Finding The Right MicroRNA (miRNA) Assays - Ask TaqMan #31

Are you working with microRNA, and are you having trouble measuring them due to their small size?
Today, we are going to talk about the different microRNA assays available, and which is the right one for your application.

MicroRNAs are small and around 22 nucleotide noncoding RNAs that regulate gene expression. Due to their small size, the measurement of miRNAs presents some special challenges. miRNAs cannot use the same RT chemistry as other RNAs and require a special strategy to ensure specificity. Also, there are many miRNAs families that are highly homologous and differ by only a single base, highlighting the need for miRNA assays that are highly specific.
To overcome these challenges, we offer two different types of microRNA assays that use the TaqMan chemistry: the TaqMan miRNA assays and the TaqMan Advanced miRNA assays.
First, let’s start with the TaqMan miRNA assays. These assays are based on an individual stem-loop RT primer and a sequence- specific TaqMan® assay to accurately detect mature miRNAs.
1. After isolating total RNA, using a method that preserves small RNAs, the stem loop primer is added to the sample. Each stem loop primer binds specifically to an individual mature miRNA target. During reverse transcription, only the target mature miRNA is converted to cDNA, ensuring that this miRNA is the only one that will proceed to qPCR.
2. If desired, for low expressing miRNAs or samples that limited, we recommend the usage of a miRNA-specific preamplification step (or pre-Amp). This pre-Amp step will amplify the number of targets in the sample prior to qPCR and allow for quantitation of smaller input starting amounts. However, this step is optional.
3. During qPCR, the TaqMan® MGB probe anneals specifically to a complementary sequence between the forward and reverse primer sites.
The DNA polymerase cleaves only probes that are hybridized to the target. Cleavage separates the reporter dye from the quencher dye, resulting in increased fluorescence by the reporter. The increase in fluorescence signal occurs only if the target sequence is complementary to the probe and is amplified during PCR, so that nonspecific amplification is not detected.

Next, let’s move on to the newest miRNA chemistry, the TaqMan Advanced miRNA assays. The TaqMan Advanced miRNA chemistry is based on a universal RT that will generate cDNA for all of the miRNA targets in the starting sample.
1. After isolation of total RNA, using a method that preserves small RNAs, Poly A polymerase is used to add a polyAdenosine tail on the 3’ end of all of the miRNAs in the sample.
2. In the next step, an oligonucleotide adaptor is ligated to the 5’ end of each of the miRNAs. Note that this reaction is dependent on the presence of a 5’ phosphate, which is only present on mature miRNAs. Any exogenous controls or spike-ins must also have this 5’ phosphate in order to be accurately quantitated.
3. A Universal RT primer binds to the polyA tail of the miRNA to reverse transcribe the RNA into cDNA. This universal RT step will convert all of the mature miRNAs in the sample to cDNA.
4. Universal amplification (miR-Amp): universal forward and reverse primers amplify the cDNA for all miRNA targets in the sample. The miR-Amp step improves quantification of low expressing miRNAs and miRNAs collected from dilute samples such as body fluids.
5. miRNA- specific primers and TaqMan probe are used to quantitate each miRNA of interest using individual qPCR assays. Note that these assays work specifically with TaqMan Advanced miRNA and cannot be used with the original TaqMan miRNA chemistry.
To summarize, the TaqMan microRNA assays use a micro-RNA-specific RT primers and as such are best for looking at 1-10 targets. There are TaqMan microRNA assays for 205 species, aligned to the latest version of miRbase. The TaqMan Advanced microRNA assays use a universal reverse transcription approach, making these assays better for situations where you are interrogating more than 10 targets. These assays are primarily focused on human microRNAs.
If you’ve got more qPCR or digital PCR questions, remember, just ask Taqman.

I’ll see you next time.

[YKKLY]

3:53 Both forward and reverse primer are drawn at the same strand of 5' adapter and universal RT primer. Primer can not hybridize like this because primer is the same direction against a template.

[randahmad]

I isolate total RNA from plasma can I use TaqMan MicroRNA Assays to synthesis cDNA to perform QRT-PCR to evaluate mRNA level of protein ?

[mykaellaandrade]

Hello, using MicroRNA transcription kit and Taq Man microRNA assay... is possible amplify off-targets on RT-qPCR?

[aminullah]

Please tell me any one that HOW many types of microRNAs profile are detected by Taqman microRNA assay kit.?Is this kit is only used for detection of a single microRNAs profile or for multiple types of microRNAs profiles.

[aminullah]

I need micro RNA 122, and 121 kit.
Which type of TaqaMan kit i used.?
Please send me their price of selected kit.

[pujakumariray]

For which one assay exogenous control is required? For taqman miRNA assays or taqman advanced miRNA assay or both

[zainabjummah]

How I can determine the SNPs of miRNA? I don't understand how they isolate DNA then perform rflp-PCR or real time-pcr can anyone help me please

[sonalidey]

If I want to creat cDNA library from all miRNA available in in plant tissue then which will be the best way to proceed plz reply

[ameetkumar]

which technique should i use for quantifying microRNA- 31

[marcuspreedy]

For the advanced miRNA assays, how do you only measure the miRNAs that you are interested in if all of the miRNAs in the sample are amplified?

[ameetkumar]

i am working on salivary microRNA 31. plz tell me regarding universal master mix, which master mix should i use taqman universal master mix with UNG or without UNG?

[Am-yohx]

what are the advantages and disadvantages of taqMan microRNA assay?

[fatimaguimaraes]

How to use the U6 to normalize? We should by a probe to U6???

[ameetkumar]

i am using taqman microrna assay.. is it necessary to use reverse transcription kit? as i studied stem loop primer is present in taqman microrna assay

[ud]

is the control prime/probe supposed to be added along with the specific primer/probe in case of PCR ( both reverse transcription and real time) ? I m.working with taqman mir124 primers and probes and using U6 snRNA as control.