Hi,
I'm isolating fungal spores from (8 day old) 10 OMA plates and dissolving in 2 ml water and placing 50 ul drops on the gel bond sheet and incubating till forms appressoria. For harvesting the samples I'm quickly freezing the water drops containing appressoria samples by liquid N2 and after scraping, transferring into tubes. Next day after isolation by Qiagen RNeasy plant mini kit the quality of RNA is very low. So, please help me how to isolate total RNA from germinated spores or appressoria for realtime PCR? How much spores do I need to incubate? Please help!
Hi Nazmiara Sabnam
I would suggest you to use Trizol. Based on my own experience, Qiagen RNeasy plant mini kit does not work well for certain types of tissue. And Trizol always give much higher RNA yield than Qiagen RNeasy kits. If you still encounter issues, I would recommend you to use the PureLink Plant RNA Reagent. I was having lots of problems with RNA extraction from potato tubers and after trying many different protocols, I found PureLink Plant RNA Reagent to work the best, the RNA quality and yield gotten was surprisedly high.
Anyhow, I hope that helps a little.
-WLD
Thanks a lot! I'll do it tomorrow. Can you please suggest me how to homogenize the fungal spores spotted on gelbond sheet??? I have a doubt, should I collect the biomass with 1 ml trizol and transfer to tubes and then dip in liquid N2 or there is no need to freeze in Liq N2, just proceed to homogenize by micropestle??? Please help!
Hi Nazmiara Sabnam,
I don't have much experience extracting RNA from fungal structures. I have, however, extracted DNA from fungal mycelium and you don't really need to use liquid N2. However, LN2 would help on a better disruption of the cells and consequently you would get a higher RNA yield. I would try without LN2 first and if you yield is not there, I would definitely use LN2 for a better tissue maceration.
I hope this helps a little.
Cheers,
-WLD
Hi...
I have isolated following the method mentioned above by Trizol without liq N2 and I have got RNA but the quantity is too low for realtime PCR. I think the homogenization is not properly done using micropestle. Can I use sonication or if u can suggest something else.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biota
- Eukaryota
- Fungi