Setting Up Reverse Transcription Quantitative-PCR Experiments

Quantitative real-time
PCR
(q
RT-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.