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Old 03-01-2012, 06:02 AM   #1
ramsemm
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Location: USA

Join Date: Jan 2012
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Default tRNA and rRNA depletion

Hi,

I am interested in creating cDNA libraries from soil rhizosphere samples, but I (and my lab) have no experience with this process. I have found products for generating ds-cDNA from RNA (looking at using the SuperScript ds-cDNA Synthesis Kit from Life Technologies, unless anyone knows of a better/cheaper one), but I am trying to figure out what I can do before this to reduce the tRNA and rRNA in my samples. I do not want to enrich specifically for eukaryotes (plants, fungi, or otherwise) or prokaryotes because I want to look at mRNA being produced from everything.

I found a Terminator 5' phosphate dependent exonuclease from Epicentre that will target the 16S, 18S, 23S, and 28S rRNAs, but it will not remove tRNA or 5S rRNA, which seem to be very abundant. I am sure this must be something people need to do routinely, and I was wondering if anyone had any advice for what to do?

Thank you in advance!
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Old 06-15-2018, 01:43 AM   #2
luiscunhamx
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Location: Portugal

Join Date: Nov 2011
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Default

Quote:
Originally Posted by ramsemm View Post
Hi,

I am interested in creating cDNA libraries from soil rhizosphere samples, but I (and my lab) have no experience with this process. I have found products for generating ds-cDNA from RNA (looking at using the SuperScript ds-cDNA Synthesis Kit from Life Technologies, unless anyone knows of a better/cheaper one), but I am trying to figure out what I can do before this to reduce the tRNA and rRNA in my samples. I do not want to enrich specifically for eukaryotes (plants, fungi, or otherwise) or prokaryotes because I want to look at mRNA being produced from everything.

I found a Terminator 5' phosphate dependent exonuclease from Epicentre that will target the 16S, 18S, 23S, and 28S rRNAs, but it will not remove tRNA or 5S rRNA, which seem to be very abundant. I am sure this must be something people need to do routinely, and I was wondering if anyone had any advice for what to do?

Thank you in advance!
Hey hey, were you able to find a solution? I am dealing with the same problem. In my case, the issue is that I have a huge number of libraries to prepare and ribo depletion options (RiboZero, RiboMInus, etc) become forbiddingly expensive. Therefore, I am considering using the Terminator 5' phosphate-dependent exonuclease and just get deep coverage of each sample. Still, cannot find convincing evidence about the process efficiency. Have you tried that method? Any insights will be greatly appreciated.

Cheers

Luis
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