Composition(30 tests/kits): Cell washing solution(R1):1ML
RNA extraction solution(R2): 100 μL
RNA purification solution(R3): 100 μL
DNA Enzyme terminating solution(R4): 100 μL
Composition(30 tests/kits): Cell washing solution(R1): 3.5 mL
RNA extraction solution(R2): 350 μL
RNA purification solution(R3): 350 μL
DNA Enzyme terminating solution(R4): 350 μL
Purpose: The kit can be used to extract, enrich and purify RNA from single cell and microscale cells. The extracted RNA can be used to detect the expression of the target gene directly by RT-PCR. It can also be used to synthesize the first-strand cDNA (complementary DNA)(<=100ng) by reverse transcription. The synthesized cDNA can be used for PCR amplification of the target gene, further construction of the cDNA library, or single-cell RNA sequencing analysis.
Sample requirements: The sample cells should be uncontaminated and with normal morphological characteristics of cell structure. The kit is mainly suitable for single oocyte, sperm cell, oosperm and embryo cell. It is also used for cast-off cells in urine, sputum or secretion, and single cell extracted from tissue biopsy, surgical specimens or cell culture.
Using protocol: 1. Cell cleaning: In the ultra clean workbench, the suspension cells were cleaned for 3 times with R1 solution which was pre-cooled at 4 degree.
2. Acquisition of single cell and extraction of RNA
(1) Under the microscope, a cell was sucked into a 0.2 mL PCR centrifugal tube containing 2μL R2 solution by oral suction.
Note: The key to obtaining a single intact cell is to operate quickly and accurately in order to minimize expression changes in cell and avoid contamination of exogenous RNA due to external stimuli and prolonged operation.
(2) Centrifuge for 15 seconds at 4 ˚C and 100g .
(3) Freeze the centrifuge tube in liquid nitrogen and thaw at room temperature. Repeat this three times.
3. RNA Purification
(1) Add 0.5 μL R3 solution to the tube. Put it in PCR instrument and incubate for 30 minutes at 37 ˚C.
(2) Add 0.5 μL R4 solution to the tube. Put it in PCR instrument and incubate for 2 minutes at 95 ˚C. Then put it on ice immediately for rapid cooling 2 minutes.
(3) Centrifuge for 1 minutes at 4 ˚C and 100g.
(4) The purified RNA was stored at -80˚C or directly for PCR amplification.
Attentions: 1. This product is used for research only.
2. RNA extraction should be standardized to avoid RNA contamination or degradation caused by external environment.
3.Aliquot the product for storage and avoid freezing and thawing repeatedly.