Products

PAXgene Blood miRNA Kit(SpinColumn)

Packing Specification：

For research use only. Not for use in medicine, clinical diagnosis, food, cosmetics or other applications.

Product Introduction:
This kit is designed for the isolation and purification of intracellular RNA and miRNA from whole blood stabilized in PAXgene Blood RNA Tubes. It eliminates the need for harmful components such as Trizol LS, phenol, and chloroform. After blood collection, PAXgene Blood RNA Tubes can stabilize samples at 18–25°C for up to 3 days, and at –20°C or –70°C for at least 60 months.

The PAXgene Blood RNA system includes PAXgene Blood RNA Tubes (supplied by BD, Catalog No. 762165) for blood collection, stabilization, and transportation, as well as the PAXgene Blood RNA Kit for separation and purification based on silica membrane technology and spin column method. Purification can be performed manually using a centrifuge or automatically on the QIAcube Fully Automated Nucleic Acid Purification Instrument. This product delivers excellent performance, ensuring highly reliable RNA purification.

Application Scope:
Compatible with PAXgene Blood RNA Tubes. Suitable for isolating and purifying intracellular RNA and miRNA from whole blood preserved in the collection tubes.

Operation Steps:
Pre-Experiment Preparation:
1.Preheat the incubator or water bath to 55°C in advance.
2.Prepare DNase I digestion working solution in advance according to the number of samples. The reaction system is as follows:

Note: DNase I is highly sensitive to physical denaturation. Do not vortex vigorously; mix gently by inverting the tube.

Operation Steps (10 mL Starting Volume):
1.Centrifuge the PAXgene Blood RNA Tube (BRT) at 3,000-5,000 x g for 10 minutes using a round-bottom horizontal rotor centrifuge. Tubes may rupture if other types of rotors are used.
2.Aspirate and discard the supernatant. If pouring off the supernatant, operate carefully to avoid losing the cell pellet at the bottom. Blot residual whole blood lysate from the tube mouth with clean absorbent paper.
3.Add 4 mL of RNase-free H₂O and vortex to fully resuspend the cell pellet.
4.Centrifuge the PAXgene Blood RNA Tube (BRT) at 3,000-5,000 x g for 10 minutes using a round-bottom horizontal rotor centrifuge. Carefully pour off or aspirate the supernatant. Incomplete removal of the supernatant will inhibit lysis, dilute the lysate, and thus affect the conditions for RNA binding to the membrane.
5.Add 350 μL of Resuspension Buffer (BR1). Resuspend the cell pellet at the bottom of the tube by pipetting with a 1 mL blue pipette tip, then vortex to fully resuspend the cell particles.
6.Transfer the sample to a new 1.5 mL EP tube. Add 300 μL of Binding Buffer (BR2) and 40 μL of Proteinase K (PK) solution, then vortex for 5 seconds to mix thoroughly.
7.Incubate at 55-60°C with shaking at 400~1,400 rpm for 10-12 minutes using a shaking incubator. If no shaking incubator is available, gently invert the microcentrifuge tube 2-3 times during incubation.
8.Pipette the entire lysate (approximately 700 μL) into a Shredder Spin Column (yellow gasket). Centrifuge at 13,000 rpm for 2 minutes. RNA will be present in the filtrate of the collection tube.
9.Carefully aspirate the supernatant from the bottom of the collection tube, avoiding the precipitate. Transfer the filtrate to a new 1.5 mL EP tube, add an equal volume of isopropanol (approximately 700 μL), and mix gently by vortexing.
10.Pipette 700 μL of the sample into an RNA Spin Column (white gasket). Centrifuge at 13,000 rpm for 1 minute and discard the filtrate.
11.Pipette the remaining sample into the same RNA Spin Column. Centrifuge at 13,000 rpm for 1 minute and discard the filtrate.
12.Add 350 μL of Wash Solution 1 (BR3) (ensure absolute ethanol has been added before use). Centrifuge at 13,000 rpm for 1 minute and discard the filtrate.
13.Add 100 μL of the pre-prepared DNase I digestion working solution to the center of the adsorption membrane. Incubate at room temperature for 15 minutes.
14.Add 350 μL of Wash Solution 1 (BR3). Centrifuge at 13,000 rpm for 1 minute and discard the filtrate.
15.Add 500 μL of Wash Solution 2 (BR4) (ensure absolute ethanol has been added before use). Centrifuge at 13,000 rpm for 1 minute and discard the filtrate.
16.Add another 500 μL of Wash Solution 2 (BR4). Centrifuge at 13,000 rpm for 1 minute and discard the filtrate.
17.Place the RNA Spin Column back into the empty collection tube. Centrifuge at 13,000 rpm for 2 minutes to completely remove residual washing solution (residual ethanol in the washing solution may inhibit downstream reactions).
18.Transfer the RNA Spin Column to a new RNase-free microcentrifuge tube. Add 30-50 μL of RNFW (RNase-free H₂O) (heating in a 70-90°C water bath can improve yield) to the center of the adsorption membrane according to the expected RNA yield. Incubate at room temperature for 1 minute, then centrifuge at 13,000 rpm for 1 minute.
Remarks:For higher miRNA concentration: Reapply the first eluate to the adsorption column and repeat Step 18.For expected RNA yield >30 μg: Add another 30-50 μL of RNase-free water and repeat Step 18, then combine the two eluates.Eluting twice yields higher concentration RNA. Combining eluates from two separate elutions increases RNA yield by 15-30% compared to a single elution but results in lower concentration. Choose the method based on experimental needs.

