RNA Detection

3.6.2 RNA Fragmentation
Quality Control

1. Prepare your samples by mixing 5μL of RNA marker (green
   cap vial) with 1μL of your fragmented RNA samples.


2. With the rest of gel–dye mix, load 9μL in the well marked “G”
   surrounded with a black circle and proceed as described in
   Subheading3.5 fromsteps 9to 19.


3. Analyze the results obtained, the overall mean size of the RNA
   fragments should be around 50–100 nts.

3.7 End-Repair 1. Combine 16μL of your treated RNA samples in a PCR tubes
with 2μL of phosphatase buffer, 1μL of RiboLock RNase
Inhibitor, and 1μL of Antarctic Phosphatase.

2. Mix by pipetting up and down.


3. Incubate the PCR tubes for 30 min at 37C then for 5 min at
   70 C to inactivate the phosphatase and store for indefinite
   hold at 4C in a thermal cycler.


4. Add the following components to the previous mix: 17μLof
   RNase-free water, 5μL of PNK buffer, 5μLofATP,1μLof
   RiboLock RNase Inhibitor, 2μL of PNK enzyme.


5. Incubate in a thermal cycler for 1 h at 37C and immediately
   proceed to the next step.

3.8 RNA Purification All the reagents except ethanol used for RNA purification are part
of RNeasy MinElute Cleanup kit.

1. Transfer the sample to a new 1.5 mL tube, and to adjust the
   final volume to 100μL, add 50μL of RNase-free water.


2. Add 350μL of RLT buffer, mix by vortexing.


3. Add 675μL of 96% ethanol and mix by inverting the tube up
   and down (seeNote 16).


4. Transfer 700μL of the sample to an RNeasy MinElute spin
   column (stored at 4 C until use). Centrifuge for 30 s at
   8000 g.


5. Repeat thestep 4 with the rest of the sample. Then, add
   500 μL of RPE buffer to the column. Centrifuge for 30 s at
   8000 g.


6. Discard the flow-through. Add 750μL of 80% ethanol. Centri-
   fuge for 2 min at 8000g.


7. Transfer the column to a new collection tube and centrifuge at
   full speed for 5 min with the lid open.


8. Transfer the column to a new 1.5 mL tube (provided with the
   kit). Add 10μL of RNase-free water in the center of the column
   filter. Wait for 1 min.


9. Centrifuge at full speed for 1 min to elute. The recovered
   volume is about 9μL.

Quantification of 2^0 - O-Me by RiboMethSeq 41