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how to detect dsRNA ?

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how to detect dsRNA ?

Postby deuce » Fri Feb 09, 2007 6:39 am

Let say two complementary 500nt ssRNAs are transcriped simutaniously in cells. How to detect the dsRNA? How to distinguish dsRNA from ssRNA? Does trizol work for dsRNA extraction? Is there any RNase able to digest
ssRNA specifically but not dsRNA? Can Northern distinguish the two species? Thanks
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Postby ping » Fri Feb 09, 2007 6:42 pm

Nuclease S1 cleaves only ss RNA and DNA,if this could help you.
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Postby Ultrashogun » Sat Feb 10, 2007 12:28 pm

dsDNA absorbs less light in the UV spectrum.
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Postby ytzan » Sun Feb 11, 2007 1:32 am

DsRNA is resistant to RNAse degradation (RNAse A ot T1) in buffers with high salt concentration (>0.2 NaCl). dsRNA is co-purified with ssRNA but the amounts are minimal. The nature of dsRNA (more bases/turn than dsDNA) make it very stable. Denaturation is very difficult and boiling may not do it unless you include DMSO. The best solution is HgCH3OH (very toxic though).
When you transfer the RNA to the membrane the helix may open due to the charge of the membrane. So I suppose you may be able to detect it - not sure though
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Postby dipjyoti » Sun Feb 18, 2007 10:47 am

Double-stranded RNA (or dsRNA) is RNA with two complementary strands, similar to the DNA found in all "higher" cells. dsRNA forms the genetic material of some viruses. In eukaryotes, it acts as a trigger to initiate the process of RNA interference and is present as an intermediate step in the formation of siRNAs (small interfering RNAs). siRNAs are often confused with miRNAs; siRNAs are double-stranded, whereas miRNAs are single-stranded. Although initially single stranded there are regions of intra-molecular association causing hairpin structures in pre-miRNAs; immature miRNAs. Very recently, dsRNA has been found to induce gene expression at transcriptional level, a phenomenon named "small RNA induced gene activation RNAa". Such dsRNA is called "small activating RNA (saRNA)".

Double-stranded (ds) RNA, a common component of virus-infected cells, is a potent inducer of the type I interferon and other cellular genes. For identifying the full repertoire of human dsRNA-regulated genes, a cDNA microarray hybridization screening was conducted using mRNA from dsRNA-treated GRE cells. Because these cells lack all type I interferon genes, the possibility of gene induction by autocrine actions of interferon was eliminated.
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Postby dipjyoti » Sun Feb 18, 2007 10:51 am

Do you mean single standard RNA viruses or siRNA or ssDNA?
Thank You.
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Postby MrMistery » Sun Feb 18, 2007 11:47 am

Very good info on dsRNA dipjyoti. Very helpful for understanding the process in general.
"As a biologist, I firmly believe that when you're dead, you're dead. Except for what you live behind in history. That's the only afterlife" - J. Craig Venter
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