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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
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