Our experience indicates that the efficacy of RNA isolation from samples with low RNA content (e.g. 10k cells) is drastically lower (up to 10 times) when no co-precipitate is added. We use glycogen as co-precipitate and add it directly after Qiazol lysis (miRNAeasy kit). The importance of using co-precipitates during RNA extraction from samples with low amount of total RNA was also demonstrated by an independent research group, which reported that some microRNAs (especially with low GC content) were selectively lost during extraction from a small number of cells [Kim, Y.K., et al. (2012) Short Structured RNAs with Low GC Content Are Selectively Lost during Extraction from a Small Number of Cells. Molecular cell 46, 893-895]. Consistently, adding a bulk RNA as a co-precipitate prevented the lost of small RNAs during extraction. You may also want to monitor the extraction efficacy by adding synthetic RNA (or miRNA) after cells lysis with Qiazol and measure it with qPCR. We observe almost 100% recovery of synthetic cel-miR-39 when glycogen is added as co-precipitate. Glycogen can be directly added to the Qiazol reagent before lysis - 1.2 mkl of glycogen (10 mg/ml) for 700 mkl of Qiazol. Good luck!

Try TRIZOL with glycogen. I used it for RNA extraction in samples from Laser-Captured Microscopy.

Try the method taht we used with just 10 cells

J. García-López and J. del Mazo “Functional Dynamics of MicroRNA Biogenesis During Preimplantation Mouse Development” BBA-Gene Regulary Mechanisms Biochimica et Biophysica Acta; Gene Regulatory Mechanisms 1819 :847–854 (2012).

Use Invitrogen TriZOL. It works fine.

Tri-reagent will give you bad quality with so low amount of cells. Therefore, I would not use it. I am doing RNA isolation of 10k-100k FACS-separated primary cells and I am using the RNeasy Mini Plus kit in combination with the QIAshredder columns from Qiagen to break the cells. The Plus kit in contrast to the normal one contains an DNA eliminating column and I always receive with this method (according to manufactors instructions) 100 ng/100k cells (respectively 50 ng/50k cells). I compared at the beginning the Micro and Mini kit and noticed that the Micro Kit is loosing more material during the process when I started with the same amount of cells (compared both kits with each three different low amounts - 5k, 10k, 50k - of cell culture cells). I elute at the end in two time 30 µL. It is a bit less concentrated but good quality. Please read the instructions from the Mini Plus Kit and let me know if you have any further questions. I hope this helps! Good luck! Nicole

PS: Did you used the Kit without the QIAshredder columns to break the cells?

Hi guys,

I'm sorry but I'm afraid I'll be the guy to bring bad news to all of you. Please take a look in this letter, which has been followed by a total/partial retraction paper in "Molecular Cell". Molecular Cell 46, 893–895, June 29, 2012

I really don't know anything about the cells you guys are using. However let me share some issues I've been facing here.

Basically the issues are:

Using TRIZOL to extract total RNA, nuclear RNA and cytoplasmic RNA from Hela, U2OS and SAOS-2 cell is great very easy and clean.

However, I've been facing huge problems trying to extract RNA from MCF10A, muscle cells and other very stable cell lines. basically what we thing is happening is:

Trizol is great to extract RNA from messed up cell, such as Hela (cancer cells, for instance). On the other hand, is not accurate enough to extract RNA from very normal cells lines (MACF10A, for instance).

In this case we've been using hot acidic phenol and it has been working fine.

I'd like to stress that the same issue has been identified by another group here at Umassmed, which may suggest it's not related to me (or my hands).

;-)

After reading that retraction, mentioned before I realized that Trizol can be great or really bad, depending on the cell type I'm willing to use as well as on the kind of RNA I'm willing to isolate.

Hopefully I brought something important to some of you guys.

Plus, I really would like to know if some of you guys have been facing something like that.

So Rakesh, I'd ask you before anything what kind of cells are you using for that?

Best wishes,

@lex

Nicole: I've used the shredder couple of times, but I thought it did not help me. When you said MicroKit from Qiagen was losing more RNA than MiniKit, I agree with that conclusion from my experience. Do you re-spin the 30uL extraction through the same filter or you do 15uL each two times on the same column?

Alex: I'm using human cancer cell lines and also primary cells. It looks like RNA quantity varies with cell lines too, but I thought cancer cells would have high transcription rate, may be not in the case of my cell lines.

Olga: I'll try the good ole method of Trizol.

Thanks all.

I am doing once 30 µL warmed RNase-free water (55°C), let it sit for like 5 min at room temperature on the column before I centrifuge and then put another 30 µL warmed water on the column, let it sit for 5 min again and centrifuge again. So, the total will be 60 µL with roughly 1-4 ng/µL. You can also use the first eluate and load it a second time on the column. Alternatively, you can just do once 30 µL. When I measured both elutions steps individually, I received with the second elution step still 10% of the amount of my first step.

Our experience indicates that the efficacy of RNA isolation from samples with low RNA content (e.g. 10k cells) is drastically lower (up to 10 times) when no co-precipitate is added. We use glycogen as co-precipitate and add it directly after Qiazol lysis (miRNAeasy kit). The importance of using co-precipitates during RNA extraction from samples with low amount of total RNA was also demonstrated by an independent research group, which reported that some microRNAs (especially with low GC content) were selectively lost during extraction from a small number of cells [Kim, Y.K., et al. (2012) Short Structured RNAs with Low GC Content Are Selectively Lost during Extraction from a Small Number of Cells. Molecular cell 46, 893-895]. Consistently, adding a bulk RNA as a co-precipitate prevented the lost of small RNAs during extraction. You may also want to monitor the extraction efficacy by adding synthetic RNA (or miRNA) after cells lysis with Qiazol and measure it with qPCR. We observe almost 100% recovery of synthetic cel-miR-39 when glycogen is added as co-precipitate. Glycogen can be directly added to the Qiazol reagent before lysis - 1.2 mkl of glycogen (10 mg/ml) for 700 mkl of Qiazol. Good luck!

I am working with oocytes and embryos and using the Picopure kit is our standard procedure.

For low amount of cells, the cell to CT kit is very very usefull (we used it for microdisscetion), also sometimes yeast RNA may be added to improve precipitation of RNA but it depends on the next experiement.

For more informations, i will suggest:

http://www.invitrogen.com/site/us/en/home/References/Ambion-Tech-Support/rna-isolation/general-articles/ten-ways-to-improve-your-rna-isolation.html

I got the same problem as you. I would recommend incubating your spin column after adding 30-50ul RNase free water, right before the first elution. Most of the time, RNA is just attached to the membrane and doesn't like to go through. Heating would be very helpful. This may increase your RNA yield to 2ug/ul.

I faced the same problem while working with sorted Side population cells. Leave the different buffers used in various step for 2-3 minutes in the spin column. Also such low number of cells use glycogen for better precipitation. Trizol will give better yield but the quality will be poor (especially the 260/230) and so you can use this for microarray experiments.

Hi all,

I used Trizol method with tissues and also in cells. Low amount of Trizol than recommended amounts could cause low quality RNA. Also taking out the aqueous layer could be tricky, you have to be very careful not to drag anything from interface or from the bottom layer- sometimes happens when using a pipet tips. I usually do follow up DNAse digestion and column purification step

using QIAGEN kits

Some down stream enzymatic reactions can be affected when using glycogen as a carrier. So very low RNA amount I am not sure about this procedure.

Chithra