Easy-to-use kit for studying RBP-RNA interactions

December 23, 2014

University of Louisville
Medicine
Graduate student

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Company:

EMD Millipore

Product Name:

EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit

Catalog Number:

17-701

One of the goals of my research is to identify proteins that interact with a specific mRNA. We had previously identified candidate proteins that could be interacting with our transcript of interest, so I ordered this kit in order to determine whether the protein-RNA interaction was actually occurring or not. I had previously ordered antibodies against the two candidate proteins for doing Western blots, and so this kit seemed like a straight-forward method for looking at protein-RNA interactions without necessitating the purchase of additional materials. I selected this product specifically because it seemed easy and straight-forward to use, and I liked the incorporation of magnetic beads into the immunoprecipitation process. While this kit comes with reagents for RNA isolation, I chose to use filter columns instead, and obtained a good RNA yield following the IP.

Experimental Design and Results Summary

Application

Determine protein-RNA interaction through IP followed by qPCR

Starting Material

Cell culture - opossum kidney cells

Protocol Overview

OK cells were grown to confluence, serum-starved overnight, and treated. Following treatment, cells were rinsed twice with ice-cold PBS, scraped into 10 mL of PBS, and pelleted. PBS was removed, and cells were lysed using the kit lysis buffer and overnight storage at -80C. The following day, the lysate was centrifuged and supernatant collected. Beads-antibody complexes were prepared as described in the protocol, with the exception of incubating for 1.5h instead of 30m before rinsing and adding OK cell lysates. Lysate-beads suspensions were rotated at 4C overnight. Beads were rinsed the next morning, and proteins were digested in a 55C water bath for 30m with proteinase K digestion buffer. Supernatant was removed from beads, and acid phenol chloroform added for RNA isolation. RNA isolation from this point forward was conducted according to mirVana RNA isolation protocol. RNA was eluted from columns with 20 uL water.

Tips

You need a LOT of starting material in order to get good PCR results. I used 3 T-75 flasks per condition, but 4 would have been better. You also need a LOT of antibody - I tried to use less than the recommended amount, and the experiment didn't work, so don't skimp.

Results Summary

With this kit, I was able to detect positive protein-RNA interaction for both of the proteins that I tested. After I isolated RNA, I used RT-qPCR to detect the presence of the transcript of interest. Reactions in which the Ct difference between positive and negative controls was greater than 5 were considered positive for protein-RNA interactions.

Additional Notes

None

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Summary

The Good

Obtained good results; confirmed proposed protein-RNA interaction

The Bad

Kit only comes with enough material for 12 reactions, and a lot of antibody is needed, so this experiment tends to be a little pricy

The Bottom Line

Don't skimp on reagents, use plenty of starting material, and make sure your antibody is good for IP reactions. A little pricy, but fairly easy to use and gives you solid data. Definitely worth it.

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