In that retracted paper they have used the Trizol method, which is a good RNA extraction method when looking at general mRNA expression of bigger sequences. When you deal with small-RNA there are kits which are developed for the isolation of their particular small feature. I would go for the miRNeasy kit from Qiagen. It is a bit more expensive, but you will give you highly pure RNA does not impact on the amount of RNA obtained. A huge plus is that you can also do an on-column DNAse I digestion, to get rid of contaminating DNA as well. I have used this isolation method for RNA used in qPCR, northern blot and microarray experiments, and this RNA isolation method have yielded good results for all these methods. I have also tried PureLink miRNA isolation kit from invitrogen, and it also seems to work fine for small RNAs.

I hope this could be of some help to you.

Good luck!

/Sebastian

mmmmm very interesting indeed. Lets see what happens next when people realise and incorporate this in their way of working. Thx a lot for sharing

In that retracted paper they have used the Trizol method, which is a good RNA extraction method when looking at general mRNA expression of bigger sequences. When you deal with small-RNA there are kits which are developed for the isolation of their particular small feature. I would go for the miRNeasy kit from Qiagen. It is a bit more expensive, but you will give you highly pure RNA does not impact on the amount of RNA obtained. A huge plus is that you can also do an on-column DNAse I digestion, to get rid of contaminating DNA as well. I have used this isolation method for RNA used in qPCR, northern blot and microarray experiments, and this RNA isolation method have yielded good results for all these methods. I have also tried PureLink miRNA isolation kit from invitrogen, and it also seems to work fine for small RNAs.

I hope this could be of some help to you.

Good luck!

/Sebastian

I use the same as Sebastian, or at times u can modify the general protocol for rna extraction kit by Qiagen. I add 100% ethanol (1.5 X) on my buffer RLT treated cells kept overnight at -80.

following this I only use buffer RPE (2x washes). this gives me good yeild for mirna and rna.

let me know if u want detailed protocol.

I also use miRNeasy kit (Qiagen)

The GC dependency was observed for low RNA amount extractions based on Trizol/Comczinsky and could be correlated with the self folding tendency of the miRNA. Mg2+ addition lowered the bias most likley by enhanced binding of the miRNA to concomitant RNA acting as carrier while EDTA/EGTA had the reverse effect. It is hypothesized that the precipitation step of selfpaired (GC rich) miRNAs is less efficent.

hope this helps to understand the problem a bit further.

all the best

Jörg

Rafal, your point is very interesting because I currently use the Qiagen universal primer system and I also got problems to comfirm some microarray expression results with qPCR... I will use the Applied system and I will tell you the results... :-)

We have tested both Qiagens and Ambions miRNA extraction kits (and others) and they both seems to be working ok, theres no difference in results between them in our case. When it comes to RT kits for miRNAs, you also have Exiqons kit, which is fairly good and it also uses a universal primer for all miRNAs. For Applied Biosystems (Life technologies) I can really recommend ordering their customized RT pools, where you can choose which miRNAs you want to perform RT for (in a single reaction) or you can also order their 384 RT primer mixes which includes universal primers for 384 different miRNAs. I have been using both of these systems from Applied and it works really good and you dont have to perform several RT reactions for each sample, which means that you save alot of material as well.

This is a very interesting case.

1.,

The first take-home message is that proper collecting and treating the samples is of fundamental importance. It can spoil your several years' time and money if you work on biassed or degraded or otherwise not representative samples. This step is usually overlooked but now we who follow this thread know exactly how important it is.

2.,

A warning for the editors is a good idea. Too bad that it is only for helping them discarding the years' data. Generally I usually think, though, that it is much better to prevent problems to happen than to try to solve them later. Therefore I guess these discussion threads are very helpful and extremely good to have. Try to spread a word in every possible way to those who are starting such a project to be careful on time.

3.,

With respect to Universal Primers I often wonder if I have two genes in a bacterial operon, having identical induction levels, would I get the same qPCR results of the two? Or will I have bias according to the lenghts of the genes or according to which of them is upstream of the other one?

Thank you all for interesting advices, and please, continue posting comments. Thus, we all can gain insights toward safe isolation strategies and generate robust data.

Very interesting discussion regarding TRIZOL, stem-loop and poly-A tail method. I have previously performed miRNA isolation from serum via TRIZOL LS and then used self-designed stem loop primers, miRNA specific forward primer and universal reverse primers. The results were okay. I am not sure if TRIZOL have same effect for serum, tissue and cells when it comes to miRNA extraction and for low abundance miRNAs. Anybody with similar experience? I have not yet tried stem-loop method from AMBION but when it comes to large sample size and expression profiling of multiple miRNA via real time PCR, poly-A methods seems to be economical and easier to perform. Any ideas or opinions on if we can pool multiple stem-loop primers for different miRNAs during cDNA synthesis step. Will this lead to simultaneous cDNA synthesis of different miRNAs in a single reaction?

@ Emanuel

It would be great if you could please provide link to that methodology article. I am not sure if or how good pooling stem loop primers will work in my condition as I am looking to make cDNA of more than 12 miRNA (few of them differ by only one base) via stem loop method, if possible in a single reaction. Apart from stem loop method kits, Ambion now also offers NCode VILO kits that are based on Poly-A tail method. Anyone had experience with these?

Today, in your opinion, what are the top 3 best kits to isolate small RNA molecules?

I think the key point of the Kim publication that has been retracted is the celular density. When working with a low number of cells, perhaps they should be treated the simple as if it was a more challenging simple like plasma or any other biofluid.

Another key point is that  they extracted total RNA using trizol and the manufacturer's protocol. But this protocolo is only suitable for mRNA extraction and must be modified to optimize the precipitation of small RNA molecules.

Personally, I've used both the miRNEasy mini Kit from Qiagen (that is based in trizol extraction but column purification) and the modified protocol of Trizol and I have obtained comparable results. However, in celular experiments I always use high density samples.

Apart from that, I've used miRvana parís Kit (ambion) and miRCury RNA extraction kit (Exiqon) and I prefer the last one, esppecially for highly challenging samples.

Lidia

@ Mustafa @sebastien, what was the Bioanalyzer data you got after miRNeasy extraction. Would you like to share detailed protocol? I am also using miRNeasy kit from Qiagen and never worked for me. Thank you very much