Fig. 1

Effects of the modified Northern protocol on the performance of Northern blot analysis. a Germinating wheat was vernalized at 0~2 ºC for 0, 10, 20, and 30 d, respectively. The total RNA was isolated and an equal amount of total RNA (50 µg) was resolved using the formaldehyde-agarose gel containing 12% formaldehyde. Gel treatment, transfer of separated RNAs to a positively charged nylon membrane, fixation of transferred RNAs on the membrane, and prehybridization and hybridization were performed, as described in Methods. The traditional posthybridization washes under high and low stringency sequentially for scheduled time were performed according to Clark [20]. The probe used for detection was an Aox1 cDNA from N. tabacum. Hybridization with 18 S was used as an internal control. The exposure times for the detecting Aox1 and 18 S were 5 d and 30 min, respectively. b An equal amount of total RNA (20 µg) from vernalized germinating wheat at 0~2 ºC for 0, 10, 20, and 30 d, respectively, was loaded to analyze the level of Aox1 transcripts. Quantitatively controlled moderate-stringency washes were performed, as described in Materials and methods. The exposure times for the detection of Aox1 and 18 S were 2 d and 30 min, respectively. The values below the blot denote the fold-change relative to the germinating wheat without vernalization (0 d), standardized to the 18 S rRNA content. This experiment was performed twice with similar results. c An equal amount of total RNA (20 µg) from the leaves of a chlorophyll reduced mutant of B. napus (MT) and its wild type (WT) grown in the field at two-leaf stage was subjected to RNA gel blot analysis following the modified protocol, as described in Materials and methods. Eight different probes were used to rehybridize to the same blot. The experiment performed twice, and the quantification of the hybridization signals from the autoradiographs showed that there was no significant difference in the expression of these genes investigated between the mutant and the wildtype oilseed rape