For my project, I need to extract RNA from muscle tissue of EEL. I am trying but the yield is low and Purity not good. The 260:230 ratio is always below 1.50 and 260:280 ratio ranges 1.6-1.86 and yield is around 120-140 ng/µl. In brief, I followed, 1 ml of TRIzol for 30-50mg of tissue for homogenization, incubated for 5 mins at room.T. Then 200µl of chloroform used for phase separation (centrifuged at 12000g for 15 min at 4oC), used 500 µl isopropanol for pelleting (incubated 10 mins at Room.T) then centrifuged at 12000g for 10 min at 4oC, then used 75% ethanol for washing pellet (centrifuged at 7500g for 5 min at 4oC) then incubated at 55oC in water bath for 10-15 mins after mixing with 20µl of MLQW.
Many Thanks......
Hello Md Moudud Islam
The protocol seems okay. But you need to consider the points provided below which you may have missed.
1. Tissue may not have been fully homogenized.
2. Tissue may not have been immediately processed or frozen after removal from the fish.
3. Tissue may not have been completely disrupted. If centrifugation is done prior to adding chloroform, there should be a white mucus-like pellet. If there is a tan-colored precipitate, this is indicative that not all the cells have been lysed.
4. If a mortar and pestle has been used to powder the tissue, RNA may stick non-specifically to the mortar and pestle. It may be better to use a glass homogenizer and teflon pestle. Add TRIzol to the homogenizer, then add the frozen tissue and homogenize.
5. Homogenizing for too long and too continuously in a small volume (say 1 ml) may cause heating of the sample and this may result in degradation of RNA in the tissue. Please note that the tissue should be cooled during homogenization, and homogenization should be done in on-off cycle.
6. Use 100mg of tissue sample per 1ml of TRIzol.
7. You may collect the upper aqueous phase carefully without disturbing the interphase. You may collect 80% of the upper aqueous phase and leave behind 20% so that you do not contaminate the aqueous phase.
8. You may try two 75% ethanol washing steps instead of a single washing to remove any remaining impurities in the form of water-soluble salts. It may help to improve RNA purity.
9. Instead of MLQW, use TE. If sample is dissolved in water, the ratio may be low due to the acidity of water or the low ion content in the water. The ratio can go up if the sample is dissolved in TE and the spectrophotometer is zeroed with TE.
Hope these suggestions help to increase the yield as well as the purity of RNA.
Best.
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