Hello,
I'm trying to capture viral RNA from my total RNA fraction so i've extracted RNA with trizol and digest with DNase.
Then I've design 7 RNA-probes of 40 nucleotide each to span all the genome of my virus. Those probes are tagged with biotine in 5' end with a spacer bone between sequence and biotine.
I use streptavidin coated beads (My one C1 from Thermofischer)
I've tried to hybridize my probes on my RNA of interest at 55°C for 30 minutes but it seems to not be sufficient. The T°M of my probes rank from 62.6 to 69°C. Can I increase the hybridization temperature to 60 degrees for examples to be more specific to my sequence of interest? Is 30 min enough or should I increase the time also ?
If some of you have already experimented this kind of RNA pull down, please give me your advice ! Thank you !
The protocol is lengthy so please follow this researchgate link and the other oneArticle RNA Pulldown Protocol for In Vitro Detection and Identificat...
https://www.jove.com/video/57379/rna-pull-down-procedure-to-identify-rna-targets-long-non-coding
- The short answer is yes you can increase your annealing temp to 60C and no hybridising beyond 30 minutes will not aid
- How do you clean up your viral RNA having extracted with Trizol and DNAse digested
- I wonder if the issue is reaction components and specifically salts interfering with efficient hybridisation ?
- I would Trizol extract, followed by chloroform. Followed by ethanol ppt. DNAse digest and then ethanol ppt and 70% wash again before mixing with your streptavidin coated beads
Thank you both for your answers !
Laurence,
- I perform acid phenol purification after DNase digestion.
- My hybridation buffer (2X) is composed of Tris-HCl 20 mM pH 6,6 ; EDTA 10 mM ; LiCl 1M
- I will try to increase the annealing temperature. My beads were very sticky to the wall of my eppendorf so I added 0,05% of Tween-20 to my buffer.
I will let you know if this improves the capture
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