Dear Experts,
I am working to identify/confirm RNA that binds to my protein of interest through RNA-ChIP assay. However, i have no experience at all in ChIP assay and its not establish in my lab.
I had obtained RNA-ChIP protocol from Terzi et al.,published in the plant journal 2009. As i understand that fixation and sonication step is crucial in successful ChIP assay, after fixation and sonication step, i used water bath Bioruptor from Comso bio to sonicate my samples 5 times in ice water with 10s on and 6s off with 1 min break during each cycle, Then, i load my sonicated samples into agarose gel to see if there is concentrated band fragment around 300-500bp. However, i could not see any concentrated fragment at all at these size even the smear from unsonicated samples looks gone in my sonicated samples as shown in attached gel pictures.
So any one could give me suggestion if my sonication steps do not work? and how can i teckle these situtation? like using different sonicator machine like rod based machine instead of water bath or increase my sonication time? and is there any steps is important in RNA-ChIP assay? Your advise are much appreciated. Thank you!
Hi,
Did you reversed the cross-linking before loading on the agarose gel? Smear at the bottom off the gel is not sheared DNA but degraded RNA. What was the sample volume per sonication? What kind of tube did you use? How many cells per sample? What is the composition of the sonication buffer, How long did you fix cells,
Sorry for asking a lot of questions, but all these parameters affect significantly the shearing efficiency.
H Dr Irina
Thanks for your responses. First of all, i have to tell i am working on arabidopsis plant. No. i did not reverse cross-linking my samples prior to agarose gel loading. I load 5ul of samples directly from the sonicated sample. I am aware that protein bound to my RNA could affect the loading thus loading after reserve cross-linking is better indication?
i used 300ul each sample for sonication, with normal eppendorf tube(SPL LIFESCIENCE). about the cell i have no idea because i dint count the cell.
Composition f the sonication buffer as as follow:
50mM Tris/HCI pH8.0, 10mM EDTA pH8.0, 1% w/v SDS, 0.2mM PMSF, one complete mini tablet for 5ml solution + 160u/ml RNAse inhibitor (1.2ul of 40U/ul RNAse inhibitor for 300ul of sonication buffer)
For the fixation process, i add 1% formaldehyde to conical tubes containing samples to 35ml and immediately apply vacuum for 7mins, then release vacuum and reapply for 8more mins. Then i add 1.1ml 2M glycine, apply vacuum for 1min, release vacuum and reapply for 4more mins.
Hi,
Thanks for these details.
2 points are really critical. First is about cross-linking reversal. This step is absolutely necessary to correctly assess the size of fragments. Without it, the size of fragments is biased because proteins bound to nucleic acids slow considerably the migration.
The second point s about tubes, used for sonication. Bioruptor is a close-tube sonicator. Ultrasound has to pass through a tube wall to reach samples. Be aware that ultrasound transmission vary considerably depending on plastics nature and the tube shape. It is highly recommended to sue specific validated tubes. You can find information about recommended tubes at https://www.diagenode.com/documents/bioruptor-organigram-tubes. Please pay attention that depending on BR model you are using, tube might be different.
I hope that will help you.
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