| Product Description | This kit is used to extract miRNAs from various animal tissues, plant tissues and cells. The extracted miRNA molecule is complete and high purity, which is suitable for various molecular biology experiments such as Northern blot, real timepcr, miRNA microarray chip, in situ hybridization, RNase protection assay, etc Composition: 
Scope of application： Nucleic acid extraction and purification Instruction：
一、Experimental preparation：
1.All reagents were prepared with DEPC-treated solvents. Please use RNase-free tip and centrifuge tube to avoid RNA degradation by RNase during extraction.
2.70 % ethanol, -20C pre-cooling.
二、Operational procedure：
There is a slight difference in the operation of miRNA extraction from different samples. The specific steps are as follows :
【 Extraction of miRNA from animal tissues】
1.Take 20-40 mg tissue, transfer to a pre-cooled mortar, and add liquid nitrogen to grind into powder.
Please click below to describe the amount of organization used :
①RNA-rich tissue ( e.g. liver ) : no more than 30 mg
②Tissues with low RNA content ( e.g., muscle ) : no more than 100 mg
③When the amount of tissue used was less than 20 mg : the amount of R-I, R-II and isopropanol used was halved.
④When the amount of tissue used was more than 40 mg : the use of R-I, R-II and isopropanol increased proportionally.
2.Add 400 ul Buffer R-I, repeatedly aspirate 8-10 times with a syringe equipped with a 21-25 needle, and transfer to a 1.5 m | centrifuge tube ( provided in the kit ).
3.Add 150 μl BufferR-1l, swirl for 15-30 s, centrifuge at 12,000 X g for 5 min. [ Centrifugation at 4 °C is recommended ]
4.Take the supernatant to 1.5ml centrifuge tube, add 180 u anhydrous ethanol, mix evenly.
The preparation tube was placed in a 2 m | centrifuge tube ( provided in the kit ), the mixture in step 4 was transferred to the preparation tube, and 12,000 X g was centrifuged for 1 min. [ 1 Centrifugation at 4 °C is recommended ; 2 miRNA in the filtrate, pay attention to preserve the filtrate. ]
Abandon the preparation tube, add 500μl isopropanol to the filtrate, and mix evenly.
7.12,000Xg centrifuged for 10 min, discard the supernatant.
8.Add 700μl 70 % ethanol ( pre-cooled at -20 °C ), centrifuged at 12,000Xg for 5min.
The supernatant was discarded and dried at room temperature for 5-10 min.
10.70 ul Buffer TE ( nucdease-free ) or RNase-free water was added to the centrifuge tube to elute miRNA.
【 Extraction of miRNA from plant tissue 】
1.Take 30-150 mg tissue, transfer to a pre-cooled mortar, and add liquid nitrogen to grind into powder.
Please click below to describe the amount of organization used :
①Plant leaves : usually 10-80 mg
② Plant fiber tissue : usually 100-150 mg
③When the amount of plant leaf tissue was less than 30 mg : the amount of R-I, R-II and isopropyl alcohol used was halved.
④When the amount of plant leaf tissue was more than 80 mg : the use of R-I, R-II and isopropanol increased proportionally.
⑤When the amount of plant fiber tissue was more than 150 mg : the use of R-I, R-II and isopropanol increased proportionally.
2.Add 400 ul BufferR-I, use a syringe with a 21-25 needle to repeatedly suck 8-10 times, and transfer to a 1.5mI centrifuge tube ( provided in the kit )..
3.Add 150 ul Buffer R-1I, vortex oscillation 15-30 s, 12.000 x g centrifugation 5 min. [ Centrifugation at 4 °C is recommended ]
4.Take the supernatant to 1.5ml centrifuge tube, add 180 mountain anhydrous ethanol, mix evenly.
The preparation tube was placed in a 2 mI centrifuge tube ( provided in the kit ), the mixture in step 4 was transferred to the preparation tube, and 12.000 xg was centrifuged for 1 min. It is recommended to centrifuge at 4 °C ; 2 miRNA in the filtrate, pay attention to preserve the filtrate. ]
Abandon the preparation tube, add 500μl isopropanol to the filtrate, and mix evenly.
7.12,000xg high heart for 10 min, discard the supernatant.
8.Add 700 ul 70 % ethanol ( -20 °C precooling ), 12,000 xg centrifuge for 5 min.
9.The supernatant was discarded and dried at room temperature for 5-10 min.
10.70 ul Buffer TE ( nucdease-free ) or RNase-free water was added to the centrifuge tube to elute miRNA.
【miRNA extraction from cells】
Steps 1-3 According to the different ways of cell culture, two experimental methods, a or b, can be selected.
a. Suspension cultured animal cells or cell suspension obtained from petri dishes or culture flasks or freshly isolated animal tissue single cell suspension :
1a.Collect 2X 10 * -1X 10 ' cells, centrifuge 2,000Xg for 5 min, discard the supernatant ;
2a. Add 400 μl Buffer R-I, repeatedly draw 8-10 times with a syringe containing 21-25 needles, and transfer to a 1.5 mI centrifuge tube ( provided in the kit ) ;
3a. Add 150μl Buffer R1I, vortex oscillation 15-30s, 12.000Xg centrifugal 5min. [ build at 4 °C centrifugal ].
b. Cells cultured on 96-well L, 24-well, 12-well or 6-well plates :
Cells were collected from 96-well, 24-well, 12-well or 6-well culture plates, and the medium was discarded as much as possible, and 400 u / well Buffer R-I was added to each well, and the pipette gun was used to blow up and down 8-10 times ;
2b.Transfer the above cell suspension to a 1.5ml centrifuge tube ( provided in the kit ), and repeatedly draw 8-10 times with a syringe containing 21-25 needles ;
3b. Add 150 μl Bufflr R-II, swirl for 15-30 s, centrifuge for 5 min at 12,000 × g. [ Recommended at 4 °C ]
4.Take the supernatant to 1.5ml centrifuge tube, add 180 mountain anhydrous ethanol, mixing evenly.
5.The preparation tube was placed in a 2 ml centrifuge tube ( provided in the kit ), the mixture in step 4 was transferred to the preparation tube, and centrifuged at 12.000 Xg for 1 min. [ 1 Centrifugation at 4 °C is recommended ; 2 miRNA in the filtrate, pay attention to preserve the filtrate. ]
6.Abandon the preparation tube, add 500 u of isopropanol to the filtrate, and mix evenly.
7.12,000Xg high heart for 10 min, discard the supernatant.
8.Add 700μ70 % ethanol ( pre-cooled at − 20 °C ), centrifuged at 12,000 × g for 5 min.
9.Abandon the supernatant, dry at room temperature for 5 - 10 min.
10.70 ul Bufer TE ( nucdease-free ) or RNase-free water was added to the centrifuge tube to elute mRNA. 三、Flow chart 
Matters needing attention： Buffer R-I contains irritating compounds, when operating to wear latex gloves and glasses, to avoid contamination of the skin, eyes and clothes, be careful not to inhale the nose and mouth. If the skin, eyes, to immediately rinse with a lot of water or saline, if necessary, seek medical advice. |
|---|
L2325105