How do I isolate RNA from Eiseia fetida? I have been trying to isolate RNA from E. fetida using Trizol and Kit based method without any success whatsoever. Kindly suggest me the best way out....
Sir, Thank you for your kind reply. By saying that 'without any success whatsoever', I mean to say that the RNA i usually get is of good quality (as visualised by 26/280 or 260/230 ratio), but when i run on 1% gel, it comes as a smear. I have not yet been able to obtain two distinct bands of RNA at 28 and 18 S
your RNA is degrated. The 260/280 doesn't show degradation, since degrated RNA also abosorbs. You should start over with new material, all RNAse free, tubes , water, fresh gloves everytime you touch something outside the materials ( nitrile gloves), new tips also RNAse free, wash pipets with ETOH at first, wash everything. I sterilized all material before (autoclaved twice), i mean the bottles containing chloroform etc ( i use TRIZOL extraction).
i hope it helps and sorry for bad english!!!
Agree with Artur, Manash and Agustina! RNA degradation might be a problem. You can either store your samples at -80*C freezer or or homogenize the samples with a bead ruptor with TRIzol immediately. TRIzol combine with column method should work very well in your application. I don't know what kit you are using right now. But, you might consider assembling your own kits with BULK RNA spin columns, such as RNA Tini spin column (EZC107) or mini spin column (ZECR101). All these columns are compatible with any silica based RNA isolation kits (e.g. RNeasy etc...), so you can use leftover solutions from the kits or just buy bulk solutions to assemble your own kits. If your samples are viscous, you should use QIAshredder or EZshredder to remove insoluble debris and reduce viscosity before loading the sample to the column.
Here is the brief description for the procedures: TRIzol (or Columnzol) extraction-->load the supernatant (containing DNA/RNA) to RNA mini or Tini spin columns-->washing with RW1 and RPE from RNeasy kit (you can buy these solution in bulk) or just simply wash the column with 80% ethanol instead-->elute the RNA from the column with DNAse/RNase free water or TE.
Please click here for protocol details using columnzol as example (you can replace it with TRIzol or QIAzol: http://www.enzymax.net/columns/RNA_isolation_column_zol.htm or http://www.enzymax.net/columns/EZCR305-Column-zol%20RNA%20isolation%20Kit.pdf
Here is the website for bulk RNA mini spin column and Tini spin column (this is so far the smallest column on the market, it can be used for RNA/DNA isolation from few cells or single colony, also can be used for RNA/DNA concentration, the elution volume can be as low as 3 ul): http://www.enzymax.net/columns/mini_column_RNA.htm
http://www.enzymax.net/columns/tini_spin_RNA_column.htm
Here is the website for bulk shredder spin column:
http://www.enzymax.net/columns/shredder_spin_column.htm
Good luck to your research. Please let me know if I can be of any further assistance!
Here I present to you the RNA extracted via kit method and trizol method and run on 1% agarose gel
Sir,
The buffer that has been used is TAE, marker is DNA 1kb ladder, run for half an hour at 60 V at room temperature...these were the conditions. I fully understand that I am in trouble and I am thankful that you are trying to help me. But I am a novice in RNA-working...so am actually very inexperienced I understand that RNA should be run in urea gel but last time I did it in plain agarose gel.
Yes I need this RNA for RT-PCR.
regards,
manash
I am not expert and specialise on this. My theory is to separate RNA is similar to surgeons or scientists to remove an organ from a human or animal body. the only difference is it is RNA which needs more sophisticated equipment and more caution.
the theory is to separate them just like cutting a loaf of bread or a pork chop. they need the same idea and similar method but with innovative theory and technology.
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