KR211-miRcute Plus miRNA First-Strand cDNA Kit-171207

miRcute Plus miRNA FirstStrand cDNA Kit
For first-strand cDNA synthesis from miRNA

miRcute Plus miRNA First-Strand cDNA Kit
Cat. No. 4992786/4992909

Storage
Upon receiving this product, please immediately store it at -30~-15°C.
When it is taken out from -30~-15°C, keep the miRNA RT enzyme mix on
ice. Thaw the 2× miRNA RT reaction buffer and mix it upside down slightly
after thawing. The solution should be used when it’s completely uniform.
The product can be stored for 1 year at -30~-15°C.

Introduction
miRcute Plus miRNA First-strand cDNA Kit adds Poly(A) to the 3’-terminal
of miRNA and synthesizes the first-strand cDNA based on Poly(A) modified
miRNA through oligo(dT)-universal tag primed reverse transcription.
miRcute Plus miRNA First-strand cDNA Kit has only two components,
which simplifies the operation procedure and reduces the possibility of
operation error. miRNA RT Enzyme Mix in this product contains the E.coli
Poly(A) Polymerase, Reverse transcriptase and RNase Inhibitor. The E.coli
Poly(A) Polymerase not only has higher polymerase efficiency and A tailing
efficiency, but also can specifically recognize single strand miRNA, avoiding
the further reverse transcription reaction of microRNA precursors which
has double strand structures. Reverse transcriptase has been modified at
the molecule level to remove the activity of RNase H, which increases the
affinity of RNA template, making the reverse transcriptional reaction of
miRNA a higher efficiency and sensitivity.
The 2× miRNA RT Reaction Buffer contains all the materials and primers
used in miRNAs A-tailing and reverse transcription, which could guarantee
the A-tailing and reverse transcription reaction of miRNAs are carried on at
the same time and make the process more efficiently.
Note: This kit has to be used with miRcute Plus miRNA qPCR Kit (SYBR
Green) (Cat. no. 4992887/4992779).

Kit Highlights

1. Combined the A-tailing reaction and reverse transcription in one tube,
   which will save operation time and reduce operation steps.
2. The E.coliPoly(A) Polymerase not only has higher polymerase efficiency
   and A tailing efficiency, but also can specifically recognize single strand
   miRNA, avoiding the further reverse transcription reaction of microRNA
   precursors which has double strand structures.
3. The detection rate of low abundance miRNA is greatly improved by
   simplified operation procedure.
4. This kit can be used for reverse transcriptional reaction of miRNA
   extracted from almost all materials. The dosage range of the templates
   can reach 20 pg-2 μg (mass range) and 10 fM-100 pM (concentration
   range).

Protocol

1. Fully thaw RNA template, 2× miRNA RT Reaction Buffer and RNase-Free
   ddH2
   O. Centrifuge transiently and put all of them on ice.
2. Prepare a reaction solution according to the following table (all the steps
   should be operated on ice).

3. Mix gently by pipetting the reaction solution and briefly centrifuge to
   remove drops from the wall of the tube. Incubate at 42°C for 60 min and
   then 95°C for 3 min. The details are shown in the following table:

4. The reaction solution including cDNA products can be stored at -20°C or
   used in downstream quantitative PCR directly.

Important Notes

1. Always replace to new gloves. Because the skin often contains bacteria,
   which may cause RNase pollution.
2. Use RNase-Free plastic products and tips to avoid cross-contamination.
3. RNA will not be degraded by RNase in the lysate. However, RNase-Free
   plastic and glassware should be used for the following operations after
   extraction. Bake the glassware at 150°C for 4 hours, or soak the plastic
   ware in 0.5 M NaOH for 10 minutes, then wash thoroughly with RNaseFree ddH2
   O, and then subject to autoclave to remove RNase.
4. Prepare the solution with RNase-Free ddH2O (add the ddH2O into a
   clean glass bottle, add DEPC to the final concentration of 0.1% (V/V),
   incubate overnight, then autoclave).

KR211-miRcute Plus miRNA First-Strand cDNA Kit-171207

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This product is for scientific research use only. Do not use in medicine, clinical treatment, food or cosmetics.

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