RNA isolation from Groundnut seed - How to get rid off of oil content (Aug/04/2007 )
Hey friends Wish u happy friendship day. I'm M.Sc. student in biotech.(Agriculture) currently I am working on groundnut seed dormancy gene tagging by differential display technique. I'm isolating RNA with TRI-reagent from seed but problem is after 1st centrifugation one upper layer is oily which gets vanished once treated with chloroform but i'm confused shall i take whole supernatanat or layer below oil content and again the Total RNA pellet is not clear some debris in it so sometimes is not dissolving in DEPC-treated water PLEASE I NEED U R KIND SUGGESTIONS. Thanking you.
Hi,
Trizol, which is essentially the same as TriReagent, has this step in the protocol:
"OPTIONAL: An additional isolation step may be required for samples with high content of
proteins, fat, polysaccharides or extracellular material such as muscles, fat tissue, and tuberous
parts of plants. Following homogenization, remove insoluble material from the homogenate by
centrifugation at 12,000 × g for 10 minutes at 2 to 8°C. The resulting pellet contains
extracellular membranes, polysaccharides, and high molecular weight DNA, while the
supernatant contains RNA. In samples from fat tissue, an excess of fat collects as a top layer
which should be removed. In each case, transfer the cleared homogenate solution to a fresh
tube and proceed with chloroform addition and phase separation as described."
For "fat" in the above read "oil" and it should work. I used to sue Trireagent on fat pads (>95% fat) from mice and had no problems with using this extra step.
Trizol, which is essentially the same as TriReagent, has this step in the protocol:
"OPTIONAL: An additional isolation step may be required for samples with high content of
proteins, fat, polysaccharides or extracellular material such as muscles, fat tissue, and tuberous
parts of plants. Following homogenization, remove insoluble material from the homogenate by
centrifugation at 12,000 × g for 10 minutes at 2 to 8°C. The resulting pellet contains
extracellular membranes, polysaccharides, and high molecular weight DNA, while the
supernatant contains RNA. In samples from fat tissue, an excess of fat collects as a top layer
which should be removed. In each case, transfer the cleared homogenate solution to a fresh
tube and proceed with chloroform addition and phase separation as described."
For "fat" in the above read "oil" and it should work. I used to sue Trireagent on fat pads (>95% fat) from mice and had no problems with using this extra step.
Thank you Sir. thak u very much.