miRNA First Strand cDNA Synthesis KitThis kit uses the method of adding a poly (A) tail at the 3 'end of miRNA to make miRNA have a Poly (A) tail, and then uses Oligo (dT) - Tag primers for reverse transcription reaction to finally synthesize the first strand cDNA corresponding to miRNA. The reaction process is as follows:
Reaction process of miRNA cDNA first strand synthesis kit
Two step operation, first perform Poly (A) tail addition, and then perform reverse transcription reaction.
Only 1 is needed μ The total RNA of g.
The obtained cDNA can be used to detect various miRNAs, which not only reduces errors and saves samples, but also achieves high throughput detection.
Compatible with miRNA fluorescence quantitative PCR detection kit.
Good specificity, unique miRNA primers and reaction systems avoid non-specific amplification of Pre miRNA.
Component specifications
E. Coli Poly (A) polymerase (5U/ μ L) 10 μ L
ten × Poly (A) polymerase reaction solution 50 μ L
ATP solution, 1mM 50 μ L
Oligo (dT) - Tag primer (0.5 μ G/ μ L) 25 μ L
MMLV reverse transcription (including RI) 50 μ L
four × RT Buffer (including dNTP) 125 μ L
RNase free water 1mL
Storage: -20 ℃
1、 Apply Poly (A) treatment to the 3 'end of miRNA
Add the following reagents to the reaction tube of pre cooled RNase Free on ice to a total volume of 20 μ L. Note that E. coli Poly (A) polymerase needs to be added at the end.
Ingredient dosage
Self prepared Total RNA (see note) 1 μ G
ten × Poly (A) polymerase reaction solution 2 μ L
ATP solution, 1mM 2 μ L
RNase Free water replenishment to 20 μ L
E. Coli Poly (A) polymerase (5U/ μ L) 0.4 μ L (2U)
Note: The Total RNA used in the reaction must contain miRNA. MiRNA can also be directly used (recommended dosage is 2-5 μ L. It needs to be determined based on the abundance of the target miRNA.
Gently mix the prepared reaction solution with a pipette, centrifuge briefly, and react at 37 ℃ for 60 minutes. The obtained reaction solution can be directly used for downstream experiments (note: only 2 μ L is directly used for subsequent RT reactions, with more than 2 μ For L, it is necessary to first precipitate with ethanol sodium acetate, then dissolve the added miRNA in water, and then take the required amount for RT. If not precipitated, the residual poly (A) polymerase reaction solution introduced will interfere with the RT reaction, and can also be temporarily stored at -20 ℃. If long-term storage is required, it is recommended to store at -80 ℃.
2、 Reverse transcription reaction of miRNA with Poly (A) tail
Set 20 according to the table below μ RT reaction of L system:
Ingredient dosage
Poly (A) reaction product (from previous step) 2 μ L
RNas
