Glyoxal/borate and RNase T1-based detection of inosine residues in RNA
Incubate for 45 min at 37 °C.
Dilute the reaction by adding an equal volume (100 μl) of 1 M sodium borate, pH 7.5.
Immediately precipitate with five volumes of 100% ethanol (relative to the diluted mixture; 1000 μl).
Spin at >15,000 g for 30 minutes at 4 °C.
Discard the supernatant.A white pellet of 10-20 μl is expected to have formed.
Add 1 volume of 70% (aq.)
ethanol to wash the pellet.
Directly proceed to a spin at >15,000 g for 5 minutes at 4 °C.
Discard the supernatant.
Let the pellet dry at room temperature, so that no traces of liquid remain (30-60 minutes).
Alternatively, dry under vacuum for 10 minutes (without heating).
Solubilize the pellet in 40 µl of Tris-borate buffer (10 mM Tris-HCl, pH 7.8; 1 M sodium borate, pH 7.5).
The final volume of the resuspended sample should be 47-48 µl.After adding the buffer, wait for 5-10 minutes before resuspending the pellet.
This makes the pellet less sticky and prevents sample loss.
Add RNase T1 to a desired final concentration.
If required, dilute the RNase in the Tris-borate buffer.
Use the Tris-borate buffer to complete the volume to 50 µl.Optimize the amount using the enzyme concentration gradient by monitoring the progress of the reaction by a Northern blot or RT-PCR.
If the inosine content of the investigated site is close to 100%, 1000 U of RNase T1 will cut essentially all molecules of a transcript present at ~0.5 pmol or less in 3 minutes at 37 °C.
Incubate for 3 minutes at 37 °C.
Dilute the reaction with an equal volume of ice-cold RNase-free water and place on ice.
Pause point: Proceed directly to the next step or snap-freeze the reactions in liquid nitrogen and store at -80 °C.
Extract the RNA, e.g.
using the home-made Trizol substitute (see the corresponding protocol: RNA extraction using the 'home-made' Trizol substitute).
Solubilize the pelleted RNA in 50 µl of 100 mM sodium phosphate pH 7.0.
Add an equal volume (50 μl) of 100% DMSO and mix well.
De-glyoxalate the RNA by incubating it for 3 hours at 65 °C (with gentle mixing every 15-30 minutes).
Dilute the reaction with an equal volume of ice-cold RNase-free water (100 μl).
Extract the RNA, e.g. using the home-made Trizol substitute (see the corresponding protocol: RNA extraction using the 'home-made' Trizol substitute).
Resuspend the RNA in RNase-free water.
Store at -80 °C or proceed directly to downstream analysis (Northern blot hybridization, primer extension, RT-PCR, RNA-Seq).
