Hi Juan!

I have used this method and I liked it, it has the advantage that by adding a poly-A tail to the miRNA you may from the same cDNA synthesis using oligo d(T) analyse the expression of different miRNAs.

http://www.ncbi.nlm.nih.gov/pubmed/16235564

I have used this method (stem-loop) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2225395/ as well. Worked perfectly but in this case you must to a different cDNA synthesis for each miRNA you plan to analuse since the primers for the reverse transcription are miRNA-specific.

I followed exactly their protocol in each case, in both ncases I have started from total RNA. Let me know if you have any trouble with that.

Hi Juan

Yes as you have mentioned above...expression analysis of miRNAs is similar to mRNA in principle..however due to special characteristics of miRNAs some time you may have to use protocols specific to miRNA analysis.....such as

1) You can study the precurosor miRNAs in a manner similar to mRNAs...using different approaches ..like SYBR, molecular beacons ot taqman probes...

2) for mature miRNAs (as they are small)...several approaches are in published papers and use sometimes approaches...like hairpin primers....but you'll have to RT for each miRNA you are planning to study

3). or you can enrich the small RNA fraction and use linkers to add to the total small RNAs...do a single RT and and use either specific primer/probe combination to study individual miRNA in the total pool...

Your approach will be dicided by howmany miRNAs/or pre-miRNAs you are planning to study and what are their leves

bye

First, you have to be sure that you extract the small RNA if you want to quantify the mature miRNA. It's possible with the Trizol and with several kits like miRNeasy Mini kit from Qiagen. And then, you have to do a reverse transcription using a TaqMan reverse transcription kit followed by qPCR with a Taq Man probe. You have a protocol in this article PMID: 23056458.

I hope it will help you.

Hey Juan,

In what kind of material do you want to study microRNA expression?

You might want to test some different isolation methods first, some methods enhance the small RNA fraction but they might influence the total yield of some microRNAs.

Furthermore like Ajay mentioned be sure what stge of microRNA (pri-pre, 3p or 5p) you want to investigate.

Otherwise steps are quite similar to mRNA expression.

Also worthwhile is too investigate you possible reference microRNA or small RNAs there is much debate on which molecules to use. The use of reference genes for microRNA is allmost organ specific.

Good luck,

Emiel

Facile means for quantifying microRNA expression by real-time PCR

Rui Shi and Vincent L. Chiang

North Carolina State University, Raleigh, NC, USA

BioTechniques 39:519-525 (October 2005)

Hi Juan!

I have used this method and I liked it, it has the advantage that by adding a poly-A tail to the miRNA you may from the same cDNA synthesis using oligo d(T) analyse the expression of different miRNAs.

http://www.ncbi.nlm.nih.gov/pubmed/16235564

I have used this method (stem-loop) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2225395/ as well. Worked perfectly but in this case you must to a different cDNA synthesis for each miRNA you plan to analuse since the primers for the reverse transcription are miRNA-specific.

I followed exactly their protocol in each case, in both ncases I have started from total RNA. Let me know if you have any trouble with that.

Are you evaluating the expression of isolated miRNAs or do you plan to use a method such as TaqMan Low density Arrays (TLDA) or microarrays? Some methods, like the TLDAs require that you start from total (not enriched in the small fraction) RNA, since the platform's endogenous controls for normalization before comparing expression of different samples are long RNAs. I would recommend to start isolating total RNA using Trizol and only if you need it, purify the small RNA fraction. We have been working with total RNA followed by the normal TaqMan protocol and it has worked fine. Hope it helps!

Hi Juan,

I would choose one of the two methods mentioned by Cláudia Martinho depending on the number of microRNAs to be quantified by RT-PCR. If you plan to analyse the expression of few candidate miRNAs, I would go with the second option. Using miRNA-specific RT primers avoids obtaining cDNA from non-specific targets, producing, therefore, a more reliable qPCR result. If you are interested in a microRNA expression profile or panel (over 100 miRNAs), I would choose the method based on poly-A tailing for the production of cDNA. This is in general a cheaper and easier-to-do option and would give you a good picture of the miRNome expression of your condition/cell/organism.

Good luck!

Hi,

We have used the TaqMan small RNA assay from Life Tech successfully to detect mature miRNA. You need a different assay (includes the RT primer and the TaqMan primers) for each miRNA you want to detect. The paper that corresponds to this assay is: Chen C. et al. 2005. Real-time quantification of microRNAs by stem–loop RT–PCR. NAR.

In terms of enrichment, we're using the Cells-to-CT kit from Life Tech/Ambion, which is pretty easy and fast.

Extract your total RNA with Trizol...do not use the mivana or qiagen kits as they do work as well. Taqman is expensive! Genecopoeia has all the miRNA primers you will need as well as the best priced syber green mastermixes and first-strand cDNA kit for miRNA...as they need to have a poly-A tail put on them. It's easy and cheaper, and I do this several times a week!

Thank you all for your answers.

I am going to extract the RNA from culture cells and analyse the expression of selected miRNA. Do you recommend me the TaqMan array cards or it is better other method?

Thank you in advance.

Hello Juan,

I usually extract RNA from human tissues and cell lines using the Trizol reagent (invitrogen). Their is no particular enrichment with Trizol, total RNA is extracted which can be useful for downstream applications. However, if using column purification you need to make sure the kit is adapted for small RNAs. For RT-qPCR of mature miR, I have tried two different protocols. The first, that I personally recommend, is Taqman miRNA assays (applied biosystems). Is is very specific and very easy to put in place. It relies on a stem loop amplification step during the RT that guarantees specificity. The second is made by Qiagen and with it I had many issues (lack of reproducible data, specificity, ...).

