MicroRNA and siRNA Cloning Protocol
Bartel Lab Protocol Updated: July 2005
Kinase RNA markers to very high specific activity by the following procedure:
1 µL of 1 µM RNA (44.12R or 18.113R)
Incubate for 1 hr at 37 oC.
2 µL PNK Buffer 1 µL PNK
Gel-purify labeled RNA on a 20% denaturing acrylamide gel, using glycogen as a carrier to
5 µL 6000 Ci/mmol 32P γ-ATP (3µM) 11 µL dH2O
precipitate after eluting from gel slice.
Resuspend each labeled RNA in 40 µL dH2O. Run 2 ul on a test acrylamide gel and wrap the wet test acrylamide gel. Expose to phosphorimage plate and see if you detect a strong signal after a 5 minute exposure. Generally, 3000 counts of labeled RNA is a good starting point to test. The goal is to determine the minimum amount of labeled RNA to add to your total RNA during the purification step of 18-26-mers. Minimizing the addition of marker RNAs will maximize the number of miRNA/siRNA clones in the final step.
Pour a 15% 1.5 mm denaturing polyacrylamide gel with wide wells (23mm). Prerun to warm up gel. Make sure the lane is quite flat for nice loading and resolution of markers.
Prepare an aliquot of total RNA (50-500 µg), adding trace but very high specific activity radiolabeled marker RNA and 1X volume of 8M Urea, 0.5 mM EDTA Loading Dye. Heat for 5 min in 80o C heatblock and load entire volume in one lane. Electrophorese until the BB dye reaches the bottom. Expose gel, cut out gel slice that includes both top and bottom hot markers. Elute RNAs O/N in 0.3M NaCl, precipitate in 2X volume EtOH (>2 hrs) with glycogen (1 µg/ ml). Spin down (full speed, 30 min) and resuspend in 10 µl dH2O.
: not all total RNA sources, particularly commercial total RNA sources, may contain small RNAs like miRNAs and siRNAs! If a sample of “total” RNA was purified by the popular silica matrix column procedure (i.e. Qiagen RNEasy columns), it will be significantly depleted in small RNAs. Extraction procedures like Trizol/TriReagent, however will purify all RNAs, large and small, and are the recommend methods for isolating total RNA from biological samples that will contain miRNAs/siRNAs.
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