Ultrahyb oligo

For mRNA analysis, total RNA (15 μg) in glyoxal loading dye was run on a 1% TBE-agarose gel and then transferred to a nylon membrane using the NorthernMax-Gly Kit (Life Technologies, AM1946). For sRNA analysis, total RNA (2 μg) in Loading Buffer II (Life Technologies, AM8546G) was run on a 10% TBE-urea polyacrylamide gel and then transferred to a nylon membrane (GE Healthcare Life Sciences, RPN119B). For all blotting, RNA was subjected to UV cross-linking after transfer, the blot was pre-hybridized in ULTRAhyb-Oligo (Life Technologies, AM8663) at 65 °C for at least 30 min, and then hybridized overnight at 65°C. In some cases, hybridizations were performed using DNA probes (Table S2) 5′ end labeled with an IR dye. Otherwise, northern blotting was performed with RNA probes transcribed from PCR-derived templates (Table S2) with T7 promoters by using biotin-16-UTP and T7 RNA polymerase (Promega, P2075) according to the manufacturer's instructions (see above). All blots were washed and imaged as described in the Odyssey northern blot analysis protocol (Licor).

Let V denote the volume of buffer to be used for blots, calculated as: (i) 1 ml per 10 cm² of membrane; (ii) rounded up to the nearest 0.5 ml; (iii) no less than 1.5 ml. For example, V = 3.5 ml for a membrane that is 32 cm² and V = 1.5 ml for a membrane that is 9 cm². Pre-heat hybridization buffer (HB; ULTRAhyb-Oligo, Life Technologies, catalog #AM8663) to 60–68°C for 1 h and maintain at 37°C until use. Pre-hybridize blot in (V − 0.5 ml) of HB in a hybridization bottle (Wheaton, catalog #805000 or #805021) for 30–60 min at 37°C in a rolling hybridization incubator (SciGene, catalog #1040-50-1). Prepare probe solution by adding each probe into a total volume of 0.5 ml of HB at 37°C such that each probe will be at a final concentration of 5 nM in volume V. Add the probe solution to the pre-hybridization solution and incubate blot overnight at 37°C in a rolling hybridization incubator. Remove excess probes by washing four times using pre-heated 37°C wash solutions with volume 2V per wash: (i) two low-stringency washes (2× saline sodium citrate (SSC), 0.1% sodium dodecyl sulphate (SDS); 5 min at 37°C); (ii) two high-stringency washes (0.2× SSC, 0.1% SDS; 15 min at 37°C). Wash reagents: (20× SSC: Invitrogen, catalog # 15557-044), (SDS: Life Technologies, catalog # 15525-017).