If you simply want to look at gene expression using qPCR from these cells, you can skip trying to make RNA and use "Cells-to-cDNA" kit. This might prevent loss of RNA from your samples. Best of luck.

I too had trouble isolating quality RNA from small number of cells. My cells however had a high concentration of melanin in them that appeared to be nonspecifically binding up free RNA after lysis - a bit like dropping molassas into the prep! If you think there might be something in your cells that could be having a similar effect, I recommend the Zymogen inhibitor removal columns. They really cleaned up my samples.

With the Qiagen RNeasy kit, at the very last step, I tend to do my first RNA elution in a small volume (~30 microlitrres) then do my second elution with the eluant from the first spin (same liqud goes through the column twice). This has improved my concentrations and improved my 260/280 values.

add glycogen (Invitrogen)along with isoprapanol and keep it in -20C for overnight. that will give u good quantity of total RNA

If you simply want to look at gene expression using qPCR from these cells, you can skip trying to make RNA and use "Cells-to-cDNA" kit. This might prevent loss of RNA from your samples. Best of luck.

Use cell to cDNA kit

To anyone else interested - the columns I mentioned are from Zymo Research - here's a link to the product.

http://www.zymoresearch.eu/rna-purification/rna-clean-up/rt-inhibitor-removal/onestep-pcr-inhibitor-removal-kit

Thanks for the suggestions. The RNA I extract needs to be enough to go through a Nugen amplification in order to run a microarray. I've tried glycogen and it did improve it some what.. Today I'll be using the columns SarahJayne suggested as well as a -20oC overnight precipitation step. I'm also going to try using water heated to 55oC to elute as suggested by someone else..

Thanks again!

I also managed to extract RNA (although in very small concentrations of about 1-4 ng/microliter) from as few as 1000 cells FACS-sorted. I simply put the cells in 350 microliters of buffer RLT (QIAGEN kit) with 30 microliters of 2-mercaptoethanol. I managed to get good results in detecting gene expression using qPCR. Hopefully this helps along with the other techniques mentioned.

Hi Matthew did u try all that you mentioned if so then please tell me if the glycogen with overnight step worked for you and were you able to get better yield with this idea. Also I would be happy to know if someone could get good amount of RNA by TriZol with less number of cells after sorting? Although  I know its not recommended to go for a seq after getting your RNA from TriZol but for now I really wish to see RNA as i am unable to get it from past few attempts. I used Trizol as well as qiagen kit.

Hi Diwas, I have gotten great yields with Trizol. While RNA-seq is not recommended post-Trizol, I tried a few things and found a way that works really well. If post-Trizol + Chloroform step (do not proceed to precipitation of the RNA), you collect the supernatant and then clean it up with Qiagen mirneasy micro-kit, the yield is more than enough for  RNA-seq. If the aim RNA-seq is differential gene expression, please don't forget to incorporate a DNAse step, to get cleaner RNA profiles. Best of luck.

Just a note: my lab still hasn't sent any sample to sequencing yet, but we do manage to obtain workable RNA from very few sorted cells, produced using the RNAqueuos-micro kit. We've had only partial and inconsistant succes with Trizol based purifications. Our tissue sample is dissociated into single cells, stained and sorted live on a FACS ARIA. We sort directly into 150 microliters of the RNAqueuos-micro kit lysis buffer and purify RNA according to kit's instructions.

From as little as 3000-5000 cells we start seeing good (RIN 8+) RNA using the agilent pico chip. From as little as 100~ cells we manage to obtain fairly convincing results in qRT-PCR from cDNA we produce with this RNA (Quanta qScript cDNA Synthesis Kit and Perfecta SYBR kit). Our next challenge is the RNAseq, which we are probably going to perform using the Takara-clonetech pico input stranded RNA SMARTer kit. 

However, our procedure from tissue to sorted samples (which can be frozen until RNA purification) is very lengthy. We are considering sorting fixed cells to enable more procedure flexibility. Has anybody here produced RNA from sorted fixed cells? I only know of this paper: 

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0089459  

But their method seems harsh for RNA (3 hours at 50°C!), also lengthy and I don't know if it is suitable for sample from very few cells.

Any recommendations? 

I have not been able to isolate good quality of RNA from small numbers of fixed cells.  I have also had difficulties isolating consistent RNA with Trizol, but I think the comment about longer isopropanol precipitation with glycogen carrier makes a lot of sense and worth trying.  It is unfortunate that methods have not been standardized to prevent a lot of wasted time. Good luck.

I can now add that our method has thus far yielded two sets of good qulity RNASEQ data :)

what did you use finally? can you recommend a method?

I also have trouble with RNA isolations.

I am trying to sort 5-10000 neurons from adult mouse brain.

In all of the cases I see no RNA on picochip:

I started with trizol (with glycogen/ glycoblue and O/N step in isopropanol), however I was advised not to use trizol for sorting of neurons.

I further tried kits from Agilent and Quiagen, with no success.

Do you have any advice on this?

A. don't bother trying to see RNA from that amount of cells. with the exception of certain cell types that are very sturdy and large like TAMs - even the pico chip won't help.

I validate that I have workable RNA with logical results by RT-qPCR.

B. Try the RNAqueous-micro kit from intvitrogen. 

C. the choice of library preparation kit is a significant one. Look into what Takara are offering. 

Hi Gil,

at the end have you managed to extract RNA from fixed cells? I would like to perform RNAseq from cells fixed and FACS sorted but I will have around 5000cells and I do not know if it is enough.

Thanks!

Hi Gil

I am trying to get RNA from 100- 2,000 cells post FACS sort using the RNAqueous kit you recommended. I followed the protocol exactly with no sucsess. Do you use the LCM buffer to prime the membrane? do you do a second ethanol precipitation step, and if so when? Do you elute in the heated elution buffer or in NF water?

Neither the bioanalyzer (utterly failed twice) or the tapestation is able to properly run the samples. I know some substances interfere with the running of these instruments, could there be anything in the buffers?

Also, the Qubit readings don’t really make much sense.

Do you think its worth reprecipitating in isopropanol and resuspending in NF water before measuring or, as you said before conducitng a qPCR to measure from such smal quantities?