HomeQuantitative PCR (qPCR)RNA Immunoprecipitation (RIP) FAQs

RNA Immunoprecipitation (RIP) FAQs

RNA immunoprecipitation (RIP) kits enable the discovery and analysis of a variety of chromatin-associated RNAs. These chromatin-associated RNAs often regulate gene expression and can be analyzed with applications including quantitative reverse transcription polymerase chain reaction (RT-PCR), microarray analysis (RIP-chip) and next generation sequencing (RIP-Seq).

Nuclear RIP can be performed using chromatin that has interactions stabilized by formaldehyde treatment (cross-linked) or chromatin that has not been treated with a cross-linking reagent (native). While both of these approaches are similar in that they are designed to recover chromatin associated RNA, the reagents used and the details of the protocol and types of interactions typically detected are different. Cross-linked can capture higher molecular weight complexes in in vivo configurations with possibly lower affinities. In contrast native RIP is expected to recover high affinity, more direct interactions between proteins encoded RNA binding motifs and candidate RNAs. For less well understood proteins and protein complexes often both approaches are used.

How do I maximize RNA quality during RNA-IP procedure with the cross-link protocol?

Formaldehyde can compromise DNA and RNA quality and thus the cross-linking time should be optimized to stabilize the RNA-Protein complex without damaging RNA too much. The cross-linking time can range from 2 minutes up to 30 minutes. And the reversal cross-linking time is also quite important, which can range between 45 minutes and up to 4 hours.

How can I reduce the non-specific binding in RNA immunoprecipitation assay?

You can try blocking your beads with yeast RNA or 1% BSA in the blocking solution, if the beads are not pre-blocked with BSA. Also, Magna Nuclear RIP™ (Native) RIP kit (Product No. 17-10522) or Magna Nuclear RIP™ (Cross-Linked) (Product No. 17-10520) helps greatly lower non-specific binding and increase signal-to-noise ratios.

When should I do cross-linking RIP ?

If you do not know whether a protein-RNA interaction is direct or not, cross-linking is recommended. It also depends on the experimental conditions whether you can detect any protein binding or not, it is recommended to do cross-linking to ensure a protein-RNA interaction is maintained under your IP conditions. After the protein-RNA interaction is verified under your IP conditions, then native IP conditions can be tested.

Should I be concerned about obtaining more total RNA from my negative control than my sample?

This is probably a good indicator that your RIP is specific. It is not uncommon to see a higher recovery of RNA (5-10-fold) in the negative control than the sample. What matters is what proportion of that total RNA pool is relevant to you. In other words, you might have a lot more different and irrelevant RNAs in your negative control, whereas a few of them in your IP are specific and relevant and are present in bigger quantities.

When doing qPCR analysis using the RIP RNAs, how do I make a ratio as RIP/Input? Should I use Ct value or do I have to calibrate to some genes?

Create a standard curve from serial dilutions of a template DNA (or cDNA) and then, using the standard curve, you can calculate a relative value for input and RIP samples and then calculate a ratio.