RNA isolation - (Apr/07/2015 )

Hi,

 

I am trying to purify RNA from plant cell extract. I used TRI reagent from sigma, isolated RNA and loaded on formaldehyde gel. I always get streak instead of clear bands. As control I also loaded other RNA where I got nice bands. The ratio of 260/280 is 1.75, which is very less. I believe there is lot of protein contamination in my RNA sample that is the reason of streaking and also for low 260/280 ratio. But do you think there can be some other reason also? How can I increase my 260/280 ratio and get good integrity of RNA?

 

P.S. we don't know what type of RNA is in our sample, it could be mRNA, tRNA, tRFs etc. 

 

Thanks

Hi

 

I haven't done very much RNA isolation but noticed noone had replied to your comment, so I will try to make some suggestions (and hopefully this will lead to better suggestions than mine).

 

Streaking could be because the RNA concentration is too high so try loading a dilution series onto the gel. However, the lowest ratio should be around 1.9 so there does seem to be some contaimination which could be protein or DNA. What you should do about it depends upon what you want to do with the RNA. You could (not in order):

 

1: Put the RNA sample through another another cleaning step. You will lose RNA which is not great if you're looking at expression or planning to sequence it but as purity is important you have to weight up minimising RNA loss against getting a pure enough sample. If you just want it for making something like RNA probes than a few more cleaning steps its probably okay. Not sure about your kit but other kits I've used in the past does have an optional/ additional cleaning step.   

 

2. Plants are tricky for RNA extraction (naughty cell walls) so different plant samples require different, sometimes modified techniques so check the literature to makes sure you're following a protocol for your tissue and species.

 

3. Read through the protocol for your current kit very carefully. I know that sounds stupid but sometimes when things aren't working I've reread the protocol and realised that I'd missed a step or was using the wrong tempreature for my sample etc.     

 

4. Related to 2, try a different kit and/or manufacturer. Old kits can be a the problem. Also in the past I've found that just changing to a completely different kit has fixed a problem so you could ask around and borrow a kit to test out from a colleague.

 

5. Start again (if you have the tissue), but use everything new - new kit, new chemicals, make sure everything is clean and RNAse free. Take your time, follow the protocol exactly, and if you're still having a problem than it sounds like to need a new protocol or you need to add another cleaning step. 

 

Hope that's given you some helpful suggestions.  

Thanks Kate Warner! Thats a great help. I dint know that, extracting RNA from plant will be tricky . But I have made an extract from my plant sample in some buffer and I am isolating RNA from that extract. Do you think still I should face the problem? 

 

And as you suggested that I may be loading more amount on gel, that could be one reason for streaking. But I am doing denatured gel, and staining with EtBr, anyways if I load less amount, I would not see anything . 

 

I checked for the literature, you were right, there are so many recent papers only on RNA extraction. I will go through them and with your suggestion will repeat the experiment.

 

Thanks a lot!

No worries.

 

Getting RNA from plants is much easier than it used to be. Believe you and me

 

You're grinding the samples in the buffer right? Not just sampling the buffer?

 

Personally I dislike using buffers for RNA isolation because you're never really sure that the buffer has impregnated the cell walls and some tissues are really hydrophobic - all that lignin and cuticle waxes. In our lab (and this is the standard protocol) we always freeze the samples in liquid nitrogen (LN) immediately after collection because RNA degrades really quickly. You can then store them at -80 for quite a long time. Then we finely grind the samples in LN in a pestle and mortar keeping it cool and preventing RNA degredation by adding more LN as you grind (watch youtube clips for this as its a real skill). Then we extract the RNA using a Qiagen Plant mini RNA extraction kit (really important its one for plants as the enzymes are different).   

 

Buffers are good when you have to transport the samples as airlines are more likely to let you do that in a buffer than in a LN. However, if you don't have access to LN than buffers are the only way to go and you will have to find a protocol that will work on plant samples stored in that particular buffer. People have done it before (even people in my lab with success) but I've always found that LN method gives cleaner results.

 

Okay that makes sense why you can't dilute your samples for uploading.

 

That's great! Yeah the best thing to do is check the literature. Also go to the lab's website because some helpful labs upload their protocols in more detail. 

 

No problem. Good luck with your extractions.

 

Kate