Analysis of RNA capping in vivo (RNA IP)

All steps in this protocol require working rigorously and in clean conditions to limit as much as possible the contamination of the samples with RNases.

1.   Prepare RNAs

·      Purify the RNAs of interest using any method available (ie RNeasy kit from qiagen).

·      Quantify RNA samples at OD₂₆₀ (a minimum of 5μg per sample is optimal).

2.   Prepare IPs and inputs

·      Equilibrate 5μl of Dynabeads protean A and 5μl of Dynabeads protean G in ice cold PBS + 5mg/ml BSA. Repeat 3 times. Make sure you have enough beads for the cap IPs with RNA, the control IPs (w/o antibody) with RNA and a cap IP with gDNA instead of RNA.

·      Ressuspend beads in ice cold PBS + 5mg/ml BSA, add 10μl of anti m₃G-/m⁷G-cap (H20, SYSY) per cap IP and incubate 2h at 4C (with control beads).

·      Wash 2 times with ice cold PBS + 5mg/ml BSA and ressupend beads in 250μl of IPP150 per IP + 40U of Ribolock (fermentas) (use LoBinding tuges from eppendorf).

·      Add 1.5μg of RNA per IP:cap and IP:CT.

·      Add 1.5μg of gDNA to an IP:cap.

·      Save 100ng of RNA and gDNA as inputs.

·      Incubate IPs at 4C on rotator ON and proceed with the reverse transcription of the inputs (ie using Quantitech Reverse Transcription kit from Qiagen).

3.   Wash and Harvest IPed RNAs

·      Wash beads 5 times with ice cold IPP150.

·      Ressuspend in IPP150 + 1mg/ml of proteinase K and incubate 30min at 37C agitating every 10min.

·      Collect SNT and proceed immediately with the “RNA clean up” protocol from the RNeasy kit (qiagen).

4.   Reverse transcription and qPCR

·      Use 12μl of each elution for each RT.

·      Dilute the reverse transcribed inputs 26X and make a Standard curve out of one of them.

·      Perform qPCRs with defined primer sets.

IPP150: