I extract RNA from brain tissue punches (SCN) and can get between 500-1000 ngs using the following techinique. I start with the Trizol method up to the point where you remove the aqueous phase. I then move to using an RNAeasy kit (Qiagen) after adding the ethanol mixture and going from there. If you are worried about DNA contamination (i.e. RNA seq) then you can also conduct the optional on column DNase treatment. I elute with about 30 uls of ddh20 and get enough RNA for several RT-PCR assays. I'm happy to email you more specifics if that would be helpful (just message me).

I am isolating around 400 cells totally successfully using TRIzol. I have searched for other method, but TRIzol seems to be the best one.

Maybe try again as it really should work. Of course pellet is invisible. Good luck!

I've used TRIzol for extracting total RNA from leukocytes with great success. When I tried it for platelets, it did not work, since platelets are basically anucleated cells which are derived from fragmentation of megakaryocytes. So, I tried the mRNA isolation kit from Promega, and it worked like a charm. Good luck!

For very small samples I use the XS-RNA kit from Macherey-Nagel. Works every time!

I have used the Arcturus Picopure kits for RNA isolation of laser capture microdissected samples and these work well even with samples of less than 50 cells. Colleagues have also succesfully used the FFPE Picopure kits on formaline fixed tissues.

Also the Qiagen kits have been used on similar samples with succes by some of my colleagues.

I have used Trizol as well for RNA extraction, for a relatively low number of cells, and it works quite well but I didn't use the RNA for sequencing. Which volume of reagent did you use? i never go below 500 microl, no matters if I do not respect the cell concentration/volume ratio proposed in the datasheet. Otherwise there are kits, but for column-based kits you have to take into account that you might lose miRs within the columns, in case in your exp you want to assess miRs expression as well. Good luck!

we do it for verry low quantity. We're working with bovine oocytes and embryos. We use Trizol and phaselock gel heavy to keep all the supernatant. we add linear acrylamide as a coprecipitant.

Allprep micro is for RNA, DNA and microRNA and the RNA is of good quality.

I think you must try with some kit, because may be you are having trouble with some other components from your sample like myelin.

Thanks a lot for your answers!

I extract RNA from brain tissue punches (SCN) and can get between 500-1000 ngs using the following techinique. I start with the Trizol method up to the point where you remove the aqueous phase. I then move to using an RNAeasy kit (Qiagen) after adding the ethanol mixture and going from there. If you are worried about DNA contamination (i.e. RNA seq) then you can also conduct the optional on column DNase treatment. I elute with about 30 uls of ddh20 and get enough RNA for several RT-PCR assays. I'm happy to email you more specifics if that would be helpful (just message me).

I can endorse the Arcturus kits, the M-Nagel kit and have also got decent results with an Ambion small sample kit. Qiagens RNEasy minelute also work well but havn't tried on minute samples. All these will probably work on a chunk of cells.

Some 'extraction free' products are also out there eg RNAgem with DNase step that are conducive to cDNA synthesis. You may also like to try a bead approach seperately or coupled to this reagent such as Beckman Coulters Ampure beads (RNase free version may have a different name).

RNAseq kits are also allowing much lower starting amounts. 100 ng should be OK these days. Some go even lower. If you can make double stranded cDNA you of course have the option to go into a DNA protocol. I have done this with only a few ng of total RNA and put my sample through the Thruplex kit by Rubicon. Looks OK but not sequeced the libraries yet ; )

I have had really good luck using the Qiagen's RNeasy Micro kit for small samples.

TRizol is suitable even for very small samples. For examples i used it to extract RNA from zebrafish kidney fragments... anyway to improve yield you can only adjust volumes as well as you can and try to be clean and precise when resuspending. For very small samples spin columns are not so preferable to classic protocol. IS it possible for u to put together more than one samples?

Amilcare, after a few weeks of playing around with this I came up with the same conclusions as you. I will indeed pool individuals, but I managed to get good yields finally with very small fragments using trizol with the appropriate manipulations.

I have used TRIzol, but I prefer TRI reagent from Sigma, because you can simultaneously obtain, Protein, DNA and pure RNA for real time PCR all at once in a very small sample. Try it.

All the people who say that their method "works" I would ask what was their criterion for "working". Just to have some material that amplifies in RT-PCR does not mean that it faithfully represents the biological material interrogated.

We developed an RNA isolation protocol using magnetic beads. The isolated RNA of 10 cells of a certain population provides basically the identical information as RNA from thousands or millions of cells of the same population.

Protocol and evaluation data are published in PLoS ONE:

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0014418

Dear Qi Zhang, I used the standard Trizol protocol than can be found here: http://tools.thermofisher.com/content/sfs/manuals/trizol_reagent.pdf . When handling small samples, you need to be very careful not to lose any material e.g. when manipulating pellets and supernatants. Longer incubation periods (e.g precipitation step 1c, keep at -80°C for 1h or -20°C overnight) can also sometimes help for higher yields. I wish you best of luck with your research..

Hello.

Jean,Did  you use tissue or cells? 

In our experiment we used stem cells of mouse,but we had really small amount of cells and we used RNeasy Mini Kit (Qiagen),but RNA concentration was so low,lower than min. Is there any way to get good results by using small amount of cells? 

Thank you.

Dear Jean-Nicolas Audet,

I am currently working with 1mm brain pnches and it is being impossible to me to obtain good ratios. I am obtaining 260/280 of 1.6 and 260/230 of 0.5.

May I ask you a question? What volume of trizol do you use? Taking into account that your brain punches are also of 1mm and yo put two in the same eppendorf.

Thank you in advance,

Pablo

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Dear Qi Zhang, I used the standard Trizol protocol than can be found here: http://tools.thermofisher.com/content/sfs/manuals/trizol_reagent.pdf . When handling small samples, you need to be very careful not to lose any material e.g. when manipulating pellets and supernatants. Longer incubation periods (e.g precipitation step 1c, keep at -80°C for 1h or -20°C overnight) can also sometimes help for higher yields. I wish you best of luck with your research..