I purified the RNA from whole blood by columna (Paxgene kit), and the RNA has 260/280 ratio 2.0, but the ratio 260/230 is less than 0.94. The purificación methodology includs DNAse diggestion
In the nanodrop the curve does not show a good peak at 260nm.
I need this RNA for microarrays.
u need to repeat phenol chlorofoam extraction again after DNAse diestion. reprecipitate RNA in smaller volume and increase the number of ethanol washes. 260/230 ration would improve significantly.
Hello María,
To selectively precipitate total RNA, add 0.3 volume of 8 M LiCl and store the tubes overnight at 4°C. Collect the RNA pellet by centrifugation at 14,000 rpm for 30 min at 4°C, then wash the pellet with ice cold 70% EtOH, air-dry for 10 min and dissolve in an appropriate volume of DEPC-treated water. Store RNA at –80°C. See page 131 in the attached publication.
Hope it helps.
Article Rapid and Efficient Isolation of High Quality Nucleic Acids ...
I've been collecting RNA and doing micro-arrays for 3 years now, the best methodology I found was to extract RNA using Phenol chloroform method and then cleaning up the collected RNA using Quiagen kit:RNeasy MinElute Cleanup Kit. Here is the link to their website: https://www.qiagen.com/us/shop/sample-technologies/rna-sample-technologies/rna-cleanup/rneasy-minelute-cleanup-kit/
I collected good quality RNA from around 100 K sorted cells successfully. Good luck
Thanks at all for your suggestions,
I apreciarte your time and share your experience. After my RNA re-purification I will inform my experience.
Tnahks a lot
Dear Laurence,
Thanks a lot for all your recomendations. I just prefiere to continue the RNA re-purificación by column thinking in do not to expose too much my RNA and avoide degradation by the manipulación process.
Thanks a lot
Dear Laurence,
I repurify my RNA simples (8) by column and the ratio 260/280 were more than 2.0, while the ratio 260/230 in 6 samples was >1.0 but in 2 was
Dear Laurence,
Again thanks for your help, i am goin to analyze RNA integrity and send you the picture for your opinion.
Thanks
Dear Laurence,
I analyzed the RNA integrity and the results were very bad since the RIN values were 1-3.6. All RNA samples were degradated. I learned that ratio 260/280 and 260/230 are very important but the integrity analysis are necesary before began microarrays analysis.
Sincerily thanks a lot for all your recomendations :)
Dear Laurence,
Thanks for share all your experience. I red all the information and is clear how the DNase treatment is important in the RNA purificación method. Let me tell you information respect the RNA samples, a PhD student in our laboratory purified them with the PAXgene blood RNA kit from whole blood. Sincerily I do not know if she had problems in the procedure. But in conclusión the RNA quality was not good.
We used the PAXgene blood RNA System and nucleic acid purificación kit (PAXgene Blood RNA kit)-PreAnalytix Quiagen, and according the recomendaciones all the procedure is at room tempeture and we use the RNase-free DNase (Quiagen) and the enzyme is removed in wash steps.
Finally, i am goin to follow your recomendations.
Thanks for your time
Hi Laurence,
I am going to take all your recomendations, and thanks a lot¡¡¡¡
Saludos desde México :)
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