Animal Tissue& Cell RNA Extraction Kit

 

Product Name	Cat.No.	Spec.
Animal Tissue& Cell RNA Extraction Kit	G3640-50T	50T

 

 

The kit is a broad-spectrum kit for the purification of RNA from animal tissues and cultured cells by column method. The kit adopts a special lysis buffer system, without the need for organic reagents such as phenol, chloroform and other organic reagents extraction, and through the gDNA Eraser Spin Column (removal of genomic DNA) and RNA Spin Column (binding of RNA), respectively, can be quickly extracted from 5-20 mg of animal tissues, 106-107 freshly cultured cells of high purity RNA, and the whole process can be completed in only 30 min. The obtained RNA can be directly used in various molecular biology experiments such as Northern blotting, spot blotting, mRNA purification, in vitro translation, RNase protection assays, RT-PCR, Real Time RT-PCR and construction of cDNA libraries.

 

 

Proteinase K, DNase and DTT Solution should be shipped with wet ice, and stored at -20℃; Other reagents are shipped and stored at room temperature for up to 12 months.

 

 

Component Number	Component	G3640-50T
G3640-1	Buffer RL1	30 mL
G3640-2	Buffer RW1	12 mL(Add 18 mL of anhydrous ethanol before use)
G3640-3	Buffer RW2	20 mL(Add 80 mL of anhydrous ethanol before use)
G3640-4	DTT Solution	1.2 mL
G3640-5	Proteinase K	500 μL
G3640-6	DNase	250 μL
G3640-7	10×DNase Buffer	500 μL
G3640-8	Nuclease-free Water	12 mL
G3640-9	gDNA Eraser Spin Columns	50 set
G3640-10	RNA Spin Columns	50 set
Manual		One copy

 

 

1. If a precipitate appear in Buffer RL1, warm it at 37℃ to dissolve the precipitate. Use it after cooling down to room temperature.

2. Before operation, add appropriate volume of DTT Solution into Buffer RL1 to a final concentration of 4% ( Add 40 μL DTT Solution per 1 mL of Buffer RL1. It should be prepared immediately before use. This mixture can be stored at 4℃ for one month.)

3. Before using Buffer RW1 and RW2, please check if the specified amount of 100% ethanol is added.

4. Prepare 60% isopropanol (ready to use) in advance according to the amount of sample to be extracted, e.g., 4mL of Nuclease-free water (G4700 recommended) to 6mL of isopropanol.

5. Please pre-cool the grinder if grinding tissue with a grinder.

Initial addition of different samples and the usage of Buffer RL1

Initial addition of sample	Usage of Buffer RL1
Adherent cultured cells (the diameter of petri dish < 6 cm)	350 µL
Adherent cultured cells (the diameter of the petri dish is 6~10 cm)	600 µL
Suspension cultured cells < 5×106	350 µL
5×106~1×107 Suspension cultured cells	600 µL
5~20 mg animal tissue	500 µL

Table 1 Initial addition of different samples and the usage of Buffer RL1

 

 

1. Samples Preparation

a. Animal tissue: Transfer 2-20 mg of fresh or cryopreserved animal tissue or animal tissue preserved in RNAsolid Tissue RNA Stabilization Solution (recommend G3019) to a 1.5 mL Nuclease-free tube or special grinding tube with containing 2-3 3 mm grinding beads (recommend G0203-150G) and 500 uL Buffer RL1 (please confirm that DTT has been added before use). Thoroughly grind the tissue to homogenization (if the tissue is not thoroughly homogenized, it will affect the yield and quality of RNA) using a Tissue Homogenizer (KZ-5F-3D recommended) at low temperature, add 10 µL of Proteinase K, mix well, and then incubate at 56°C for 10-15 min and centrifuge at 12,000 rpm for 5 min at 4°C.

b. Transfer 2-20 mg of fresh or cryopreserved animal tissue or animal tissue preserved in RNAsolid Tissue RNA Stabilization Solution (recommend G3019) to a 1.5 mL Nuclease-free tube or special grinding tube with 500 uL Buffer RL1 (please confirm that DTT Solution has been added before use). Put the centrifuge tube in an ice bath and use an electric grinder to grind the tissue thoroughly to homogenate (if the tissue is not thoroughly homogenized, it will affect the yield and quality of RNA). Add 10 µL of Proteinase K, mix well, and then incubate at 56°C for 10-15 min and centrifuge at 12,000 rpm for 5 min at 4°C.

c. Transfer 2-20 mg of fresh or cryopreserved animal tissue or animal tissue preserved in RNAsolid Tissue RNA Stabilization Solution (recommend G3019) to a mortar pre-cooled with liquid nitrogen and grind the tissue with a pestle, adding liquid nitrogen continuously until it is powdered (if the tissue is not thoroughly homogenized, it will affect the yield and quality of RNA). Then transfer the ground powder sample to a 1.5 mL Nuclease-free centrifuge tube containing 500 µL Buffer RL1 (please confirm that DTT Solution has been added before use) and mix repeatedly. Add 10 µL of Proteinase K, mix well, and then incubate at 56°C for 10-15 min and centrifuge at 12,000 rpm for 5 min at 4°C.

