation factor 1α (EF-1α )] frequently used housekeeping genes in rice. The expression stability of the five selected housekeeping genes varied considerably in different tissues (seedlings, vegetative and reproductive stages) in a given stress condition. The analysis allowed us to choose a set of two candidates (ACT1 and EF-1α ) that showed more uniform expression and are also suitable for the validation of weakly expressed genes (≥0.5 fold), identified through microarray analysis.