TaqMan™ MicroRNA Reverse Transcription Kit, 200 reactions - FAQs

How long can I store the cDNA from my reverse transcription step?

You can store your cDNA at 2-6 degrees C for up to 24 hours. For long-term storage, store the cDNA at -15 to -25 degrees C and add EDTA to a final concentration of 1 mM to prevent degradation.

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.

How many tubes of the MultiScribe reverse transcriptase enzyme do I get in the TaqMan MicroRNA Reverse Transcription Kit (4366597)?

1 tube containing 1 mL of MultiScribe Reverse Transcriptase at 50 U/uL. This is sufficient for 1,000 reactions.

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

The TaqMan MicroRNA Reverse Transcription Kit (Cat. No. 4366596) does not include the 5x RT primer mentioned in the protocol. How do I perform reverse transcription?

The 5x RT primer is specific for the miRNA for which you have purchased the TaqMan MicroRNA assay. The 5x RT primers do not come with the above reverse transcription kit, rather with the TaqMan MicroRNA Assay (Cat. No. 4427975). This product includes the following components:

• 1 tube containing RT primer at concentration of 5X (XS and S sizes), 20X (M size), or 60X (L size)
• 1 tube containing a 20X (XS, S, and M sizes) or 60X (L size) mix of pre-formulated assay (1 probe and 2 primers).

I want to purchase TaqMan Array Human MicroRNA A Cards v2.0 (Cat. No. 4398965). What other reagents do I need to purchase in addition to these cards?

In order to perform reverse transcription on these cards, you will need the following:

• TaqMan MicroRNA Reverse Transcription Kit (Cat. No. 4366596)
• Megaplex RT Primers pools (for human Type A cards Cat. No. 4399966, for Type B cards Cat. No. 4444281)

Can I purchase the MultiScribe Reverse Transcriptase from the TaqMan® MicroRNA Reverse Transcription Kit (Cat. No 4366596) separately?

Yes, the MultiScribe Reverse Transcriptase (Cat. No. 4311235) can be purchased as a standalone product. Please see the following link.

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