Description
Product description
This product utilizes the poly(A) tailing method to synthesize the first-strand cDNA of miRNA. The enzymes and buffer system included in the product have been meticulously optimized to ensure that both the Poly(A) tailing at the 3' end of miRNA and the reverse transcription process can be carried out efficiently and simultaneously.
For downstream qPCR applications, this product requires only the design of specific forward primers. In combination with the universal reverse primer provided in the kit, it enables the detection of miRNA in the sample. Additionally, the kit includes universal forward and reverse primers for U6 that are applicable to human, rat, and mouse samples, allowing for the generation of good standard curves over a wide range.
This product is recommended for use with the miRNA qPCR Dye Premix (Cat#N132039) to achieve optimal experimental results.
Specifications
|
Catalog Number
|
N132068E
|
N132068S
|
|
Specifications
|
10 T
|
50 T
|
Components
|
Catalog Number
|
Component Name
|
N132068E
|
N132068S
|
|
N132068-A
|
miRNA RT Enzyme Mix
|
17.5 μL
|
87.5 μL
|
|
N132068-B
|
2×miRNA RT Buffer
|
50 μL
|
250 μL
|
|
N132068-C
|
RNase-free H2O
|
2 mL
|
10 mL
|
|
N132068-D
|
Universal Reverse Primer (10 μM)
|
800 μL
|
4 mL
|
|
N132068-E
|
U6 Forward Primer (10 μM)
|
100 μL
|
500 μL
|
|
N132068-F
|
U6 Reverse Primer (10 μM)
|
100 μL
|
500 μL
|
【Note】Component C of the 4×cDNA Synthesis SuperMix contains the gDNA Digester terminator.
Storage
Transport with ice packs. Store at -20℃. Valid for 12 months.
Notes
1. Before use, thaw the components completely and gently mix them before use.
2. During the experiment, use RNase-free consumables to avoid unnecessary losses that may affect the experimental results.
3. Avoid repeated freeze-thaw cycles of the product. When preparing the mixture, protect it from strong light.
4. For your safety and health, please wear a lab coat and disposable gloves when operating.
5. For Research Use Only.
Instructions
1. Reaction System Preparation
Thaw Component A (miRNA RT Enzyme Mix) and Component B (2× miRNA RT Buffer) at room temperature. After thawing, gently invert to mix and place on ice. Then, prepare the reaction system according to the table below:
|
Component
|
Volume（μL）
|
Final concentration
|
|
2×miRNA RT buffer
|
5 μL
|
1×
|
|
miRNA RT enzyme mix
|
1.75 μL
|
-
|
|
RNA
|
-
|
X
|
|
RNase-free H2O
|
Up to 10
|
-
|
【Note】For RNA samples, the concentration range of total RNA and extracted miRNA should be between 10 pg and 2 μg. The minimum number of miRNA copies that can be synthesized is 60 copies. The input volume should not exceed 3.25 μL.
2. Reverse Transcription Procedure
Gently mix the prepared reaction mixture using a pipette. Perform the reverse transcription reaction for miRNA according to the program in the table below:
|
Temperature
|
Time
|
Remarks
|
|
37℃
|
50 min
|
miRNA Poly(A) Tailing and Reverse Transcription Process
|
|
85℃
|
5 sec
|
The process of enzyme inactivation
|
【Note】 The reverse transcription product can be directly used for downstream qPCR detection. To avoid inhibition of the qPCR reaction by the reverse transcription system, the product can be diluted 10-1000 times before use. If the downstream experiment will not be performed immediately, the product can be stored at -20°C. For long-term storage, it is recommended to aliquot and store at -80°C to avoid repeated freeze-thaw cycles.
