Tailing miRNA First-Strand Synthesis Kit

CAT: MF0201

price：$275  for 25T (negotiable if bulk purchase)

Storage temperature: -20℃

Product composition

Component	MF0201
miRNA RT Enzyme Mix	50 ul
2 × miRT Reaction Mix	250 ul
RNase free H2O	1 ml

Product Description

miRNA First Strand Synthesis Kit This kit uses the principle of Poly(A) tailing. First, a Poly(A) tail is added to the 3' end of the miRNA, and then the Anchored oligo(dT)-universal tag universal reverse transcription primer is used for reverse transcription reaction, and finally the first strand of cDNA corresponding to the miRNA is generated. This kit uses a special optimized premix to combine Poly(A) tailing and reverse transcription into one step, which simplifies the operation steps and improves the efficiency of Poly(A) tailing and reverse transcription. The kit can effectively prepare the first strand of cDNA corresponding to miRNA from 20pg-2ug of total RNA. The cDNA synthesized once can detect multiple microRNAs, saving samples and costs.

Note: This kit must be used in conjunction with the miRNA Enhanced Fluorescence Quantification Detection Kit (MR0201)

Operation steps

I. Poly (A) tailing and reverse transcription reaction (first-strand synthesis) at the miRNA 3' end

1. Thaw 2 × miRNA RT Reaction Mix and mix well. Put the miRT Enzyme Mix on ice for later use. Add the following reagents to a total volume of 20μl (add miRNA RT Enzyme Mix last).
 

Components	Volume	Final Concentration
Total RNA	x μl	Up to 2μg
2 × miRT Reaction Mix	10 μl	1 ×
miRNA RT Enzyme Mix	2 μl (see details in Note)	-
RNase free H2O to final volume	20 μl	-

Note:

(1) miRNA RT Enzyme Mix is ​​very viscous, and the solution is easily adsorbed to the tube wall and the outside of the pipette tip, resulting in loss. Please centrifuge before use and avoid loss due to adhesion to the outer wall of the pipette tip. The enzyme in the Enzyme Mix is ​​in excess, and even if 1.8μl is used each time, it will not affect the effect.

(2) The total RNA used in the reaction must include small molecule RNA (miRNA). Enriched miRNA can also be used in this process. Pure miRNA cannot be directly quantified by spectrophotometer. It is recommended to add 2μl ~5μl directly. The amount of addition can be determined according to the abundance of the target miRNA, but for low-abundance miRNA samples (such as serum plasma extracts), a maximum volume of 8μl can be added directly.

2. Gently mix the above-prepared reaction solution with a pipette, centrifuge briefly, and react at 42℃ for 60 min.

3. Heat at 85℃ for 5 seconds to inactivate E.coli Poly(A) Polymerase and Labscript Hˉ RTase. The synthesized cDNA reaction solution can be stored at -20℃; it can also be directly used for downstream PCR or fluorescence quantitative PCR detection.

2. Perform quantitative PCR according to the LABLEAD miRNA Enhanced Fluorescence Quantification Kit (Cat. No.: MR0201).

Note: When the cDNA template obtained according to the above steps is used for downstream PCR or fluorescence quantitative PCR detection, the usage amount can be selected according to the actual situation. For special detection systems, high-content cDNA templates are prone to non-specific amplification. The cDNA can be appropriately diluted (5-10 times or 100 times) according to the abundance of the detected miRNA before use. If non-specific amplification bands are found, or the melting curve shows non-specific amplification, it often indicates that the cDNA template is excessive. You can try to dilute the above cDNA template dozens to hundreds or even thousands of times before use.