Dear Michael,

we are often working with cells isolated by laser capture microdissection. Here, we have to deal with very low cell numbers (several hundred). For RNA isolation we routinely use the column based Qiagen RNeasy Micro Kit and it works very well in our hands. I think what is much more critical is to prevent cell lysis and RNA degradation upstream in your experiment. Instead of storing the cells in RNA Later I would suggest to directly resuspend the cells in the lysis buffer according to the protocol (from the RNA isolation kit) and immediately put them to -80°C until you will isolate the RNA.

Best regards and good luck with your experiments,

Monika

Low cell #s are a problem with the standard RNeasy mini kit. However,, Qiagen's RNeasy micro kits typically yield about 1-2 micrograms of RNA starting from 60-80K cells. This should be adequate for microarray. Worse case, just run duplicate conditions and pool.

I have done RNA extractions from semi confluent 24-wells which would correspond to around 100-150K cells. I used Trizol and performed two chloroform precipitations and got satisfying yields. I don't recommend using columns as their RNA binding ability does not guarantee you will retain all small RNAs. These columns enrich your sample in RNAs from a specific size range but smaller RNAs are inevitably lost. The is literature indicating a bias of different column technologies towards specific sequences.

Neel is right about RNA size. Columns are fine for most mRNAs. Not a good choice for snRNAs, snoRNA, or microRNAs.

I have also isolated RNA from 30,000 Kupffer cells using RNAzol (just like Trizol) with chloroform precipitation followed by isopropanol and ethanol washing. Whereas kits with column didnt give me much RNA.

Neel is right about columns and Trizol. I worked with retinae from mouse embryos day 13. The yield was low, but when you collect the whole RNA-phase by holding the tube in an angle of about 20 degree and stepwise pipetting the RNA-phase in new tube. When the DNA-phase gets in or at the tip of your pipette, put this fraction back and centrifuge again. After precipitation with 2-propanole remove stepwise the supernatant: i) with 1ml tip till ca. 150µl is left, ii) with 200µl tip till a drop is left and iii) with 10µl tip the rest. After every step you should centrifuge your samples at 12,000-16,000g for 2-5min. Always look at your RNA pellet! When something is in your tip, put it back and centrifuge again! The same procedure after the Ethanol-step. You will not increase your yield dramatically, but you will get more RNA than using by column or standard Trizol protocol... and everything on ice!

We have isolated RNA from cells 10 times less than that by using Qiagen RNA isolation kit and it worked. We used Laser captured micro dissected cells even for this purpose and compared several kits and manual protocols. Qiagen tursn out to bee the best when you compare both qaulity and qualtity but you will loose some RNA when compared to the manual methods. But this is better in quality so it makes sense than having much quantity with less quality.

Since you've already stored in 500 uL RNAlater you've diluted your RNA a lot (too much) already... For Trizol, with that few number of cells, 25 to 40 uL of nuclease-free water would be added in the end (to the small, small, non-visible RNA pellet) to get a reasonable yield of RNA/uL. But, this, diluted 1:10 with more nuclease-free water and RNAseOUT (to a final 0.5 U/uL) should work well for you as a starting concentration of RNA for RT-qPCR and/or LCM-RT-qPCR. Figuring 10-15 pg of RNA per [eucaryotic] cell isolated... this should get you in a nice range (if adding 3.6 uL of described sample in a 15 uL RT-qPCR). The use of an RNase inhibitor (such as RNAseOUT is always key)... no need for columns here - just strategic use of numbers and RNAseOUT-like reagents. Trizol/Qiazol works very well. Depending on cell type and inhibitors; is a concern sometimes, but with such small number of cells - inhibitors become a mute point in most of these cases. Reprecipitation and reconcentration of samples should be avoided here - as you are inviting RT-qPCR inhibition possibilities through the front door.

Since you used the 500 uL RNALlater -- you are already at ~1 cell's worth of RNA in your qPCR - and thus might be suffering from too much dilution.

Obul has good ideas above as well. Be careful to not overdilute samples...

And use some math to find out where you are at ng/uL-wise (during RT-qPCR) and also what this "ng/uL" value means in terms of numbers of cells' RNA represented per RT-qPCReaction.

Qiagen RNA isolation kit. Add RNA inhibitor in sorted cells to keep RNA from degradation

We routinely use RNAzol from MRC in combination with the DirectZol kit from Zymo to isolate RNA from 10-50K cells. We get great yields with high purity and excellent RIN values.

I have used Trizol combined with Norgen RNA Clean-up and micro concentration kit successfully on very small tissue samples for micro-array. The good thing about Norgen kit is that you will get all sizes for RNA in the end (unlike qiagen where you have to switch kits for smaller size RNA). Good luck.

Why dont your try to amplify?. You can label RNA for microarrays (mRNA) starting with as little as 10 ng of total RNA, using Agilent kit (Low Input Quick Amp Labeling kit), or 100 ng using Invitrogen kit (Superscript RNA amplification system)

I agree with Anna, that microarray is the best method.

