Total RNA was extracted using the TRIzol reagent (Invitrogen) according to manufacturer’s instructions, whereas from caryopses

Total RNA was extracted using the TRIzol reagent (Invitrogen) according to manufacturer’s instructions, whereas from caryopses it was isolated by a LiCl based method. The resulting RNA was treated with RNase-free DNase I (Promega) according to the manufacturer’s protocol. Following digestion, nucleotides were removed from RNA using a G50 sepharose buffer exchange column (Amersham). Absence of genomic DNA contamination in DNase I-treated samples was checked by PCR using a primer pair (5’-CTTATGCAATCCAATGATGG-3’ and 5’-TCTATGGTTCTGAAGAGGACC-3’) designed to amplify an intron sequence of a gene encoding typical PDI and located on chromosome 4A [1]. When a single DNase treatment did not completely remove interfering genomic DNA, a second DNase incubation was performed to eliminate any detectable DNA. Synthesis of cDNA from the DNase I treated RNA was only performed when the genomic control amplifications scored negative. RNA concentration and integrity were checked with a UV/VIS spectrophotometer Lambda 3B (Perkin Elmer) before and after DNase I digestion. The quality of RNA samples was also assessed by electrophoresis on 1% formaldehyde agarose gels. First-strand cDNA was synthesized from 3 μg of RNA by the Expand TM