详情
Tailing miRNA First-Strand Synthesis Kit
CAT: MF***1
price：$**5 for *5T (negotiable if bulk purchase)
Storage temperature: **0℃
Product composition
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Component
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MF***1
|
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miRNA RT Enzyme Mix
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*0 ul
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|
2 × miRT Reaction Mix
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2*0 ul
|
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RNase free H2O
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1 ml
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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
*0pg*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 (MR***1)
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 *0μl (add miRNA RT Enzyme Mix last).
|
Components
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Volume
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Final Concentration
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Total RNA
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x μl
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Up to 2μg
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2 × miRT Reaction Mix
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*0 μl
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1 ×
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miRNA RT Enzyme Mix
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2 μl
(see details in Note)
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-
|
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RNase free H2O to final volume
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*0 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 *2℃ for *0 min.
3. Heat at *5℃ for 5 seconds to inactivate E.coli Poly(A)
Polymerase and Labscript Hˉ RTase. The synthesized cDNA reaction
solution can be stored at **0℃; 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.: MR***1).
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 (***0 times or **0 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.
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China |
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