Chromatin Isolation by RNA Purification (ChIRP) troubleshooting tips

Step Potential Problems Experimental Suggestions
Cross-linking Not enough or too much cross-linking
  • Always use fresh unopened glutaraldehyde.
  • The amount of glutaraldehyde and time of cross-linking may be determined empirically. Conduct a time course at a fixed glutaraldehyde concentration and/or investigate a range of glutaraldehyde concentrations for a fixed time.
Cell Lysis Inefficient disruption of cells
  • It is important to have sufficient Lysis Buffer for the weight of cells processed. Follow the guidelines in this protocol.
Chromatin Shearing Not enough/too much sonication
  • If fragments are too large or too small, optimize sonication conditions using approach outline in appendix A to obtain appropriate size fragments.
Denaturation of proteins from overheating sample
  • Keep the water bath cold during sonication. Shorten the duration of each sonication pulse and increase the number of sonication pulses. Allow sufficient time for sample to cool between pulses.
ChIRP Probe doesn’t retrieve RNA in the lysate
  • Confirm the probe design
  • Check the expression of the target RNA. Target RNA should show Ct value of lower than 23 per 100 ng of total RNA by qRT-PCR.
  • Use Millipore ChIRP control probes
  • Perform ChIRP with different amount of probes for a fixed amount of lysate or vice versa.
  • Consider using more or less lysate (chromatin)
Insufficient quantity of Streptavidin Magnetic Beads.
  • The magnetic beads settle to the bottom of the tube over time. Make sure the magnetic beads are well mixed prior to beads withdrawal.
  • Carefully aspirate beads when using vacuum aspirator and use a high strength neodymium magnetic rack such as the Millipore Cat. # 20-400 Magna GrIP Rack.
  • Use more beads.
Washing High background due to insufficient washing
  • Ensure buffer is pre-warmed ChIRP washes is  are at 37 °C
  • Increase number of washes.
Low signal due to aspiration of the beads
  • Carefully remove supernatant and make sure there are no beads in the supernatant prior to removing it.
  • Use rack with magnets capable of firmly holding beads in place (e.g. Magna GrIP Rack Cat. #. 20-400)
Elution and Proteinase K digestion Incomplete elution
  • When performing elution, make sure that the temperatures are correct. Proteinase K will be inactivated by prolonged incubation at temperatures above 65°C.
RNA Purification Protein Contamination
  • Avoid the interphase when extracting RNA using Trizol® extractions.
Low RNA yield
  • Most ChIRP reactions do not yield measureable amounts of RNA. Sub nanogram quantities of RNAs can however be detected by qRT-PCR.
  • If RNAs are not detectable following cDNA synthesis, consult ChIRP 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 probe designs are correct.
  • Confirm the expression of the RNA in the cell by qRT-PCR.
PCR No PCR product from Positive Control ChIRP samples
  • Increase amount of ChIRP 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.
  • Perform PCR reaction with melting curve assessment to confirm amplification conditions and ability of primers to generate a single DNA product.
  • Confirm the probe designs are correct.
High background level with negative control ChIRP samples Insufficient wash after ChIRP. Increase the time of beads washing. More stringent washing may be achieved by adding optimally determined concentration of formamide or by increasing temperature.

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