Operation Steps (4 mL Starting Volume):
This protocol is intended for users without a round-bottom horizontal rotor centrifuge, or those who need to preserve samples for total RNA extraction or other applications.
1.Take two 2 mL centrifuge tubes and add 2 mL of whole blood sample from the PAXgene Blood RNA Tube (BRT) to each. Centrifuge at 13,000 rpm for 2 minutes using a high-speed benchtop centrifuge.
2.Carefully pour off the supernatant, avoiding the cell pellet at the bottom. Blot residual whole blood lysate from the tube mouth with clean absorbent paper.
3.Add 1 mL of RNase-free H₂O to each tube. Resuspend the cell pellet fully by pipetting with a 1 mL blue pipette tip, then combine the resuspended cells from both tubes into one tube.
4.Centrifuge at 13,000 rpm for 1 minute. Carefully pour off or aspirate the supernatant. Incomplete removal of the supernatant will inhibit lysis, dilute the lysate, and thus affect the conditions for RNA binding to the membrane.
5.Add 350 μL of Resuspension Buffer (BR1). Resuspend the cell pellet at the bottom of the tube by pipetting with a 1 mL blue pipette tip, then vortex to fully resuspend the cell particles.
6.Add 300 μL of Binding Buffer (BR2) and 20-40 μL of Proteinase K solution, then vortex for 5 seconds to mix thoroughly.
7.Incubate at 55-60°C with shaking at 400~1,400 rpm for 10-12 minutes using a shaking incubator. If no shaking incubator is available, gently invert the microcentrifuge tube 2-3 times during incubation.
8.Pipette the entire lysate (approximately 700 μL) into a Shredder Spin Column (yellow gasket). Centrifuge at 13,000 rpm for 2 minutes. RNA will be present in the filtrate of the collection tube.
9.Carefully aspirate the supernatant from the bottom of the collection tube, avoiding the precipitate. Transfer the filtrate to a new 1.5 mL microcentrifuge tube, add an equal volume of isopropanol (approximately 700 μL), and mix gently by vortexing.
10.Pipette 700 μL of the mixture into an RNA Spin Column (white gasket). Centrifuge at 13,000 rpm for 1 minute and discard the filtrate.
11.Pipette the remaining mixture into the same RNA Spin Column. Centrifuge at 13,000 rpm for 1 minute and discard the filtrate.
12.Add 350 μL of Wash Solution 1 (BR3) (ensure absolute ethanol has been added before use). Centrifuge at 13,000 rpm for 1 minute and discard the filtrate.
13.Add 100 μL of the pre-prepared DNase I digestion working solution to the center of the adsorption membrane. Incubate at room temperature for 15 minutes.
14.Add 350 μL of Wash Solution 1 (BR3). Centrifuge at 13,000 rpm for 1 minute and discard the filtrate.
15.Add 500 μL of Wash Solution 2 (BR4) (ensure absolute ethanol has been added before use). Centrifuge at 13,000 rpm for 1 minute and discard the filtrate.
16.Add another 500 μL of Wash Solution 2 (BR4). Centrifuge at 13,000 rpm for 1 minute and discard the filtrate.
17.Place the RNA Spin Column back into the empty collection tube. Centrifuge at 13,000 rpm for 2 minutes to completely remove residual washing solution (residual ethanol in the washing solution may inhibit downstream reactions).
18.Transfer the RNA Spin Column to a new RNase-free microcentrifuge tube. Add 30-50 μL of RNFW (RNase-free H₂O) (heating in a 70-90°C water bath can improve yield) to the center of the adsorption membrane according to the expected miRNA yield. Incubate at room temperature for 1 minute, then centrifuge at 13,000 rpm for 1 minute.
Remarks:For higher RNA concentration: Reapply the first eluate to the adsorption column and repeat Step 18.For expected RNA yield >30 μg: Add another 30-50 μL of RNase-free water and repeat Step 18, then combine the two eluates.Eluting twice yields higher concentration RNA. Combining eluates from two separate elutions increases RNA yield by 15-30% compared to a single elution but results in lower concentration. Choose the method based on experimental needs.