I would buy it if you show a positive (some RNAs known or expected to bind to the 1.9 Kb RNA) and negative (some RNAs that do NOT bind to the 1.9 Kb RNA) controls.

Maybe you should try with a higher acrylamide concentration, around 6-8%, but let it run more time, in order to see a better and more clear displacement.

As personal experience, I run 12% denaturing PAGE with 7M urea for resolving RNA of around 340bp in a chamber of 15X16cm at 400v and 15W in order to heat the gel (to around 50 or 60°C) for help the secondary structures to denaturate.

In addition to this, you should run the gel without samples during 15-20min at the same running conditions for pre-heating it and to dissolve urea in the wells, previous to sample loading.

The only thing is that I am not used to work with radioactive marks, so I don't know how they react with heat...

This brief explanation may be useful for you to resolve this methodological complication you are facing now, as well as the use of positive and negative standards and running controls that I strongly recommend you to use.

BR,

Carlos Díaz

Thanks for the suggestions guys!... I should mention that mutations to my 260 mer of interest can disrupt the observed interaction (new result this morning). Also the 1.9 kb interaction partner is coming from crude human cell lysate, which contains many other RNAs. Hence my apprehension that the cross-link product may not be what I think it is. Am working on specific/non-specific competitor experiments now. My only worry about using a higher % gel and running longer is that the un-shifted 260 mer will run off the bottom before the 1.9 kb RNA runs appreciably. Pretty sure that denaturation during the run is about as good as it can be. Samples are heated @ 65C for 10 min in 7 M urea/50 mM EDTA before run. Gel is pre-run for ~20 min. (until stick on thermometer strip on plate reads 60C).

" the 1.9 kb interaction partner is coming from crude human cell lysate, which contains many other RNAs. Hence my apprehension that the cross-link product may not be what I think it is."

In this case the interpretation of your results is much more complicated. Besides, I am not sure you are using the appropriate technique. Would not it be better to pull-down the 1.9 kb RNA from the cell extract using a biotinilyted 260nt RNA probe? you can then reveal the 1.9 kb RNA by either Northern blot or quantitative RT-PCR.

I think it is very reasonable to believe that you are getting the appropriate crosslink, if the right controls are done as mentioned above. In addition, I would run your samples on a sequencing gel for the best separation and resolution, although 4% would be too low, maybe 6%. Also, try making your radiolabeled probe with a little bit of biotinylated UTP in the transcription reaction, then you can follow with Northern or qPCR to make sure you are specifically pulling out the right RNA. Or better yet, gel purify the crosslinked product and do qPCR to show that its there, with the appropriate controls, which would be a gel slice from the control lanes at the same size and a gel slice of the input lane at the 1.9 kb band. The more controls the better...

Pull down with biotinylated RNA is on the to do list (I am also interested in what proteins may be interacting...). Had to convince the PI (and myself) that making a 5'OH, 3' biotin RNA and fishing in cell lysate was worthwhile. Gel also gives a nice visual 'here it is' figure for quick checks of conditions/RNA mutants. Show gel will be single thick sequencing run.

Keith/Michele... Know I am gonna end up in blot/qRT-PCR land eventually. How about this for a quick identity check on the 1.9 kb guy in the mean time. I know what its sequence should be. Therefore I should be able to specifically cleave it with a complementary DNA oligo (but not an irrelevant or scrambled one) and RNAse H. Poof... cross-link product runs lower on the gel.

PCR is faster, more specific and more reliable in my opinion. couple dollars for primers, then run on agarose gel. But if you want to prove it for the specific band in the gel, RNase H and DNA oligo should work. do it after the crosslink reaction and do it to the control 1.9kb lane RNA only too, if you can.

I agree with Keith. It should be straightforward to cut the presumptive crosslink band from the gel and amplify sequences from both cross linked RNAs. A control gel band wants to be included as well to deal with PCR contamination issues

Hi George

fropm what you describe, your X-link product is likely to be branched (Y shaped). It is well established that such branched RNAs migrate very differently form linear ones, and do not well align with size standards. A relatively well established trick to verify the non-canonical shape is to analyze the migration relative to a size standard in gels of varying concentrations (that has been suggested already) and of varying ratios of PAA/Bis in the acrylamide mix.You should be able to dig out literature by using "lariat" as a key word (interemdiate in splicing and often seen on gels in the coreesponding literature)

Regards, Mark

Your product can certainly run much higher than a linear molecule. If you want to be sure of what it is, purify it and do RT for both RNAs of interest (individually of course). You should get RT stops at crosslinking sites, thereby mapping the sites at the same time. You will need multiple priming sites for the 1.9 kb RNA. You will need to have control RT reactions of non-crosslinked material to recognize natural RT stops that are not due to crosslinking.

If you do PCR with the crosslink intact, the PCR enzyme may not be able to get through the entire RNA.

Oh - and you can run a stacking gel with a higher percentage on the bottom, lower on the top to separate really big things without running your 260-mer off the gel.