9. Blood Total RNA Extraction Kit, 50T $250

Description

Product Description

This kit is designed for the extraction of total RNA from fresh blood samples. By utilizing a novel centrifugal adsorption column combined with unique lysis reagents, high-purity total RNA can be efficiently recovered from blood without the need for reagents such as phenol or chloroform. With a processing time of only 1 hour, the kit enables rapid extraction of total RNA from less than 1.5 mL of whole blood while effectively removing impurities and proteins. The extracted RNA can be directly used in various molecular biology experiments such as RT-PCR, RT-qPCR, Northern blotting, in vitro translation, RNase protection analysis, and molecular cloning.

Storage and Transportation

DNase, DTT Solution should be transported with wet ice and stored at -20°C. Other reagents can be transported at room temperature and stored at room temperature. The kit has a shelf life of 12 months.

Preparation before Use

1. If Buffer RL has precipitation, heat it at 37°C to dissolve and use it after it has returned to room temperature.
2. Prior to use, add DTT Solution to Buffer RL to achieve a final concentration of 4%. For example, add 40 µL of DTT Solution to 1 mL of Buffer RL. It is recommended to prepare the lysis buffer fresh and use it immediately. Buffer RL with added DTT Solution can be stored at 4°C for one month.
3. Before use, add 18 mL of ethanol to Buffer RW1 and 72 mL of ethanol to Buffer RW2.
4. Prepare 70% ethanol using DEPC water before use.

Procedure

1. Dilute 10× Red cell Lysis Buffer with nuclease-free water to obtain a 1× working solution (e.g., if the blood sample volume is 200 µL, dilute 140 µL of 10× Red cell Lysis Buffer).
2. Add 1 volume of blood sample to 5 volumes of 1× Red cell Lysis Buffer and mix thoroughly. For example, if the blood sample volume is 200 µL, add 1 mL of 1× Red cell Lysis Buffer.
3. Incubate on ice for 15 minutes, gently invert the tube 2-3 times during incubation. When the solution becomes translucent, it indicates complete lysis of red blood cells. Depending on the degree of blood sample lysis, the incubation time can be extended to 20 minutes.
4. Centrifuge at 3,000 rpm, 4°C for 10 minutes. Carefully remove the supernatant without disturbing the white cell pellet.
5. Add 2 times the volume of the blood sample of 1× Red cell Lysis Buffer to the white cell pellet (e.g., if the blood sample volume is 200 µL, add 400 µL of 1× Red cell Lysis Buffer) and resuspend the white cell pellet.
6. Centrifuge at 3,000 rpm, 4°C for 10 minutes. Carefully remove the supernatant without disturbing the white cell pellet.
7. Add Buffer RL to the resuspended white cell pellet according to the volume of the processed blood sample (ensure DTT Solution has been added). For blood sample volumes less than 0.5 mL, add 400 µL of Buffer RL. For blood sample volumes between 0.5 mL and 1.5 mL, add 600 µL of Buffer RL.
8. Transfer the resulting solution to a gDNA Eraser Spin Column, centrifuge at 12,000 rpm, room temperature for 2 minutes, discard the gDNA Eraser Spin Column, and collect the filtrate.
9. Add an equal volume of 70% ethanol to the filtrate (precipitation may occur at this stage) and mix by pipetting.
10. Transfer the mixture (including the precipitate) to an RNA Spin Column. If the volume exceeds 600 µL, add it in batches, but do not exceed 600 µL at a time.
11. Centrifuge at 12,000 rpm, room temperature for 30 seconds, discard the waste liquid, and place the RNA Spin Column back into the Collection Tube.
12. Add 500 μL of Buffer RW1 to the RNA Spin Column, centrifuge at 12,000 rpm, room temperature for 30 seconds, discard the waste liquid, and place the RNA Spin Column back into the Collection Tube.
13. Add 500 μL of Buffer RW2 to the RNA Spin Column (add Buffer RW2 along the wall of the RNA Spin Column to help wash off salt residues on the column wall), centrifuge at 12,000 rpm, room temperature for 30 seconds, discard the waste liquid, and place the RNA Spin Column back into the Collection Tube.
14. DNase digestion (optional):

a) Prepare the DNase reaction mixture by mixing 5 µL of 10× DNase Buffer, 5 µL of DNase, and 40 µL of nuclease-free water in a nuclease-free centrifuge tube.

b) Add 50 μL of the DNase reaction mixture to the center of the RNA Spin Column and incubate at room temperature for 15 minutes.

c) Add 600 μL of Buffer RW2 to the RNA Spin Column, centrifuge at 12,000 rpm, room temperature for 30 seconds, discard the waste liquid, and place the RNA Spin Column back into the Collection Tube.

15. Repeat step 13.
16. Centrifuge at 12,000 rpm, room temperature for 2 minutes to remove residual liquid.
17. Place the RNA Spin Column in a new 1.5 mL nuclease-free centrifuge tube and let it sit at room temperature for 3-5 minutes to allow complete evaporation of ethanol from the RNA Spin Column.
18. Add 30-50 μL of nuclease-free water to the center of the RNA Spin Column, let it sit at room temperature for 5 minutes, centrifuge at 12,000 rpm, room temperature for 2 minutes to elute the RNA. If a higher concentration of RNA is desired, the first eluate can be re-applied to the RNA Spin Column, let it sit at room temperature for 5 minutes, centrifuge at 12,000 rpm, room temperature for 2 minutes, and collect the RNA again.

Notes

1. Carefully read the product manual before starting the procedure.
2. The maximum sample processing volume for this kit is 1.5 mL (approximately 1×107 white blood cells). If the white blood cell content in the blood is high, reduce the blood volume proportionally.
3. This kit is suitable for extracting RNA from blood preserved with common anticoagulants such as sodium heparin, EDTA, and sodium citrate.
4. Wear a lab coat, disposable gloves, and a mask during the experiment. Avoid speaking and use nuclease-free tips and centrifuge tubes.
5. Use a dedicated RNA workstation and electrophoresis equipment.

This product is for research use only and is not intended for clinical diagnostics.