Total RNA was isolated from 8-week-old flower buds using TRIzol reagent (Life Technologies) according to the manufacturer's instructions. RNA was treated with DNase Max (QIAGEN) when RNAs were used in reverse transcription (RT)-PCR or quantitative RT-PCR (qRT-PCR) assays. Mitochondrial mRNA abundances were measured by qRT-PCR. They were calculated using the comparative ΔΔCt method after normalization to the nuclear 18S ribosomal RNA as previously described in (15 (link)). Two biological and three technical repeats were performed for these analyses.
For RNA gel blotting, 10 μg of total RNA was electrophoretically separated in formaldehyde-containing (1.5% [w/v]) agarose gels and transferred onto nylon membranes (Genescreen) as described previously (10 (link)). Hybridization probes were generated by PCR amplification using gene-specific primers (listed inSupplementary Table S1 ) and radiolabeled using the Prime A Gene labeling kit (Promega) according to the manufacturer's recommendations.
For RNA gel blotting, 10 μg of total RNA was electrophoretically separated in formaldehyde-containing (1.5% [w/v]) agarose gels and transferred onto nylon membranes (Genescreen) as described previously (10 (link)). Hybridization probes were generated by PCR amplification using gene-specific primers (listed in
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