Northern Blot for small RNAs

 

 

Electrophoresis and transfer:

 Dry 20 ug of RNA with the speed vac and resuspend in 10ul ambion gel loading buffer II.

Gel preparation: 15 % TBE/Urea/polyacrylamide gel

1X 2X 3X
4.2g 8.4g 12.6g Urea
1ml 2ml 3ml 10X TBE
3.75ml 7.5ml 11.25ml 40% Acrylamide 19:1

Heat the mix at 37 °C to dissolve.

Adjust vol. to 10 mL per gel

Add 80 uL APS 10 % and 8 uL temed per gel

Heat samples 5min @65°C

Run at 200 V in 0.5 X TBE until the first blue dye is at the bottom of the gel. (1h)

Stain with EtBr (1ul/25ml MQH2O, 2X H2O wash) for 15 min and take a picture for loading control.

Transfer on Hybond XL membrane with the BioRad semi-dry apparatus in 0.5 X TBE at 0.4 A for 50 min.

UV-crosslink membrane X2 and store wrapped at 4C until ready to probe.

Probe preparation (star fire kit):

 Annealing:

1 uL starfire probe (0.5 uM)

1 uL template oligo

1 uL starfire reaction buffer 10X

Incubate 1 min at 95 °C

Remove the tube from heat and allow the reaction to cool to RT (5 min). Briefly spin the reaction.

Labeling:

Add 3 uL of α32P dATP and 1 uL of exo- Klenow DNA polymerase

Incubate 1 hour at RT.

Add 40 uL of stop buffer

Prep oligo columns – 1min @1000g, discard FT

Purification:

Load entire labeling reaction (47ul) onto Sephadex G25 oligo spin columns (Roche) and centrifuge 4min at 1000g.

Before use heat probe at 65 °C 5 min and keep on ice.

Hybridization: 

Pre-hybridization is done at 65°C for 1 h with 6 mL of Ambion UltraHyb oligo buffer.

Add 50 uL of starfire probe to the pre-hybridization buffer and shift the temperature of the oven to RT. The blot is then allowed to hybridize O/N.

The actual temperature of the oven the next morning is about 32 °C.

Washing and exposure: 

Washes are performed in the oven at RT

2X 30 min washes in 0.5 % SDS

1X 15 min wash in 1X SSC, 0.2 % SDS

Membranes are exposed O/N or longer depending on the signal!

Source: Wu et al. Mol Cell 2010