d. Suspension cells: Transfer the suspension culture cells together with the culture medium into a 1.5 mL Nuclease-free centrifuge tube, centrifuge at 1,000 rpm for 5 min, collect the suspension culture cells (106-107), gently aspirate and discard the supernatant, add Buffer RL1 (recommended addition in Table 1) to the cell precipitate (please confirm that DTT Solution has been added before use), and pipette and blow evenly until there is no obvious precipitate. Then add 10 µL Proteinase K, mix well and incubate at 56℃ for 10-15 min, centrifuge at 12,000 rpm and 4℃ for 5 min.

e. Adherent cells: Remove the growth medium from the cells and then wash with 1×PBS buffer(pH 7.4). Add Buffer RL1 (recommended addition in Table 1) to the cultured cells (please confirm that DTT Solution has been added before use), gently blow with a pipette to make the cells fall off completely, and transfer the lysate containing the cells to 1.5 mL Nuclease-free centrifuge tubes. Add 10 µL of Proteinase K, mix well, and then incubate at 56°C for 10-15 min and centrifuge at 12,000 rpm for 5 min at 4°C.

2. Transfer the supernatant of the animal tissue or cell lysate from the first step into a gDNA Eraser Spin Column (to avoid aspiration of precipitate) and centrifuge at 12,000 rpm for 1 min at room temperature.

3. Dicard the gDNA Eraser Spin Column, retain the filtrate in Collection Tube.

4. Add an equal volume of 60% isopropanol to the flow-through in the collection tube from the above step (precipitation may occur at this point). Mix well with a liquid pipette.

5. Transfer all of the mixture (including precipitate) into the RNA Spin Column, adding no more than 600 µL at a time, or in batches if more than 600 µL.

6. Centrifuge at 12,000 rpm for 30 seconds at room temperature. Discard the waste liquid, and reinsert the RNA Spin Column in the same collection tube.

7. Add 500 μL Buffer RW1 to the RNA Spin Column. Centrifuge at 12,000 rpm for 30 seconds at room temperature, and discard thewaste liquid.

2. Add 600 μL Buffer RW2 to the RNA Spin Column (Please add Buffer RW2 along the RNA Spin Column pipe wall to help flush the residual salt on the pipe wall). Centrifuge at 12,000 rpm for 30 seconds at room temperature, and discard the waste liquid.

8. (Optional) DNase Digestion: The reagents in this kit and the gDNA Eraser Spin Column effectively remove most of the genomic DNA, and the extracted RNA contains very little genomic DNA. For some tissues with high genomic DNA content (e.g., liver, kidney, spleen, etc.) or for subsequent experiments with more stringent requirements for RNA purity, DNase digestion can be selectively performed on the RNA Spin Column adsorption column membrane.

a) Preparation of DNase reaction solution: 5 µL of 10× DNase Buffer, 5 µL of DNase, and 40 µL of Nuclease-free Water were mixed in a new 1.5 mL Nuclease-free centrifuge tube.

b) Add 50 μL of DNase reaction solution to the center of the RNA Spin Column and let it stand at room temperature for 15 min.

c) Add 500 μL of Buffer RW2 to the RNA Spin Column, centrifuge at 12,000 rpm for 30 s at room temperature, discard the waste liquid, and return the column to the collection tube.

9. Repeat step 8.

10. Place the RNA Spin Column in a Collection Tube and centrifuge at 12,000 rpm for 2 min at room temperature to remove residual liquid.

11. The RNA Spin Column was placed in a new 1.5 mL Nuclease-free centrifuge tube and left at room temperature for 3-5 min to allow complete volatilization of the ethanol remaining in the RNA Spin Column.

11. Add 50-100 μL Nuclease-Free Water to the center of the RNA Spin Column. Incubate at room temperature for 5 minutes. Centrifuge the Column for 2 minutes at 12,000 rpm at room temperature to elute the RNA. To obtain a higher concentration of RNA, you can also add the first eluate back into the RNA Spin Column, let it stand for 5 min at room temperature, centrifuge at 12,000 rpm for 2 min at room temperature, and collect the RNA again.

 

 

1. Please read the Product Manual carefully before use.

2. Try to use freshly cultured cells or tissue as much as possible. RNA solid tissue RNA stabilization solution (recommend Cat. NO.: G3019) is recommended for rapid RNase inhibition and stable preservation of RNA from tissue samples if cells or tissues require long-term preservation.

3. For your safety and health, please wear safety glasses, gloves, or protective clothing. Avoid speech, use Nuclease-free tips, centrifuge tubes to avoid cross-contamination.

4. A special operating bench for RNA extraction with electrophoresis equipment should be used.

Supplementary Table

The amount of total RNA extracted from different tissues and cells by this kit is shown in the table below. The RNA yield extracted from tissues or cells is related to samples freshness or growth status.

 

The following table is for reference only.

Sample species	Sample names	RNA yield
Animal tissues	Mouse liver	30-45 μg/10 mg
	Mouse spleen	20-30 μg/10 mg
	Mouse lung	10-20 μg/10 mg
	Mouse kidney	20-30 μg/10 mg
	Mouse heart	5-10 μg/10 mg
	Mouse thymus	10-20 μg/10 mg
	Mouse brain	5-10 μg/10 mg
	Mouse pancreas	5-15 μg/10 mg
	Rat muscle	2-4 μg/10 mg
	Mung bean leaves	15-20 μg/100 mg
Cells	Hela	8-15 μg/106 cells

 

For Research Use Only!