Hi, Lakshmi Narasimha, we add NaCl to reach 0.5M. The dsRNA would be more stable, under this cncentration after RNase A treatment.

Hi Songquing

see the links for some informative articles on your problem

1) http://web.natur.cuni.cz/flegr/prepbio.php

2) http://www.sciencedirect.com/science/article/pii/S0166093408002012

further I am not very sure but a 'density gradient ultracentrifugation' may also be able to separate dsRNA from ssRNA...check this also with some experts

bye

Thank you, Ajay Saini~

You may need a better RNase A preparation, sounds like RNase III is in there.

Thank you, Frank~ I solved this problem by adding more NaCl.

@songqing, can you please let me know the basic principle behind adding more NaCL?

Thank you

Hi Songquing,

Please try LiCl up to 2-3 M after Trizol extraction. ssRNA (~400 bases) should be precipitated quantitatively.

Also do not treat dsRNA by RNase A at salt concentration lower than 300 mM.

Yuri

Hi, Lakshmi Narasimha, we add NaCl to reach 0.5M. The dsRNA would be more stable, under this cncentration after RNase A treatment.

Yes, you are right, thank you, Yuri Drygin~

Thanks Yuri and Songqing for providing this useful information

Hi!

use the http://web.natur.cuni.cz/flegr/prepbio.php method mention above, is very good!, but don't precipitate the dsRNA with etanol, you will get a amonium sulphate plus dsRNA pellet very dificult to remove, use isopropanol.

or you can use the celulose metho , dilute your total dsRNA in STE buffer + 15-18% etanol, add to celulose in the same buffer , wash 3 to 4 times and ellute you dsRNA with STE ( no ethanol). Search sciencdirect or springerlink or Biotechniques for dsRNA + CElulose purification methods for details!

to remove ssRNA from dsRNA with RNASE use SSC 2x or ssc 4x buffer (High salt buffer,--- > NaCl) in this buffer Rnase digest ssRNA and but don't digest dsRNA. Test several RNAse concentrations and optimized in ssc 2x to 4 x buffer.

and finally good luck! :)

Hi Songqing Wu

I am also facing same problem

https://www.researchgate.net/post/How_to_get_a_stable_intact_double_stranded_RNA_dsRNA_after_treatment_with_RNase_A

I actually want to know the RNase A treatment conditions (NaCl concentration, RNase A concentration, incubation time and temperature). I am using Thermo Scientific RNase A, DNase and Protease-free #EN0531.

Thanks.