I am trying to in vitro transcribe some fluorescein labeled ssRNA. And I want to purify the ssRNA from the free NTPs and the enzymes (RNA polymerase and DNase I). Due to the characteristic of the label, fluorescein, these RNAs cannot be purified by Phenol-Chloroform method, and the manufactory of the labeling kit suggested me to do a Guanidine Hydrochloride purification. But I can only find a lot of protocols for RNA extraction from tissues or cells, but not from the RNA transcription mixture. Do you have a working protocol of Guanidine Hydrochloride purification for synthesized RNA? Thanks!
Hi there,
Macherey Nagel sells kits for RNA purification based on chaotropic agent treatment and suitable for both cells and acellular samples.
https://www.mn-net.com/media/pdf/b0/51/ee/Instruction-NucleoSpin-RNA.pdf
If you want to further separate RNAs based on RNA size:
https://www.mn-net.com/media/pdf/85/84/e0/Instruction-NucleoBond-RNA-DNA-CB-AXG.pdf
Hi Rudian,
I have worked with plant RNA viroid extraction using GuHCl. CCCVd RNA usually found to be present in low concentrations.
Not sure if you can apply that method.
You can prepare these buffers in-house and give it a go.
https://digital.library.adelaide.edu.au/dspace/bitstream/2440/37756/10/02whole.pdf
10.33687/phytopath.001.01.0020
Hi Rudian,
We used Guanidine hydrochloride with phenol chloroform Isoamyl alcohol to isolate microRNA. However, give a try to extract fluorescein labeled ssRNA without phenol. Use Guanidine hydrochloride, 2M sodium acetate along with chloroform isoamyl alcohol mixture to precipitate nucleic acids. For that, centrifuge the mixture for 10 mins at 12000 g at 4°C. Then transfer 70-80% of the supernatant to a new tube and add equal volume of ice-cold isopropanol. Centrifuge it for 10 mins at 12000 g at 4°C. Now transfer 70-80% of the supernatant from the above step into a new tube. Add equal amount of ice-cold isopropanol to it and store at 4°C for an hour or sometimes store at 4 °C for overnight. Then centrifuged for 10 mins and the wash the pellet with 75% ethanol prepared with DEPC treated water. If possible, check it in a PAGE gel to see the RNA bands. We used sequencing gel electrophoresis to visualize small nucleic acid segments. I hope this will help you in some way.
Akila Wijerathna Yapa Thanks! It is a great method. However, I'm looking for a method for in vitro transcriptions. Still, thanks!
Aravindan Kalyanasundaram Thanks for your method. I will first try the MEGAclear kit from Thermo. If the kit does not work, I will try your method. Thank you!
Dominique Liger Thank you. My supervisor and I decide to first try MEGAclear Kit from Thermo, because I used that kit before, and the technical support said it should adapt to our case.
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