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 ) .
