Methylated RIP (meRIP) troubleshooting tips

Step Potential Problems Experimental Suggestions
RNA fragmentation Not enough/too much fragmentation
  • If fragments are too large or too small, optimize fragmentation conditions.
Immuno- precipitation Antibody doesn’t immune- precipitate methylated RNA
  • Titrate antibody and RNA to determine most effective immunoprecipitation conditions by performing IP using a dilution series of antibody with a fixed amount of RNA.
  • Increase incubation time of the antibody of interest with RNA to overnight at 4°C.
Insufficient quantity of magnetic beads in immune- precipitation
  • The magnetic beads settle to the bottom of the tube over time. Verify that the Magna ChIP Protein A/G magnetic beads are well mixed prior to removing the appropriate volume for IP.
  • Carefully aspirate liquids when using vacuum aspirator and use a high strength neodymium magnetic rack such as the Milliporesigma’s Cat. # 20- 400 Magna GrIP Rack to ensure Magna ChIP Protein A/G magnetic beads are tightly held against the wall of the microcentrifuge tube.
RNA Purification Low RNA yield
  • Most MeRIP reactions can expect yields on the order of tens to hundreds of nanograms from 5 µg of mRNA or 300 µg of total RNA depends on the cell line of tissue of origin.
  • If RNAs are not detectable consult immunoprecipitation step troubleshooting above.
RNA degraded
  • Use RNase inhibitor in solutions as recommended in this protocol. Make certain that all work conditions are RNase-free and RNases are not being introduced.
  • Follow the guidelines in the RNase control section before the Detailed Protocol section.
  • Use RNase-inactivating reagents to ensure work area and materials are RNase-free.
No RNA detected
  • Confirm the quality of RNA before and after fragmentation
PCR No PCR product from Positive Control assay
  • Increase amount of MeRIP sample used for PCR reaction up to 10% of total reaction volume.
  • Ensure amplification reaction program is correctly set on thermal cycler.
  • Re-examine primers for correct Tm. (If own primers are designed)
  • Perform PCR reaction with melting curve assessment to confirm amplification conditions and ability of primers to generate a single DNA product
High  background level with negative control assay
  • Insufficient wash after immunoprecipitation. Increase the time of beads washing. Too long immunoprecipitation incubation time. Shorten the incubation time.

For more information about RIP experiment guides, troubleshooting tips and supplementary protocols, please view our RNA Immunoprecipitation (RIP) page.