Dear all
I have tried 1ml Trizol to extract total RNA from crabs gills. The weight of most of my samples is + -0,03 g. We have applied this protocol for hepatopancreas samples and the results were very good. However, for gills, the efficience of RNA extraction was ok for less than 25% of samples. We are using petles for microtubes (RNAse free).
Any information you can provide me would be greatly appreciate.
Cheers
Isabella
While i have never extracted RNA from crab gills my experience with trizol extraction is if the tissue homogenization is good it is usually good for extraction efficiency. May be increase the amount of tissue samples to the microtubes for same amount of Trizol. Also how large are the pellets are you getting or even earlier what about the clear layer of RNA after the first centrifugation?
I have never extracted RNA from crab gills either, but it could be how well you're homogenizing your tissue sample in question. One way to get better RNA yields through more durable tissue is by freezing it down with liquid N2, grinding it up into a powder, and then resuspend in 1ml TRIzol. However, with this method sterile technique needs to be a constant thought.
Cheers,
This article may prove helpful to you...
Article Transcriptome analysis and characterisation of gill-expresse...
In this they used the Trizol method for RNA extraction but further purify it the RNeasy Midi Kit. So, may be because of contamination you are not getting RNA in all the samples.
Good luck.
I think its the same way of extracting RNA of any tissue. 1 cubic cm from tissue in 3ml trizol
Dear Isabella C. Bordon
I have never extracted RNA from crab gills either, but already used "silica-based columns (Aurum Total RNA mini kit, Bio-Rad)" which I think should be cheaper than Trizol, the result was great. The final elution volume is very important to get proper yield for example I have use it for the tissue 0.003 g with 40 ul elution buffer. Sometimes changing the kit might be helpful.
Hope to could make it anyway.
Best regards,
Salman
Silica based membrane is quit cheaber and yield high quantity of RNA with a good purification. I recommend to use the same protocol of Dr Salman but try to minimize the elution buffer to 20-30 ul for higher yield. Some time I replace the elution buffer with molecular biology water as a lysate.
Many Thanks to you all!
I will consider your suggestions for my new samples ( I've just collected them).
Please Salman Malakpour and Mahmoud Mabrok : if you have published any paper that explains the use of sillica based membrane in details , please let me know. I've never tried it and I really want to learn how to use.
Cheers!
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