TaqMan™ Universal Master Mix II, with UNG, 1 x 5 mL - FAQs

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

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 Thermo Fisher Scientific 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 Thermo Fisher Scientific portal are empty. An example of this is Assay “hsa-miR-505” (Assay ID 001049).

What is the TaqMan MicroRNA Assay?

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.

What is mechanism of the TaqMan MicroRNA Assays?

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).

Why are miRNAs so important?

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.

What are miRNAs?

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.

What Applied Biosystems reagents and instruments can I use with TaqMan Gene Expression Assays?

The TaqMan Gene Expression Assays are optimized to work with one of the TaqMan qPCR Master Mixes that include TaqMan Universal PCR Master Mix, No AmpErase UNG(P/N 4324018), TaqMan Universal PCR Master Mix with AmpErase UNG (P/N 4304437), TaqMan Universal PCR Master Mix II, No AmpErase UNG (P/N 4440040), TaqMan Universal PCR Master Mix II, with AmpErase UNG (P/N 4440038) or TaqMan Gene Expression Master Mix (P/N 4369016) and with your cDNA. It is recommended to use the High Capacity cDNA Archive Kit (P/N 4322171) for converting your RNA to cDNA. TaqMan Gene Expression Assays are capable of being run on all Applied Biosystems Real-Time PCR Systems models including 7000, 7300, 7500, 7500 Fast, 7700, 7900HT, StepOne, StepOnePlus and ViiA 7 Instruments.

Can dU-containing DNA produced by amplification with dUTP be cut by restriction enzymes?

You will need to confirm with the manufacturer of the restriction enzyme you are using to find out whether or not it can recognize dUTP-containing DNA.

Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.