RNA extraction from ta desert plant using RNeasy kit is giving us a very low concentration -below the range-.
The main problem is that desert plant are really rich on polysaccharides, polyphenols and other various secondary metabolites such as alkaloids, flavonoids, terpenes and tannins which usually hamper the DNA and RNA isolation procedure.
How to improve the RNA isolation protocol of 'Rneasy kit' to over come this difficulty?
Any methods, tips or notes are appreciated in advance.
Hi.
To you somehow clog your tubes in the kit. When working with polysaccharide rich samples i encountered the same problem, low or no RNA. You might try this protocol with a CTAB precipitation step http://www.ncbi.nlm.nih.gov/pubmed/17952668. It works quite fine in most cases.
I agree. Use a CTAB protocol. I used 3x CTAB for apple tissues. worked fine although beeing very viscous.
Try following ... (partly german, you will find the the corresponding german substance names)
Extraction:
========
-750 µl CTAB extraction buffer +32 µl β-Mercaptoethanol, heat to 65°C
-crush plant material with liquid nitrogen (imho the best crushing method)
- use 0,1 mg (store it in liquid nitrogene also for short term storage (several seconds))
- add the prepared extracion buffer, shake strongly, complete resuspending
- incubate (1000 rpm, 65 °C, 10 min)
- +1 Vol Chloroform –Isoamylalkohol (24:1)
- invert (10 min)
- centrifuge (10 min, 12000g, 4°C)
- take aquaous phase (upper phase) --> new eppi
- +1 Vol Chloroform –Isoamylalkohol (24:1)
- invert (10 min)
- centrifuge (10 min, 12000g, 4°C)
- take aquaous phase (upper phase) --> new eppi
- + 0,5 Vol LiCl-Lsg (8M)
- store (4°C, over night)
Clean RNA/resolve
=========
- centrifuge (30 min, 12 000g, 4°C)
-discard liquid
- + 500 µl 0,05% SDS, invert
- + 500 µl Cloroform –Isoamylalkohol (24:1)
- invert (30 sec by hand)
- centifuge (10 min, 12 000g, 4°C)
- take aquaous phase (upper phase) --> new eppi
- +2 Vol 100 % EtOH, invert
-store (2h, -20°C)
- centifuge (30 min, 12 000 g, 4°C)
repeat 3 times: [wash pellet (70 % EtOH, 500 µl, -20°C) , centrifuge (5 min, 12 000 g, 4°C)
wash once (100 % EtOH, 500 µl, -20°C)
- centrifuge10 min, 12 000 g, 4°C)
-dry pellet (5 min SpeedVac)
+30 µl H2O (5 min resolve) - use ddH2O or DEPC H2O, no elution buffer or stuff, downstream apps might be disordered)
This RNA is stable over years in -80 °C freezer.
Additionally:
==========
upscaling in 50ml falcons did not work properly, maybe RNA sticked at the walls.
alternatively: do the procedure in 4 eppis simultanously. using 16 eppis simultanously is possible, and during the 10 min centrifugation step you can manage 16 more if you have routine ...
We use Trizol for onion leaves- this gives you much greater yields than RNeasy and is much cheaper. We also routinely perform a Caesium Chloride precipitation after the Trizol extraction. This gives you very good quality clean DNA. We have also used this method for other roots and shoots of other plants as well as insects and fungi.
Thank you all for your collaboration, really valuable notes are being posted.
Unfortunately, the kit we are using is RNeasy and it isn't possible to switch to trizol or CTAB.
Anyways your notes will be taken in consideration in our future work :) Bless You All.
Thank you all for your collaboration, really valuable notes are being posted.
Unfortunately, it isn't possible for us to switch from RNeasy to trizol or CTAB.
Anyways your notes will be taken in consideration in our future work :) Bless You All.
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