RNase 4 (NEB #M1284) Digestion Protocol

Optimized for 10 μg of RNA

1. In a 0.2 ml PCR tube, prepare a mixture of 10 µg RNA in 3 M urea to a final 10 µl volume. (Note #1)
   
   
2. In a thermal cycler, heat the RNA/ urea mixture at 90°C for 5 minutes followed by quick cooling to 25°C.
   
   
3. In a 1.5 mL centrifuge tube dilute the denatured RNA mixture into 20 μL of 1.5 X NEBuffer™ r1.1, for a final 30 µl volume.
   
   
4. Add 1 μL of RNase 4 stock (50 U/µl ). (Note #2)
   
   
5. Incubate the digest for 1 h at 37°C.
   
   
6. Stop the RNase 4 digest with 1 μL of NEB RNase inhibitor, murine (NEB #M0314) followed by incubation at room temperature for 10 minutes. (Note #3)
   
   
7. Proceed to downstream sample preparation for LC-MS/MS analysis. (Note #4)
   
   

Note #1: A specified volume of RNA (<6.25 µl)  can be combined with 3.75 µl of 8 M Urea and brought to a final 10 µl with nuclease-free water.

Note #2: 50 U of RNase 4 is optimized for the digestion of 10 µg of an unmodified 5000 nucleotide transcript.  More RNase 4 may be required to achieve optimal digestion of longer sequences or RNA-containing chemical modifications. For digestion of lesser quantities or short oligonucleotide sequences, further dilution of the RNase4 stock may be necessary. Dilution in 1X NEBuffer™ r1.1 is recommended.

Note #3: Both RNase inhibitor, Human Placenta (NEB #M0307) or Murine (NEB #M0314) inhibit RNase 4 activity. 

Note #4: RNase 4 Product fragments will contain a mixture of linear 3´-P and cyclic 2´, 3´ P termini.