Hi. I extracted RNA from soleus muscle (7-10mg) in mouse by using Quiazol reagents. And another method was that I extracted RNA by RNAeasy kit. The result showed that total RNA was 6 ug and 2ug; ratio 260/280 was 1.86 and 2.0 respectively. However, 260/230 ratio for both method were about 0.5. As far I known, that caused by salt contamination. How can I improve the quality of RNA? Can I use this RNA for Reverse Transcription and Realtime PCR?
I addition to the suggestions given here, I think you could use this RNA in a RT reaction. If you have the chance, run it also on an agarose gel and check the integrity of the RNA on a Bioanalyzer. And then if all these tests are good, then don't worry too much about this low 260/230 ratio (which should be close to 2 by the way, not 1.5).
If you are using the Qiagen RNAeasy mini kit and repeating the extraction you could try to do one or two more washing steps with the RPE buffer. Salts are in the RW1 buffer and RPE is used to remove them.
If you want to try desalting the already extracted RNA you can try an ethanol precipitation of RNA. If you google it you will certainly find a protocol for it.
If I remember correctly the ideal ratio should be 1.5 or close to it, so it would be beneficial to improve it prior to reverse transcription.
I have previously had this problem and solved it with an overnight 2.5M Lithium Chloride precipitation. Decreased my concentration by about 20-25% (which was completely acceptable for my sample) but increased quality massively. Both the science and the practicalities are discussed here:
http://www.lifetechnologies.com/uk/en/home/references/ambion-tech-support/rna-isolation/general-articles/the-use-of-licl-precipitation-for-rna-purification.html
I agree with both Christian and Philip. The only concerns you should be aware of with Philips approach is that if you're looking for low expression products or products with a relatively short half-life then a loss of 20 - 25% yield will be a huge problem. You don't lose 20 - 25% evenly across the board so replication might prove a problem in repeat experiments not using the same RNA isolation products or detection in general if all your product is lost.
I'd first go with the additional wash-steps and for those which have already been extracted try ethanol precipitation.
Also contamination (salt or otherwise) is a problem for downstream application but this doesn't say terribly much about the quality of your RNA template if you've just used a Nanodrop or similar spectrophotometric assessment. Try micro-capillary electrophoresis with the Experion or similar for a better idea of the quality of your RNA extract. Since cleaning up might remove contamination but not necessarily allow for decent results in downstream applications if your extracted RNA is degraded.
Agree with Nathaniel Re: My suggested approach. It's great if you've got a huge amount of RNA and will normalise concentrations when making cDNA anyway, but if your target is low abundance, or your sample is of low concentration or particularly precious it's probably worth more careful consideration.
I addition to the suggestions given here, I think you could use this RNA in a RT reaction. If you have the chance, run it also on an agarose gel and check the integrity of the RNA on a Bioanalyzer. And then if all these tests are good, then don't worry too much about this low 260/230 ratio (which should be close to 2 by the way, not 1.5).
You can go for ethanol precipitation of RNA, that will remove all the salt. I use it when ever i have this kind of problems. But the total RNA yield will decrease to 20-30%.
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