RNA extraction and Northern blot hybridization and quantification

RNA was extracted from cells as described previously (Chomczynski and Sacchi, 1987 ). RNA concentrations were determined by measuring absorbance at 260 nm with a Nanodrop apparatus. Equal amounts (3 µg) of total RNA were separated by electrophoresis on 1.5% agarose, 2% formaldehyde denaturing gels in 1X MOPS buffer and then transferred by capillarity to nylon membranes (Hybond; Amersham) using 20X SSC as transferring buffer. Membranes were dried and hybridized to ³²P-labeled probes. PCR DNA fragments of PGK1, SCR1, or RPL11B genes were labeled using the Megaprime DNA labeling system (Amersham). Hybridization was performed at 65°C using 2.5 × 10⁷ cpm of labeled probe in 10 ml hybridization buffer (0.5 M sodium phosphate, pH 7, 1 mM EDTA, 7% SDS, bovine serum albumin [BSA] 10 mg/ml). Filters were washed twice (5 min each time) at room temperature in 2X SSC and three times at 65°C in 0.2X SSC (10 min each time). Membranes were dried and exposed on phosphor screens to record the hybridization signal. Screens were scanned using a Storm PhosphoImager (Amersham Biosciences), and the resulting images were analyzed with ImageQuant software. We used SCR1 as a reference gene for normalization. Levels are expressed relative to those found before nitrogen addition.