Hi all,
I am trying to extract RNA from snap frozen leaf tissue ~100-150mg. I am getting good yields >150ng/uL normally with 260/280 >2.1 and 260/280 >2 as well. This is a good sign, but my gels are running smeared with some faint bands.
Our -80C tripped a breaker a few weeks back and the freezer was off for ~1.5 days. When discovered the freezer was still frozen (i.e. still had ice in it). however when we turned back on the freezer it was reading 1C.
I fear that this event may have lead to the degradation of the RNA. I attached pictures of the gels below. Thoughts?
I'm not familiar with plant tissue, but I always check the quality of my RNA extracts with a Bioanalyzer or Tapestation. They give you something called an RNA integrity number (RIN) that tells you the quality of your samples.
I second the idea of submitting to Bioanalyzer. If what we can see are your target band lengths then they're visible, but smeared. So they might still be usable depending on your downstream needs. Might as well check at least a few of them through Bioanalyzer.
Dear Sean,
You indeed got RNA with pretty nice purity and concentration, according to the data you sent. However, it does not mean your RNA had high integrity after the extraction. The freezer accident theoretically could also lead to degradation if you have a lot of RNase inside of your tubes, but it's hard to say since you did not run the gel right after the extraction.
If you want to do RT-qPCR, your RNA will be still good enough, but if you want to do RNAseq, it doesn't look so promising. The gel you show has a bit smeared ladder too, try to run the gel slower and longer. I can recommend 1-1.5% agarose gel, and make it thinner.
In the case of RNAseq, I would recommend extracting RNA again if you have enough samples and it's easy to do. If you have easy access to the Tapestation or Bioanalyser - just go for one of them. The gel is not enough anyway - it can only say if your RNA is too bad, but it can't say if it's good enough.
Some tips for the RNA extraction:
- make 3 aliquots right after the extraction and freeze them all. Use one aliquot after freezing for Nanodrop and/or Quibit measurements and for running it on the gel. Keep the second aliquot as an extra and the third one you can process further.
- use liquid nitrogen during the homogenisation. It's a game-changer. Grinding with the mortar and pestle works pretty well. But If you prefer to grind samples with the steel balls, make the round of grinding very short (30s max), otherwise balls get hot. You can repeat homogenization after cooling it back. Do not put more than one ball in the tube (I use 5 mm steel balls for the plant material). Put the buffer to the sample as soon as possible. RNA will degrade if the sample is dry and without any buffer.
The biggest degradation comes during the homogenisation step - make it as fast as you can. Do not process a lot of samples together.
I wish you a lot of fun and success!
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