Dear all,
I am analyzing the degradation of RNA (samples between 1000 and 2000 bp). The higher concentration I can obtain is about 50ng/uL. I have read that the best method is to use an agarose gel, but I think that at this concentration, and after degradation, I will see nothing in the gel. Does anyone has experience on this kind of experiments?
Thanks you very much
CRISTINA
Cristina you are right, agarose method is the most suitable for the purpose. However for your concentration it is better to get your samples checked on a Bioanalyzer.
SD
If you have no access to a bioanalyser and are wanting to check total cellular or total cytoplasmic RNA, you can get away with about 200ng of RNA on a 1.2% agarose gel if you add EtBr direct to the sample. Use a dsDNA marker ladder in the 1-5kb range, and look for crisp rRNA bands with minimal degradation.
check with a nanodrop, it provides useful information for the RNA samples.
Christina, You are right : you may not see anything on your gel if degradation is extensive.
The Bioanalyzer is a good option but the nanodrop will not give you much information since it is not very sensitive to degradation. Upon degradation RNA Absorbance will slightly increase.
You also may rely on RT-qPCR. It's a good option if you have very limited amounts of RNA. With the suitable controls, this will allow you to determine the uncut proportion of any target sequence. For more details, please find the attched file I am sending you.
I use bioanalyzer to check the integrity of RNA after every extraction. What is good about bioanalyzer is that it can give you precise information about the level of degradation and all degraded RNA sizes. Moreover it gives you a number (RIN value, from 1-10) for the integrity level of your RNA, 10 being the best.
You can first, check the concentration of your RNA at a nanodrop, in which 260/230 and 230/280 values may indicate purity and integrity of your RNA samples. Then further you can check your RNA by separating on a form-amide gel (Sambrook and Meniatis book). You can also take readings of your RNA samples at a bioanalyser for further confirmation.
Bioanalyzer is likely the only option with these concentrations as it can analyze 0.5 to 1ng of RNA if you use RNA pico. A cheaper method is using qiaxel if you have one. But very low concentrations are also a problem
Yes Bioanalyzer is the best option to check the integrity of your RNA.
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