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I recently extracted RNA from developing plant leaves for the first time, as part of a very long and intensive experiment. The samples were extremely precious because of the amount of effort that went into obtaining them (harvesting thousands of miniscule leaves, one from each plant, to get the required mass).

I extracted the RNA with TRIzol and chloroform. Nanodrop showed excellent yield as you would expect for actively growing young tissue, but some of the samples had really low 260/230 ratios. I know this suggests phenol or salt contamination, but what can I do to clean the samples without losing any of the precious RNA? And how can I avoid the contamination in the future?

Rik Smith-Unna
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2 Answers2

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You can clean up phenol by washing with choloroform, and then doing an isopropanol precipitation followed by a 75% EtOH wash (let me know if you'd like an exact protocol).

To avoid contamination (and sample loss), you have to be meticulous in your pipetting (which you'll get better with practice). You can always use those phase-lock tubes which basically jams a solid gel between the aqueous and organic phases so it's a lot easier to pipette.

jp89
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  • Which bit of pipetting is the crucial part? Is it the removal of the clear upper phase after centrifugation? If so - do most people leave a bit of the upper phase behind so as to avoid contamination? I was trying to get every last drop! – Rik Smith-Unna Feb 03 '12 at 18:11
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    @RichardSmith Yeah the pipetting of the organic/aqueous phase is crucial. It depends on (1) how good you are at pipetting, and (2) how "precious" your samples are. Of course, if you're more meticulous about getting as much sample you can get, there's a tradeoff between yield vs. purity. If I can spare some sample, I usually leave just a tiny bit to spare myself from touching the junk in the interphase between the two layers. – jp89 Feb 03 '12 at 18:15
  • I must not be as good at pipetting as I thought! Thanks very much for your answer. I'll make sure I get better at pipetting and in the meantime I'll leave a safe amount of the aqueous phase behind. – Rik Smith-Unna Feb 03 '12 at 18:19
  • @RichardSmith It might've not been completely your fault. The standard procedure is to clean up the RNA with isopropanol and Ethanol, which it didn't seem like you did. Once you do that, you should get super clean RNA even if there was a bump in your pipetting. – jp89 Feb 03 '12 at 18:24
  • I did clean with isopropanol and Ethanol, and then Nanodropped afterwards. I take full responsibility! – Rik Smith-Unna Feb 03 '12 at 18:26
  • @jp89, From what I understand the IPA and Ethanol cleans only effectively take care of the salts. The Phenol Choloroform extraction does a much better job with the removal of proteins. Possibly you could take some of the interphase and clean again. The phenol will affect your columns and affect your yields. – bobthejoe Feb 03 '12 at 20:46
  • Also to get rid of all traces of phenol, try extracting with ether. – jp89 Feb 08 '12 at 03:23
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An ethanol precipitation should work. But I have had great success using the Qiagen RNA cleanup columns, which are in my opinion easier. Here is a URL to see the RNA cleanup columns Qiagen offers: http://www.qiagen.com/products/rnacleanupconcentration/default.aspx

Also in the future you should consider using PhaseLock tubes:

http://www.5prime.com/products/nucleic-acid-purification/organic-nucleic-acid-extraction/phase-lock-gel-.aspx

That saved me from having your exact problem many times over.

Chris Plaisier
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