I am doing RNA immunoprecipitation, using the RNA TRAP technology, with which you can capture actively translating ribosomes with the L10 ribosomal subunit fused with GFP, using GFP antibody coated magnetic beads. Right now I'm testing it on HEK293T cells transfected with L10-GFP and I am having trouble finding good reference genes / controls for qPCR.
GADPH & b-actin seem to be differentially expressed between the IP & the unbound (the supernatant of the IP) fractions, which result in very different & unreliable results when I look at enrichment of GFP between IP & unbound. For example, b-actin sometimes shows Ct value difference of 5 between IP & unbound fraction, of the sample.. Actually b-actin seems to be 'enriched' in the IP sample (so I suspect it binds to the beads..)
What do you suggest I do? Should I compare with the input sample? n
Hi, I know this topic may be a while ago. But I recently gained some knowledge from somewhere else about what should be used as RIP-qPCR controls.
Theoretically, there is "no" proper controls when assessing RIP-qPCR, since regular internal controls such as gapdh, actin, tubulin, they should normally not be bound by the RBP of interest, which in theory, should not be in your IP-ed RNAs. I got a calculation chart downloaded from Sigma to show how to calculate ChIP and RIP-qPCR fold change, which is, you have your input, anti-RBP, and anti-IgG RNAs, input here just serves as one kind of control that amplicons will be amplified using IP-ed RNAs can be amplified in your input. Then, the comparison should only be done between anti-RBP RNA and anti-IgG RNA (none of them should contain gapdh, actin or tubulin), but you know they came from the same amount of cells (you control this), then the only thing you should do is to directly test your amplicons of interest, and calculate the (fold compared to IgG) of your anti-RBP IP-ed RNAs. Since anyway in a regular qPCR, internal control genes are only used to normalize RNA amount due to different cell numbers to start with.
I dislike the term RIP (unwisely termed RNA Immuno-precipitation--because, one does not precipitate RNA using an anti-RNA antibody), but that is not your fault and I am not blaming you--I am blaming the half-literate person who first used this term.
That aside, and sorry about that; but can you use a region of the 28S rDNA for positive control RTqPCR?. Do you need to care whether GAPDH or ACTB RNA is effectively precipitated? You should go for a positive control (rDNA), a negative (I hope you can mine many expressing lncRNAs for HEK293--there have been many recent reports on lncRNA transcriptomes, but I cannot remember if they included HEK293; you can also mine an eRNA as a negative) and your experimental targets. Of course, there always lies the dangerous possibility that a ncRNA could still be translated (and thus spook your analysis). Sorry, that's biology.
However, RNAs are notorious for binding beads (yes; I have been pained personally). And, moreover, many robosomal proteins are part of the CRAPome (http://www.nature.com/nmeth/journal/v10/n8/full/nmeth.2557.html?WT.ec_id=NMETH-201308) and they pose serious problems for downstream analyses of results. So, brace yourself for a little frustration. All I can say, is: Good Luck.
Nefeli, I think that GADPH and b-actin are probably good controls, but it is your negative control (comparison) that that could be your problem. I don't think that the supernatant is necessarily a good negative control. Might you perhaps doing a simultaneous IP with beads coated with an innocuous antibody like Protein G?
RNAs sticking to beads can be an issue. In the past when I performed IPs, I pre-blocked the beads with a solution of yeast tRNA, BSA, and glycogen. I then washed the beads and added my extract. You could try this and see if it helps with the control RNA levels.
Hi, I know this topic may be a while ago. But I recently gained some knowledge from somewhere else about what should be used as RIP-qPCR controls.
Theoretically, there is "no" proper controls when assessing RIP-qPCR, since regular internal controls such as gapdh, actin, tubulin, they should normally not be bound by the RBP of interest, which in theory, should not be in your IP-ed RNAs. I got a calculation chart downloaded from Sigma to show how to calculate ChIP and RIP-qPCR fold change, which is, you have your input, anti-RBP, and anti-IgG RNAs, input here just serves as one kind of control that amplicons will be amplified using IP-ed RNAs can be amplified in your input. Then, the comparison should only be done between anti-RBP RNA and anti-IgG RNA (none of them should contain gapdh, actin or tubulin), but you know they came from the same amount of cells (you control this), then the only thing you should do is to directly test your amplicons of interest, and calculate the (fold compared to IgG) of your anti-RBP IP-ed RNAs. Since anyway in a regular qPCR, internal control genes are only used to normalize RNA amount due to different cell numbers to start with.
- Badges
- Science topic