Either too low or too high a260/a230
Posted 12 January 2012 - 06:19 AM
For around a month I am trying to isolate RNA from leaf samples. The amount of my samples are not very much. I used both trizol method and qiagen Rneasy kit. In trizol, I always obtain peaks at a230, no matter how long do I wait at ethanol drying, how careful do I behave at obtaining upper phase etc. In Rneasy, I have no such problem but I can't obtain good concentration of samples, and today I even obtained a260/a230 ratio at around 4.5. If I do RNA cleanup to the samples I obtained from Trizol, this time the concentration of my RNA drops dramatically. What do you suggest me to do in order to obtain pure RNA at high concentration? In the attachments you see some of my results.
Posted 12 January 2012 - 03:50 PM
Regarding trizol, you can do the phase separation for longer with the centrifuge at 4 degrees. Make sure the stay well away from the organic and aqueous bilayer when you do the chloroform extraction. Once you get the RNA, I love lithium chloride preciptiation for clean ups...it purifys RNA beautifully and always solves the issue when I have a low 260/230. It selectively precipitates RNA, (not proteins, DNA (for the most part) or Trizol). Kits that use Silica based columns are also great but a lot more expensive than trizol. I prefer RNAaqueous over RNeasy.
If concentration is an issue, bring it up in less volume...or, if its for real time, there are cDNA kits that work better for low inputs (Ive made cDNA from 200ng of RNA with no trouble, which worked great for real time).
Posted 13 January 2012 - 02:03 AM
Today, I will do both trizol and qiagen, and I will obey your suggestions at chloroform extraction phase. As far as I know, when we do LiCl precipitation, there is a loss of some of the RNA. Around how much RNA is lost during this step? We are currently a bit short on cash, so that I can use either trizol or qiagen kit now. Besides, my PI suggested me to use PBS from miRvana kit, and try to purify the RNA I have already obtained. Do you suggest me to do it?
We are already solving the RNA in 30 uL water, I am not sure if there is a problem if I make it less.
Thanks again for your answer
Posted 13 January 2012 - 07:32 AM
You can improve perfomance of these methods by using less tissue (for RNeasy, instead of 100 mg tissue/450ul buffer, use less than 50mg/450ul buffer). Also, with RNeasy, use buffer RLC instead of RLT (RLT is more likely to cause problems with plant tissues).
This method gives very good RNA for some species, but it hasn't been tested on difficult plants:
Bilgin, D.D., DeLucia, E.H., and Clough, S.J. (2009) A robust plant RNA isolation method suitable for Affymetrix GeneChip analysis and quantitative real-time RT-PCR. Nat. Protoc. 4, 333-340.
CTAB protocols are commonly used to deal with plants high in polysaccharides and they usually are inexpensive. I can give you a few references if you like.
Posted 14 January 2012 - 10:22 AM
Although we work with animal tissues, there are some (like skeletal muscle) that are fibrous. We pulverize difficult/tough samples using liquid nitrogen and a tissue pulverizer (something like this: http://www.spectruml...Pulverizer.html). This works fabulous on leaves, Ive tried it on a lark :~). It turns them into a fine powder.
Posted 27 January 2012 - 07:58 AM