Magnetic Bead-based Blood Total RNA Extraction Kit, 50 T (for Nucleic Acid Extraction)

Product Information

Product Description/Introduction

MagBind Blood Total RNA Extraction Kit uses superparamagnetic magnetic beads designed for nucleic acid extraction and purification to extract total RNA from fresh blood samples. This kit can obtain high purity RNA by lysing leukocytes to release the nucleic acid therein after removing red blood cells with specially optimized lysing solution, then adsorbing RNA under certain conditions by superparamagnetic magnetic beads with separating effect, and releasing the adsorbed RNA by the magnetic beads after changing the conditions. The extracted RNA can be directly used in various molecular biology experiments such as RT-PCR, RT-qPCR, Northern Blot, in vitro translation, RNase protection analysis and molecular cloning.

Storage and Shipping Conditions

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

Product Contents

Before starting (please read carefully)

1. If a precipitate has formed in Buffer BRL, please dissolve it by heating at 37 and use it after it returns to room temperature.

2. Add DTT Solution to Buffer BRL before use to a final concentration of 4%, i.e., 1 mL Buffer BRL to 40 µL DTT Solution. This lysate is best prepared ready to use, and Buffer BRL added to DTT Solution can be  stored at 4°C for 1 month.

3. Before use, add 18 mL of absolute ethanol to Buffer RW1, add 80 mL of absolute ethanol to Buffer RW2 and mix well separately.

4. Magnetic stands are required but not supplied in this kit.

Assay Protocol / Procedures

1. Dilution of 10×Red cell Lysis Buffer: dilute to 1×Red cell Lysis Buffer with RNase-Free ddH2O. (for example, take 140 μL of 10×Red cell Lysis Buffer if the volume of the blood sample to be treated is 200 μL).

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

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

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.

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

Note: Please do not aspirate the leukocyte precipitate.

5. Add 1× Red cell Lysis Buffer of 2 times the volume of the blood sample to the leukocyte precipitate ( e.g., if the blood sample volume is 200 µL, then 400 µL of 1×Red cell Lysis Buffer should be added) to suspend the leukocyte precipitate.

6. Centrifuge at 3,000 rpm for 10 min at 4°C to completely remove the supernatant. Do not aspirate the leukocyte precipitate 

7. Add Buffer BRL suspension leukocyte precipitate according to blood sample volume (ensure that DTT solution is added before use). If the volumn of blood sample is less than 0.5 mL, 400 μL of Buffer BRL is added; if the volumn of blood sample is 0.5-1.5 mL, add 600 μL of Buffer BRL.

8. Add 2/3 volume of isopropanol to the centrifuge tube, mix upside and down. And then add 20 μL of SweMag Beads to the mixture (SweMag Beads must be fully resuspended and evenly distributed prior to use), and mix well with pipette.

9. Stand at room temperature for 10 minutes and mix well with pipette 3-5 times during placement.

10. Place the centrifuge tube on a magnetic stand for 30 seconds. Then slowly turn the centrifuge tube up and down several times along with the magnetic stand to wash down the residual magnetic beads on the wall of the tube, and then aspirate and discard the supernatant when it is clear.

11. Add 500 µL of Buffer RW1 and pipet up and down gently to resuspend the magnetic beads. And then place the tube on a magnetic stand for 30 seconds. Then slowly turn the centrifuge tube up and down several times along with the magnetic stand to wash down the residual magnetic beads on the wall of the tube, and then aspirate and discard the supernatant when it is clear.

12. Add 500 µL of Buffer RW2, remove the magnetic stand , pipet up and down gently to resuspend the magnetic beads. Then place the tube on a magnetic stand for 30 seconds. Then slowly turn the centrifuge tube up and down several times along with the magnetic stand to wash down the residual magnetic beads on the wall of the tube, and then aspirate and discard the supernatant when it is clear.

13. Remove the magnetic stand for DNase I digestion:

a. Prepare DNase I working solution: Take 10 μL of 10×DNase Buffer, 10 μL of DNase, 80 μL of Nuclease-free Water and mix well in Nuclease-free centrifuge tube.

b. Add 100 µL of DNase I working solution to the tube containing the magnetic beads, and pipet up and down gently to resuspend the magnetic beads. Stand at room temperature for 15 minutes and mix well with every 5 min during incubation.

c. After DNase I digestion, add 600 µL of Buffer RW2 and pipet up and down gently to resuspend the magnetic beads. And then place the tube on a magnetic stand for 30 seconds. Then slowly turn the centrifuge tube up and down several times along with the magnetic stand to wash down the residual magnetic beads on the wall of the tube, and then aspirate and discard the supernatant when it is clear.

14. Repeat step 12.

15. Open the centrifuge tube lid, leave it at room temperature for 5-10 minutes to ensure complete evaporation of residual ethanol. (Do not allow them to dry out to avoid affecting the nucleic acid yield.)

16. Remove the magnetic stand, add 3050 μL of Nuclease-free Water and pipet up and down gently to resuspend the magnetic beads, stand at room temperature for 5 minutes.

17. Place the tube on a magnetic stand to collect the beads against the side of the tube, then aspirate the supernatant into a new Nuclease-free centrifuge tube to obtain high purity RNA.

Note

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), and the amount of blood used can be proportionally reduced if the blood has a high leukocyte content.

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

4. Magnetic bead suspension should avoid freezing operation during storage; magnetic beads are easy to settle, and should be sufficiently shaken to keep them in uniform suspension before use.

5. Before adding the magnetic beads, other reagents need to be mixed well.

6. The ethanol should be completely volatilized before eluting RNA to avoid residual ethanol affecting downstream experiments.

7. Do not dry the beads for a long time as this may affect the RNA elution efficiency.

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

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

For Research Use Only!

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