RNA Extraction from Sludge

[megalodon]

Hi,

I am working on getting RNA extracted from a sludge sample that we obtained from a sewage treatment plant which will then be used for an Illumina RNA-seq library. I used the FastRNA Pro Soil - Direct kit from MP Biomedicals to do the RNA extraction but haven't had much success. In total, we have 10 different sludge samples from which we extracted RNA and the extractions were split between 3 people. All 10 extractions look the same. I have the bioanalyzer result of one of them attached to this post. Does anybody have experience with RNA extractions (from soil, sludge...)? What am I looking at here? Why am I not seeing the peaks for 16S and 23S? Are my RNA samples degraded? The sludge samples frozen in liquid nitrogen right after they were taken at the sewage treatment plant and then stored at -80C until RNA extraction. We also tried the RNA extraction kit with a fresh soil sample but got the same result.

Any input on this would be greatly appreciated! Those samples are supposed to be turned into Illumina libraries and I'm not sure if that would even be worth trying.

Thank you,

Daniela

[pmiguel]

Hi Megalodon,

I don't know, but I could speculate. If you look at the non-denaturing agarose gel in the advertisement here at this site http://www.qbiogene.com/fastprep/fas...soil-dir.shtml:



You see that the 23S bands are present at a much lower intensity than the 16S bands. That could be for the trivial reason that the lower molecular weight RNAs bound so much of the ethidium bromide in the gel that the bands above were de-stained. But, if not, then it is possible that the kit somehow is less effective at capturing high molecular weight RNA than low molecular weight RNA.

Wacky gel, by the way--the MW marker is the canonical "1KB" ladder, a dsDNA MW marker. So you are looking at double stranded base-paired molecular weight markers to estimate the size of their predominantly single stranded RNA. We inadvertantly ran a similar gel on some bacterial genomic DNA samples:



The left 2 sample lanes were treated with RNAse (largely unsuccessfully) and loaded into the right 2 sample lanes. The ladder, though, is the same. The good old BRL 1 KB ladder: 1-12 kb in 1 kb increments with a calibrated amount of pBR322 digested with HinfI.

But I digress...

Perhaps, like many insects, soil bacteria have cleaved 23S RNAs? In drosophila, honey bee, hessian fly and probably many other insects there is single rRNA band at the 18S size and nothing at the 28S size because of this. There is some size heterogeneity in rRNA sizes. That combined with cleaved 23S rRNA could explain a single broad peak.

--
Phillip

[megalodon]

Hi Pmiguel,

thank you so much for taking your time to look into this and for your input! I will give MP Biomedical a call today to see if they know anything about their kit being less efficient for high molecular weight RNA.

As for cleaved 23S rRNA in bacteria: I haven't heard about this, however, that doesn't mean much! I have mainly worked with DNA so far and only just recently started working with RNA, so I still have a lot to learn!

If I do get any good feedback from MP Biomedicals, I will post it to this thread.

Thanks again,

Daniela

[pmiguel]

Hi Daniela,
I just emailed a bacteriologist here a Purdue with interest in soil bacteria. She wrote that some soil bacteria have been reported to have cleavage sites. So that could be what is going on. It might explain why the advertisement showed a non-denaturing gel--the secondary structure of the 23S rRNA may hold it together to some extent in non-denaturing conditions. Better to show the expected 2 rRNA bands to researchers expecting normal looking total RNA if you are an advertiser?

Again, mainly speculation on my part, though.

--
Phillip

[megalodon]

Hi Phillip,

thanks a lot for that information, I will ask MP Biomedicals about that as well.

Have a good weekend,

Daniela