Pri-miRNA Analysis

Workflow Features

Ideal for measuring miRNA “gene” transcription in human, mouse or rat species
Enables the regulation of miRNA biogenesis to be probed when used in combination with TaqMan® MicroRNA Assays that detect mature miRNA sequences
Adds new perspective to your understanding of miRNA expression

How It Works
MicroRNA “genes” are transcribed in the nucleus creating a long pri-miRNA transcript. The pri-miRNA sequence contains one or more stem-loop sequences, each containing mature miRNA sequences. TaqMan® Pri-miRNA Assays are designed in close proximity to each independent stem-loop sequence identified in the Sanger miRBase sequence repository (http://microrna.sanger.ac.uk/sequences/) using Applied Biosystems’ state-of-the-art bioinformatics pipeline. This approach ensures that the primary transcript containing the stem-loop of interest is being measured.

Products
Experimental Workflow: Find the Products You Need for Every Step
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Step 1: Prepare Sample

Applied Biosystems offers a variety of kits that preserve small RNAs such as miRNAs. While it is not essential to use them due to the larger size of primary miRNA transcripts, these kits do enable both pri-miRNA and mature miRNA quantitation from the same RNA sample. Each kit is ideal for miRNA analysis because they enable quantitative recovery of all RNA >10nt in length and maintenance of representative amounts of all RNA (eliminating experimental bias). To ensure accurate quantitation with TaqMan® Pri-miRNA Assays, samples must be free from genomic DNA.

Products for Step 1: Prepare Sample

mirVana™ miRNA Isolation Kit

Isolate total RNA that contains small RNAs such as miRNA, siRNA, and snRNA. These small RNAs are often lost during traditional RNA isolation methods. In addition, this kit includes reagents and instructions for the isolation of RNA that is enriched for small RNA (<200 bp), enabling more sensitive detection of low-level small RNAs.

RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE

Extract total RNA or DNA from formalin- or paraformalin-fixed, paraffin-embedded (FFPE) tissues. This kit enables the molecular analysis of archived tissue samples at both the genomic and gene expression levels. With this kit, nucleic acid is obtained that is suitable for RT-qPCR, mutation screening, and microarray analyses.

TURBO DNA-free™ Kit

TURBO™ DNase is a recombinant, engineered form of DNase I that is more efficient than wild-type DNase I at digesting trace amounts of unwanted DNA in RNA samples. The TURBO DNA-free™ Kit comes with all reagents needed to perform 50 TURBO™ DNase treatments plus a highly effective DNase Inactivation Reagent, making this kit the tool of choice for RT-PCR sample preparation.

TaqMan® MicroRNA Cells-to-CT™ Kit

Featuring a unique method for lysing cultured cells while removing genomic DNA and preserving RNA integrity, the TaqMan® MicroRNA Cells-to-CT™ Kit makes it possible to perform miRNA expression profiling directly from cultured cells without RNA purification.

Step 2: Reverse Transcribe RNA

The High Capacity RNA-to-cDNA kit provides all the necessary components to reverse transcribe long RNA into cDNA. (Note: this step can be omitted if a one-step RT-PCR protocol is used.)

Products for Step 2: Reverse Transcribe RNA

High Capacity cDNA Reverse Transcription Kit

The High Capacity cDNA Reverse Transcription Kit contains all components necessary for the quantitative conversion of up to 2 µg of total RNA in a single 20 µL reaction to single stranded cDNA. Reactions can be scaled up to 100 µL to generate 10 µg of cDNA from a single reaction.

High Capacity RNA-to-cDNA Kit

The High Capacity RNA-to-cDNA™ Kit provides high quality reverse transcription that delivers a streamlined workflow with maximum sensitivity and dynamic range.

Step 3: Set Up Singleplex TaqMan® Pri-miRNA Assay Reaction

For the final quantitation step, the cDNA product generated in step 2 is mixed with TaqMan® Gene Expression Master Mix and added to the matched TaqMan® Pri-miRNA Assay.

Products for Step 3: Set Up Singleplex TaqMan® Pri-miRNA Assay Reaction

TaqMan® Pri-miRNA Assays

TaqMan® Pri-miRNA Assays quantitate primary microRNA (pri-miRNA) transcripts, the most upstream RNA molecules in the miRNA biogenesis pathway. TaqMan® Pri-miRNA Assays are designed using Applied Biosystems’ state-of-the-art bioinformatics pipeline, delivering gold standard assay performance and data quality for pri-miRNA studies.

TaqMan® Gene Expression Master Mix

TaqMan® Gene Expression Master Mix is tailored for precise quantification by real-time PCR. It is designed for routine and challenging experiments.

Step 4: Perform Real-Time PCR Amplification

The real-time PCR can be run on any Applied Biosystems® Real-Time PCR System.

Products for Step 4: Perform Real-Time PCR Amplification

StepOnePlus™ Real-Time PCR System

The Applied Biosystems® StepOnePlus™ Real-Time PCR System, makes it simple and easy to get publication-ready high-quality real-time PCR results on day one. This remarkably simple 96-well, 4-color, real-time PCR system is designed with a user-friendly, yet powerful, interface for researchers of all experience levels.

7300 Real-Time PCR System

Get the Applied Biosystems advantage at an affordable price; the 7300 Real-Time PCR System balances exceptional quality with budget sensitivity.

7500 Real-Time PCR System

The Applied Biosystems® 7500 Real-Time PCR System is a powerful platform for labs requiring superior performance and maximum dye versatility. This third generation platform features an innovative optical system that enhances sensitivity and lets you access a broader range of fluorophores. The variable excitation capability allows greater sensitivity for longer wavelength (red) dyes.

7500 Fast Real-Time PCR System

The Applied Biosystems® 7500 Fast Real-Time PCR System offers maximum performance in the minimum time. Fully optimized for Fast cycling, the 7500 Fast System delivers high-quality results in as little as 30 minutes.

7900HT Fast Real-Time PCR System

The 7900HT Fast Real-Time PCR System is the only real-time quantitative PCR system that combines 384-well plate compatibility with fully automated robotic loading — and also offers optional Fast real-time PCR capability. Acknowledged as the gold standard in real-time PCR, the 7900HT System combined with TaqMan® Genomic Assays enables you to achieve unprecedented throughput and flexibility, allowing you to pursue projects beyond the scope of previous real-time instruments.

Step 5: Analyze Data

Integromics RealTime StatMiner® Software is a powerful, easy-to-use bioinformatics tool for quality control and differential expression analysis of real-time PCR data. The software easily imports raw data or Ct values from any Applied Biosystems® Real-Time PCR System and guides you though every step of analysis for accurate results in minutes.

Products for Step 5: Analyze Data

RealTime StatMiner® Software (optional)

RealTime StatMiner® Software from Integromics is a powerful, easy-to-use bioinformatics tool for quality control and differential expression analysis of real-time PCR data. With just a single click, the software imports raw data or Ct values from any of our real-time PCR instruments, and then guides you though every step of analysis, giving you accurate results in minutes.
Literature
Product Information
- TaqMan® Pri-miRNA Assays (Product Bulletin)