Hybridization

Gene Screen sheets carrying RNA samples as dot blots and Northern blots were presoaked in hybridization buffer（50%［v/v］formamide，3XSSC，0.1%［w/v］SDS，1X Denhardt's solution，100 μg/ml calf thymus DNA）at 37℃for 4 h，then hybridized with nick-translated 32 P-labeled plasmid DNA（106—107cpm）in 10 ml of fresh hybridization buffer at 42℃for 16 h［9］.After hybridization，the Gene Screen membranes were washed extensively in 2XSSC，0.5%（w/v）SDS，at 65℃for 1.5 h，then in 1XSSC，0.1%SDS（w/v），at 65℃for another 1.5 h.In experiments using heterologous plasmid probes for rcS and cab from pea and barley respectively，less formamide（10%）and lower temperatures were used for hybridization（37℃）and washing（42℃）for better hybridization.

The washed and dried dot and Northern blots were exposed to X-ray films at-70℃using intensifying screens for autoradiography.To assess mRNA quantitatively in the dot hybridization，several exposures of the films were scanned with a Zeineh Soft Laser Scanning Densitometer with a peak area integrator.This allowed us to estimate the level of an individual mRNA relative to total RNA which is mainly rRNA.A linear relationship was found between the scanner readings and the RNA on the Gene Screen in amounts of 5 μg and below hybridized with 32 P-labeled DNA（about 106 cpm）for a strip of 10 RNA dots.Therefore，aliquots containing identical amounts of total RNA（5 μg），monitored both by spectrophotometry（A at 260 nm）and RNA gel electrophoresis（mainly cytoplasmic 25S，18S rRNA，and plastid 16S，23S rRNA）were spotted onto Gene Screen.(https://www.daowen.com)