RNA extraction from egg allantoic fluid with high virus titer using trizol yielded from 350 to 60 ng/ul RNA but the purity is 1.4.
I managed to transfer trizol aquous phase into qiamp viral kit column and washed it twice; purity reached 1.98 but the yield decreased to about 16 ng in ul. it is recommended to start NGS with 2000 ng. Based on your experince, how can i improve the yield and the puirty of my RNA to reach the recommended concentration?
Try Qiagen's Ultrasens Virus Kit.... It's better suited for Plasma, Culture supernatants and other fluids...
Greetings,
When it comes to work with difficult samples, where you get such a low yield, you can always tweak the protocol. It's a very easy trick when you reach the part when pour IPA, mala a partition of your sample, let's say you have a total volume of 500 uL, divide them in 5 1.5 mL eppendorf Tube and add 1.5 IPA to each one. Once done, follow normal protocol, but mix them together before the final temp incubation. This way, you'll get a concentrated RNA extract, with higher purity. Good luck.
Best regards.
By the way, If you are only interested in viral RNA, you can lyse you tissue using sonicator, then concentrate your viral particles using ultracentrifugation gradient purification (iodixanol, sucrose, etc, using cushion approach, that is A discontinuous gradient from 10 to 40 or 60%. Then, you can further purify the viral particles by using 20-40-60 gradient. Once isolated, use Trizol protocol as I recomended before.
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