Blood Total RNA Extraction Kit

 

Product Name	Cat. No.	Spec.
Blood Total RNA Extraction Kit	G3636-50T	50 T

 

 

This kit is suitable for the extraction of total RNA from fresh blood samples. The new centrifugal column with unique lysis buffer can be used to efficiently recover high-purity blood total RNA without the need for phenol, chloroform and other reagents, and can rapidly extract total RNA from less than 1.5 mL of whole blood and effectively remove impurity proteins in only 1 hour. The extracted RNA can be directly used in various molecular biology experiments such as RT-PCR, RT-qPCR, Northern hybridization, in vitro translation, RNase protection analysis and molecular cloning.

 

 

DNase,DTT Solution are shipped with wet ice, stored at -20℃; Other reagents are shipped and stored at room temperature; valid for 12 months.

 

 

Component Number	Component	G3636-50T
G3636-1	10×Red cell Lysis Buffer	60 mL
G3636-2	Buffer RL	30 mL
G3636-3	Buffer RW1	12 mL (18 mL of anhydrous ethanol added before use)
G3636-4	Buffer RW2	20 mL (80 mL of anhydrous ethanol added before use)
G3636-5	DNase	250 μL
G3636-6	10×DNase Buffer	500 μL
G3636-7	DTT Solution	1.2 mL
G3636-8	Nuclease-free Water	12 mL
G3636-9	gDNA Eraser Spin Columns	50 sets
G3636-10	RNA Spin Columns	50 sets
Manual		One copy

 

 

1. If a precipitate has formed in Buffer RL, warm it at 37°C until the precipitate has fully dissolved and use it after it returns to room temperature.

2. Before operation, add DTT Solution to Buffer RL to a final concentration of 4%, i.e., 40 µL of DTT Solution to 1 mL of Buffer RL. This lysate is best used as is, and Buffer RL with DTT Solution can be left at 4°C for up to one month.

3. Before first use, add 18 mL of absolute ethanol to Buffer RW1, add 72 mL of absolute ethanol to Buffer RW2.

4. Prepare 70% ethanol with DEPC water before use.

 

 

1. Assay Protocol / Procedures

2. Dilution of 10×Red cell Lysis Buffer: dilute to 1×Red cell Lysis Buffer with Nuclease-free Water. (for example, take 140 μL of 10×Red cell Lysis Buffer if the volume of the blood sample to be treated is 200 μL).

3. Add 5 volumes of 1×Red cell Lysis Buffer to 1 volume of blood sample and mix well. (for example, add 1mL of 1×Red cell Lysis Buffer if the volume of the blood sample is 200 μL).

4. Incubate on ice for 15 min and mix upside and down for 2-3 times during incubation.

5. Note: During incubation, the mixture will become translucent, indicating red blood cells are completely lysed. Depending on the degree of lysis of the blood sample, the incubation time can be extended to 20 min.

6. Centrifuge at 3,000 rpm for 10 min at 4°C to completely remove the supernatant.

7. Note: Please do not aspirate the leukocyte pellet.

1. Add 1× Red Cell Lysis Buffer to 2 times the volume of blood sample to the leukocyte pellet (e.g., when the volume of blood sample is 200 μL, 400 μL of 1× Red Cell Lysis Buffer needs to be added) to suspend the leukocyte pellet).

2. Centrifuge at 3,000 rpm for 10 min at 4°C to completely remove the supernatant. Please do not aspirate the leukocyte pellet.

3. Resuspend leukocyte pellet by adding Buffer RL (ensure that DTT Solution is added before use). If the volumn of blood sample is less than 0.5 mL, 400 μL of Buffer RL is added; if the volumn of blood sample is 0.5-1.5 mL, add 600 μL of Buffer RL.

2. Transfer the above solution to gDNA Eraser Spin Column, centrifuge at 12,000 rpm for 2 minute at room temperature. Discard the gDNA Eraser Spin Column, keep the flow-through in the collection tube.

3. Add equal volume of 70% ethanol (any visible precipitate that may form after adding 70% ethanol) to the flow-through in the collection tube and pipet up and down to mix well.

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

5. Centrifuge at 12,000 × rpm for 30 s at room temperature. Discard the flow-through, and reinsert the RNA Spin Column in the same collection tube.

6. Add 500 μL Buffer RW1 to the RNA Spin Columns Centrifuge at 12,000 rpm for 30 seconds at room temperature, and discard the flow-through. And reinsert the RNA Spin Column in the same collection tube.

7. Add 500 μL Buffer RW2 to the the RNA Spin Columns (Buffer RW2 is added along the wall of the RNA Spin Column to help flush the residual salt). Centrifuge at 12,000 rpm for 30 seconds at room temperature, and discard the flow-through. And then, reinsert the RNA Spin Column in the same collection tube.

8. (Optional) DNase Digestion:

a. Prepare DNase working solution: Mix 5 µL of 10× DNase Buffer, 5 µL of DNase, and 40 µL of Nuclease-free Water in a Nuclease-free centrifuge tube.

b. Pipet all 50 μL of the DNase mixture onto the center of the RNA Spin Column membrane. Incubate at room temperature for 15 minutes.

9. Add 600 μL Buffer RW2 to the RNA Spin Columns. Centrifuge at 12,000 rpm for 30 seconds at room temperature, and discard the flow-through. And then, reinsert the RNA Spin Column in the same collection tube.

11. Repeat Step 13.

12. Centrifuge the Column at 12,000 rpm for 2 minutes to Remove residual liquid.

13. Discard the flow-through and the Collection Tube and insert the RNA Spin Column into a new 1.5 mL Nuclease-free centrifuge tube. Incubate at room temperature for 3-5 minutes to completely evaporate residual ethanol in RNA Spin Column.

14. Add 30-50 μL Nuclease-Free Water to the membrane 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, add the first eluate back into the RNA Spin Column, stand it for 5 minutes at room temperature and centrifuge at 12,000 rpm for 2 minutes at room temperature, and collect the RNA again.

 

 

1. Please read the Product Manual carefully before use.

2. The maximum sample throughput of this kit is 1.5 mL (WBC count 1×107), which can be proportionally reduced if the blood is high in leukocytes.

3. This kit is suitable for blood RNA purification preserved by conventional anticoagulants (including heparin sodium, EDTA, sodium citrate, etc.).

4. Wear a lab coat, disposable gloves, a mask, avoid talking, and use Nuclease-free tips and centrifuge tubes.

5. Specialized lab benches and electrophoresis equipment for RNA manipulation should be used.

 

For Research Use Only!