I am trying to isolate RNA and DNA from the tissue biopsy stored in Trizol at -80 degree for more than 6 months .
I isolated RNA using the standard Trizol method and obtained good quality and quantity of RNA (validated using Nanodrop and formaldehyde gel). However, when I tried isolating DNA from the leftover interphase using two of the commonly used protocols: (1) DNA isolation protocol from Trizol (ethanol extraction method; 0.1M sodium citrate in 10% ethanol) (2) Back extraction buffer (4 M guanidine thiocyanate; 50 mM sodium citrate; 1 M Tris, pH 8.0). In both the cases, I obtained a smear of sheared DNA. I also tried isolating DNA from similar tissue samples using Qiagen Mini Kit and the DNA obtained was again sheared. However, the DNA obtained here was in the range of 10-1 Kb (better in yield and quality than what was obtained when compared to other methods).
Since, I wish to use the DNA samples for next-generation sequencing, I have following queries: (1) I want to know which method is more suitable for DNA isolation using Trizol? (2) Whether Trizol is not a better alternative to store the tissue sample for a longer period?
Hi,
I have tried extracting DNA and RNA from patient samples stored in Trizol (after cell homogenisation, kept for more than 1 year) and the results were ok for me. So, I think samples storage using Trizol should be a good way.
For your case, did you check the DNA quality using nanodrops before you ran PCR or gel? If the nanodrops results showed good DNA quality, it means your DNA extraction method was good. Then you might need to think of way to either optimize your PCR (adjust those temperature, concentration of reagents/DNA template etc) or optimize your gel running condition (like run for longer time and change the concentration of the gel etc)..
Alternatively, if you wish to store your samples for a longer duration and if you do not trust trizol, you may bank and freeze your cells and when you are about to extract the DNA/RNA, only you homogenize the tissue using Trizol before you proceed for the DNA/RNA extraction.
Thanks Chong for the help. PCR amplification is working fine with the DNA I have isolated but I wish to send them for whole exome sequencing, thus I feel this shearing is a problem.
I would request you if you could let me know which protocol you are following either the Trizol method or the back extraction buffer (BEB) method. It would be great if you help me with it.
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