Hi Juan,

If you are planing to work with miRNA isolated form cells I would go with TLDA (TaqMan Low Density array) for fast screening and qRT-PCR to confirm your results. TLDA already has internal control included so you don't have to worry about it. If you want to isolate miRNA from serum , whole blood or plasma you will need to "spike" your sample before. I used cel-miR-39 to spike the sample but there other available. This can be used as a control for your isolation and PCR. Hope this helps you. Best regards

Hi, Ivana! I've been trying to understand how a spike-in control can fully replace an endogenous control. It is obviously not the same thing, considering that an endogenous control normalizes inter-individual expression heterogeneity. What do you have to say about this?

Spike-in controls are useful for samples where there is no reliable endogenous control, such as serum samples. By spiking in the same known amount of a standard at a very early stage of the extraction in all of your samples, you control for any possible variations in efficiency of the extraction procedure. (For serum, a good time to add the spike is at the first step of the extraction, but just after you've added a denaturant/detergent solution that will inactivate any nucleases which could degrade the spike.) By normalizing to the measure of this standard in the final prep, you will be able to determine the relative amounts of your analyte of interest in the different samples. For other kinds of samples, like cells in culture or tissue, you don't need to spike because you can use endogenous standards which do remain constant--either other small RNAs or even the same kinds of standards used for mRNA RT-qPCR.

When using cell culture and only a few miRNAs we have good and stable results using TaqMan single miRNA assays.

Please also keep the house keeper in mind! I would recommend to test for the best normalizer by using 2 to 3 different controls as endogenous control including miR-16, which is ubiquitously expressed. I would not recommend using mRNA probes, since there are of different size and you might have lost a huge proportion of small RNAs without detecting this. The use and the testing for house keepers is of importance since the control small RNA is crucial for the results of your experiments, but are cell type specific. If you wanted to compare the miRNA expression between different cell types you would need to evaluate the variance of expression of your house keeper across the cell lines. Here is an article regarding the correct selection of a house keeper for miRNA qRT-PCR experiments:

http://www.ncbi.nlm.nih.gov/pubmed/?term=18718003

A handy tool called RefFinder that uses several statistical approaches for this house keeper is located here for free:

http://www.leonxie.com/referencegene.php?type=reference

And it also has a link to the respective methods like BestKeeper, Normfinder etc.

Another possibility would be to do quantitative Northern blotting. This is still the gold standard and you can see also the pre-miR if there is a processing deficient phenotype. This method is mandatory for novel miRNAs from my perspective to exclude that it is part of other larger non-coding RNAs like tRNAs, sn/snoRNAs etc.

If you would need to analyze a large panel of miRNAs I would consider small RNA seq, since this becomes more and more cost-efficient when barcoding. Hope this helps a bit.

Depending on the number of samples and miRNAs to be analyzed, you may want to use either TLDAs (medium throughput), nCounter (high throughput, I think youu would need to contact some servicing there), or miRNAome sequencing (very high throughput and allowing for discovery as well)

Hi juan..

If u think about miRNA expression then plz go through the stem loop RT-PCR

in which the first step is to design stem loop primers specefic to ur desired miRNA then forward and universal revirse primer then RT PCR

ALL D Best....

Hi Juan,

I agree with Mohiuddin,,, since I am also working in that way,,, I usually uses trizol to isolate total RNA from cell lines. And during RT(cDNA synthesis), I uses miR specific RT primers along with random primer mix, to make sure we are not loosing house keeping genes and other genes, which can be used as endogenous control. I uses SNORD44 as endogenous control,,,This protocl works for me,,,and I have got good results,,,

Hi, we used trizol method to isolate total RNA from human PBMCs and globin gene as endogenous control and did RT-PCR using taqman mix, it worked well for us

Hi, I use Genecopoeia products instead of Taqman...way cheaper. I use Trizol to isolate total RNA and the miRNA kit to add poly-A tails and their verified primers...works well!

@ Juan Bastida

Reverse transcription quantitative PCR (qRT-PCR):

In the present study, we selected differentially expressed and functionally significant 13 miRNAs (nine conserved and four novel) for qRT-PCR. Briefly, Mir-X-miRNA qRT-PCR SYBR Kit (Clontech Laboratories, Inc., USA) was used for the qRT-PCR reactions, which has a single-step, single-tube reaction to produce the first-strand cDNA, which was then specifically and quantitatively amplified using a miRNA-specific primer and SYBR Advantage qPCR chemistry. All the qRT-PCR assays were conducted according to the MIQE guidelines [45]. U6 snRNA was used as an internal control gene for normalization. qRT-PCR was performed on Light Cycler 480 (Roche, USA) using 1:20 diluted cDNAs and SYBR Advantage Premix (Clontech Laboratories, Mountain View, USA), according to the manufacturer’s instructions. Assays were performed in triplicates for three independent biological experiments and the relative gene expression data were analyzed using 2-ΔΔCT method [46]. The values of these three independent experiments were statistically analyzed using one-way ANOVA to calculate the statistical significance.

Reference: Rebijith KB, Asokan R, Hande HR, Krishna Kumar NK (2016) The First Report of miRNAs from a Thysanopteran Insect, Thrips palmi Karny Using High-Throughput Sequencing. PLoS ONE 11(9): e0163635. https://doi.org/10.1371/journal.pone.0163635

hello I also need your help. with my post graduate student we use the miscript technology (extraction and real time PCR) of qiagen to quantify mature Mirna. I need detail of all the experimental experience and your experience in quantification.