What is mechanism of the TaqMan® MicroRNA Assays?

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There are 2 steps involved in using the TaqMan® MicroRNA Assays:

1. RT step: total RNA to cDNA using miRNA-specific RT primers from the TaqMan® MicroRNA Assays and the TaqMan® MicroRNA Reverse Transcription Kit (P/N 4366596, 200 reactions or 4366597, 1000 reactions)

2. PCR step: cDNA to PCR products using primers and probe from the TaqMan MicroRNA Assays together with TaqMan® Universal PCR Master Mix No AmpErase® UNG (P/N 4324018).

Answer Id: E2516

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0c3742a470ce0e505ab823ff7dc81afa_FAQ

What are miRNAs?

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MicroRNAs (miRNAs) are endogenous non-coding RNAs, about 22 nucleotides in length, that play an important role in post-transcriptional gene regulation in animals and plants by targeting messenger RNA (mRNA) for degradation or translational repression. The miRNA precursors are transcribed from individual genes but are not translated into proteins. The primary transcript is processed in the nucleus to give one or more hairpin precursors that is exported to the cytoplasm. The mature miRNA is excised from the hairpin precursor. The mature miRNA is the biologically active form and regulates the expression of mRNA transcripts by binding to complementary sites on target mRNAs and inhibiting translation or, when there is perfect complementarity to the target sequence, inducing mRNA cleavage. In animals, translational repression, not transcript degradation, is the dominant miRNA mode of action.

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fa2ec7387b6362de3402af6823f87303_FAQ

Why are miRNAs so important?

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MicroRNAs are believed to play an important role in post transcriptional gene regulation. A total of ~850 unique miRNAs have been discovered so far, including ~333 human miRNAs. They are relatively abundant, ranging from a few to as many as 50,000 molecules per cell. They account for approximately 1% of the predicted genes in animals and plants. The level of individual miRNAs varies dramatically across cell type and developmental stages. These differences in miRNA level are believed to be a key indicator of miRNA activity.

Recent research has implicated miRNAs in the following: cell development, differentiation, communication and cell death; DNA methylation and chromatin modification; metabolism; human cancer development; haematopoiesis; nervous system patterning; insulin secretion.

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f9442e4dbb7788744ea6e2ea036b6587_FAQ

How do the miRNA assays correspond to miRBase?

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Individual TaqMan® miRNA Assays are designed to target specific mature miRNA sequences. The mature sequences correspond to those contained in miRBase. Often several precursor miRNAs with different names and sometimes across several species will produce the identical mature miRNA sequence. Thus, one particular assay may be valid for use across several species. The Assay Index File provides the cross species key for TaqMan® MicroRNA Assays.

Answer Id: E2514

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4334cb46392bbc9e2a438cdc4cf3f0e0_FAQ

What is the TaqMan® MicroRNA Assay?

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A TaqMan® MicroRNA Assay has two components: an RT reaction containing a miRNA specific stem-loop reverse-transcription primer and a separate specific TaqMan® miRNA Assay. The RT stem-loop primer provides the specificity for the mature miRNA target; it does not detect its precursor. The formation of a RT primer/mature miRNA-chimera extends the length of the 5’ end of the miRNA. The longer RT product provides a miRNA specific cDNA template amenable to the TaqMan® assay design.

Answer Id: E2519

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823e6693839866248be4f1899e32cc45_FAQ

What does it mean when a miRNA target entry has been removed (obsoleted) from miRBase?

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miRBase continues to re-annotate the miRNA sequences as more information becomes available. A sequence that once mapped to a given species (e.g.: human) may no longer exist, so it is removed from miRBase. Accordingly, the original assay for that sequence no longer maps to an annotated miRNA, and as a result the annotation fields on the search results page on the Life Technologies portal are empty. An example of this is Assay “hsa-miR-505” (Assay ID 001049). miRBase continues to re-annotate the miRNA sequences as more information becomes available. A sequence that once mapped to a given species (e.g.: human) may no longer exist, so it is removed from miRBase. Accordingly, the original assay for that sequence no longer maps to an annotated miRNA, and as a result the annotation fields on the search results page on the Life Technologies™ portal are empty. An example of this is Assay “hsa-miR-505” (Assay ID 001049).

Answer Id: E2522

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976fa058438744d313bea903aae0ce78_FAQ

Can I use any reverse transcription kit for a TaqMan® MicroRNA RT reaction?

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No, only the TaqMan® MicroRNA Reverse Transcription Kit (Cat. No. 4366596 (200 reactions)) or Cat. No. 4366597 (1,000 reactions)) should be used. This kit was developed specifically for use with TaqMan® MicroRNA Assay reverse transcription.

Answer Id: E2768

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