Amersham hybond n blot

Poly A+ RNA was fractionated from total RNA by NucleoTrap mRNA Mini Kit (Macherey-Nagel). 5 μg of Poly A+ RNA from HCT116 WT or KO cells were separated on 1% agarose gel prepared with NorthernMax Denaturing Gel Buffer (Ambion) and run in NorthernMax Running Buffer (Ambion). RNAs were then transferred to Amersham Hybond-N+ blot (GE Healthcare) by capillary transfer in 10 x SSC and crosslinked to the blot by UV (254 nm, 120mJ/cm²).
The DNA probes were labeled with [α−32P] dCTP by Prime-It II Random Primer Labeling Kit (Stratagene) as per manufacturer’s instructions. Hybridization was carried out using ULTRAhyb Hybridization Buffer (Ambion) containing 1 × 10⁶ cpm/ml of denatured radiolabeled probes overnight at 42°C. Blots were then washed with 2 x SSC, 0.1% SDS and 0.1 x SSC, 0.1% SDS sequentially at 42°C, and developed using phosphor-imager.

For the pre-rRNA Northern, 2 μg of total RNA extracted from WI-38 cells treated with Ctr-ASO or ASO-SNUL was separated on 1% denature agarose gel prepared with NorthernMax Denaturing Gel Buffer (Ambion) and run in NorthernMax Running Buffer (Ambion). RNA was then transferred to Amersham Hybond-N+blot (GE Healthcare) by capillary transfer in 10 x SSC and crosslinked to the blot by UV (254 nm, 120mJ/cm²). The DNA probes were labeled with [α–32P] dCTP by Prime-It II Random Primer Labeling Kit (Stratagene) as per manufacturer’s instructions. Hybridization was carried out using ULTRAhyb Hybridization Buffer (Ambion) containing 1X10⁶ cpm/ml of denatured radiolabeled probes overnight at 42°C . Blots were then washed and developed using phosphor-imager.

2 μg polyA+ RNAs were isolated using NucleoTrap mRNA Mini kit for polyA+ RNA extraction (Macherey-Nagel) and separated by 1% formaldehyde agarose gel. ssRNA Ladder (New England Biolabs) was used for marker. The agarose gel was prepared using NorthernMax Denaturing Gel Buffer (Ambion) and run using NorthernMax MOPS Gel Running Buffer (Ambion). RNA gel was washed two times with nucleotide-free water for 30 min each, followed by transfer in 10x SSC buffer to Amersham Hybond-N+ blot (GE Healthcare). RNA was then fixed by UV crosslinking with 120 mJ/cm². Labeling of random-primed probes was performed with the Prime-It II Random Primer Labeling Kit (Agilent) and a mammalian expression vector containing full-length FORCP cDNA. Hybridization was done overnight at 42°C in ULTRAhyb hybridization buffer (Ambion) as per the manufacture's instructions. Blots were washed at 42°C using 2X SSC+0.1% SDS and 0.1xSSC+0.1%SDS and imaged using a Phosphorimager.