1. How do you maximize RNA quality during RNA-IP procedure with 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.
2. How to 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 (Cat. #: 17-10522) or Magna Nuclear RIP™ (Cross-Linked) (Cat. #: 17-10520) helps greatly lower non-specific binding and increase signal-to-noise ratios.
3. For RNA immunoprecipitation, when should I do cross-linking or not cross-linking?
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 proved under your IP conditions, then native IP conditions can be tested.
4. In RIP, 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.
5. When doing qPCR analysis using the RIP RNAs, how to make a ratio as RIP/Input? Should I use Ct value or do I have to calibrate to some genes?
First, you can create a standard curve from serial dilutions of a template DNA (or cDNA). Then, by using the standard curve you just created, you can calculate a relative value for input and RIP samples and then calculate a ratio.
For more information about RIP experiment guides, troubleshooting tips and supplementary protocols, please view our RNA Immunoprecipitation (RIP) page.
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