Tbe urea criterion precast gel

A549 cells in log growth phase were plated at 80% confluency in 10 cm plates. Cells were transfected with 1 nmol ON-TARGETplus siRNA SmartPool (Dharmacon) using Lipofectamine 2000 as indicated. Following a 24-h incubation, the cells were exposed to 2 Gy of IR. At 4 and 12 h post-IR cells were washed with PBS and lysed in wells with 2.5 ml of TRIzol; no-IR (0-time point) was harvested in tandem with the 12 h time point. Total RNA was extracted as per the study by Rio et al.1 50 μg of RNA was incubated with 10 U of CIP (New England Biolabs) at room temperature for 15 min. Samples were extracted with phenol, ethanol precipitated and resuspended in formamide loading buffer. Samples were separated on a 15% TBE-Urea Criterion Precast Gel (Bio-Rad). The gel was stained with ethidum bromide (used as loading control) and the RNA was transferred to Nylon Hybond-N+ (Amersham). RNA was crosslinked in a Stratagene UV Stratalinker 2400 run on the ‘optimal crosslink' setting. miR-34 northern blot was performed as previously described in the Bartel Lab (original) Northern Blotting Protocol using a DNA probe complementary to miR-34a.

A549 cells in log growth phase were washed with PBS and lysed in wells with TRIzol. Total RNA was extracted as per the study by Rio et al.1 50 μg of total RNA was treated with 10 U of Terminator 5′-Phosphate Dependent Exonuclease (Epicentre). Samples were extracted with phenol, ethanol precipitated and resuspended in formamide loading buffer. Samples were separated on a 15% TBE-Urea Criterion Precast Gel (Bio-Rad). The gel was stained with ethidum bromide (used as loading control) and the RNA was transferred to Nylon Hybond-N+ (Amersham). RNA was crosslinked in a Stratagene UV Stratalinker 2400 run on the ‘optimal crosslink' setting. Northern blot was performed as previously described in the Bartel Lab (original) Northern Blotting Protocol using a DNA probes complementary to the indicated miRNAs.