23 November 2018 2 3K Report

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?

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