Pri-miRNA Analysis Using Real-Time PCR

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://www.mirbase.org) 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.

Pri-miRNA Analysis Experimental Workflow

Life Technologies 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.

Recommended Products

		TaqMan® miRNA ABC Purification Kit The TaqMan® miRNA ABC (anti-miRNA bead capture) purification kit contains buffers and magnetic beads for single-tube isolation of specific microRNA (miRNA) from small inputs of all human sample types, including blood, serum, plasma, FFPE samples, solid tissues, cultured cells, and saliva in 75 minutes. TaqMan® miRNA ABC Purification Kit
		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. mirVana™ miRNA Isolation Kit mirVana™ miRNA Isolation Kit (without Phenol)
		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. RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE
		*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. *TURBO DNA-free™* *TURBO DNA-free™ w/Manual*
		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. TaqMan® MicroRNA Cells-to-CT™ Kit TaqMan® MicroRNA Cells-to-CT™ Kit w/Manual Cells-to-CT Bulk Lysis Reagents Cells-to-CT™ Stop Solution

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.)

Recommended Products

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.

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.

Recommended Products

		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 our state-of-the-art bioinformatics pipeline, delivering gold standard assay performance and data quality for pri-miRNA studies.
		TaqMan® Universal Master Mix II TaqMan® Universal Master Mix II offers convenience and reliability for all real-time applications including microRNA, gene expression, and genotyping studies.

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.

Recommended Products

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 through every step of analysis, giving you accurate results in minutes.

Literature

Product Information

TaqMan® Pri-miRNA Assays (Product Bulletin)