I like qiagen kits but they are expensive. Trizol is good but you need to add Glycogen to the precipitation step to make the pellet clearly visible.

I isolated RNA from small number of sorted cells (sometimes only 10 000 cells). Therefore, I used RNeasy Micro Kit for RNA isolation and it worked fine. Additionally, I cooled cells during sorting process and freezed cells in lysis buffer to prevent RNA degradation directly after sorting.

I had the same amount of cells,I mean>100,000; I used Quiagen Micro RNA plus kit I had good yield & good quality. They recently had an offer that I used (49% of for RNasy plus kit!) It was about a mount ago, Hope they still have it . you can ask them

Before precipitation with isopropanol add 1ul of 20ug/ul glycogen (molecular biology grade) to the aqueous phase. After that, add isopropanol and precipitate. Transfer samples to the freezer (-20 Celsius degree) for about 40 minutes and then centrifuge samples at 12000xg for 10 minutes. The rest steps, according to standard procedure. It worked in my case. Good luck!

Qiagen has a kit that worked for well for me when I was trying to get RNA from very young plant embryos. It is called the RNeasy Micro Kit. If you need to get RNAs of all sizes (even miRNAs), there is also the miRNeasy Micro Kit. There is a figure online showing that the miRNeasy Micro Kit can be used to extract from very small numbers of cells. See http://www.qiagen.com/Products/Catalog/Sample-Technologies/RNA-Sample-Technologies/miRNA/miRNeasy-Micro-Kit#productdetails.

Qiagen RNEasy MICRO kit is really good, but expensive. I have switched over to Omega Biotek E.Z.N.A. MICROelution kit. Side by side comparison using capillary eletrophoresis showed better RNA quality using Omega Biotek columns as compared to Qiagen ones for a fraction of the cost. They are offered through VWR. We have used as little as 10,000 cells with this kit and it worked beautifully every time. Good luck!

Dear Michael,

we are often working with cells isolated by laser capture microdissection. Here, we have to deal with very low cell numbers (several hundred). For RNA isolation we routinely use the column based Qiagen RNeasy Micro Kit and it works very well in our hands. I think what is much more critical is to prevent cell lysis and RNA degradation upstream in your experiment. Instead of storing the cells in RNA Later I would suggest to directly resuspend the cells in the lysis buffer according to the protocol (from the RNA isolation kit) and immediately put them to -80°C until you will isolate the RNA.

Best regards and good luck with your experiments,

Monika

RNeasy Mini kit may be used. In this RNA separation is based on anion exchange columns(spin columns). This will provide high quality of RNA from small number of cells.

you can read this article, I hope it is good for you

http://www.ncbi.nlm.nih.gov/pubmed/17431883

or this one

http://www.invitrogen.com/site/us/en/home/References/Ambion-Tech-Support/rna-isolation/tech-notes/recovering-rna-from-small-samples.html

We are performing isolation of RNA with purezol, is a liitle bit of time consuming, but, we can isolate a good amount of RNA

You can use Trizol method for RNA extraction using glycogen before precipitation step. This should increase your RNA yield.

Like Lauren, I have had great success working with very small amounts of cells, using the Ambion RNAqueous-Micro kit. I can get enough RNA from 1000 cells for microarray, with excellent quality (average RIN approx. 9.2). I have used the same protocol for LCM samples.

After FACS, I spin the cells down to remove the PBS, then immediately put in the Lysis Buffer. Some people then leave it there overnight, but I have had problems with degradation unless I continue the full isolation on the same day. After using the RNAqueous-Micro kit (with the instructions for LCM application), I always do the optional DNase-treatment. After I remove the supernatant, I do an additional ethanol precipitation. I put in 100% ethanol, 3M sodium acetate, and glycogen. I leave this in the -80'C freezer overnight, and finish the EtOH prep the next day. This gives me excellent 260/280 values, and my records show that I have less than a 5% loss of RNA from the EtOH ppt., and cleaner RNA, with excellent 260/280 values.

Do not be surprised when your 260/230 values are very low - this is normal when you have so little starting material. Good luck!

As mentioned by Lauren, we was using Arcturus PicoPure RNA Kit to isolate RNA of barley microspores for RNA seq. Good luck!

I learned from my colleagues that some labs who deal with very small amount of cells use a kit called PicoPure RNA Isolation kit. I am dealing with picograms of RNA and is thinking about try this kit.

I recently compared Picopure and Agilent’s RNA Nanoprep kits for recovering very low amount (5 ng, my answer previously mistakenly said 5 pg, which is not what I tested, but Agilent says it can go that low) of RNA from enzymatic reactions. The RT-qPCR didn’t show much difference but the Sanger sequencing result of samples from Nanoprep is much better. I don't have single-cell experience with those kits yet. But I am now sticking to Agilent‘s kit.