Mar 17, 2026

Public workspacePreAnalytiX PAXgene Blood RNA Kit

This protocol is a draft, published without a DOI.
  • Kristine Wylie1,
  • Hunter Olson1,
  • Jane Schrimpf1,
  • Madison Eschbach1
  • 1Washington University
  • HVP Pregnancy Postpartum
Hunter Olson
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Protocol Citation: Kristine Wylie, Hunter Olson, Jane Schrimpf, Madison Eschbach 2026. PreAnalytiX PAXgene Blood RNA Kit. protocols.io https://protocols.io/view/preanalytix-paxgene-blood-rna-kit-jwagcpabx
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 17, 2026
Last Modified: March 17, 2026
Protocol Integer ID: 313384
Funders Acknowledgements:
NIH NCCIH
Grant ID: U01 AT012970
Abstract
PreAnalytiX PAXgene Blood RNA Kit
Materials
  • Microcentrifuge
  • Pipettors (50–200 µL; 100–1000 µL)
  • Vortex
  • Shaking heat block
Troubleshooting
Before start
  • Wear a lab coat, face mask, eye protection and nitrile gloves while extracting RNA from whole blood. Working behind a plexiglass shield or wearing a face shield is optional.
  • All steps done at room temperature unless specified.
Reagent and Tube Preparation
Before using a kit, prepare DNase I stock solution by dissolving the solid DNase I in 550 µL of the RNase-Free Water provided with the kit. Take care that no DNase I is lost when opening the vial. DO NOT VORTX THE RECONSTITUTED DNASE I. Reconstituted DNase I can be stored at 4°C for up to 6 weeks. For longer term storage, divide DNase I into single-use aliquots and store at -20°C for up to 9 months. Thawed aliquots can be stored at 4°C for up to 6 weeks. Do not refreeze aliquots after thawing.
Before using a kit, prepare a working concentration of the Buffer BR4 by adding 4 volumes of 95% ethanol (provided by the user) as indicated on the bottle containing the concentrated Buffer BR4. The label on the bottle has a box that should be checked to indicate that the ethanol has been added.
Remove the PAXgene Blood RNA Tubes from the -80°C freezer and incubate overnight at room temperature to ensure complete lysis of blood cells.
Buffer BR2 may form a precipitate upon storage. If necessary, warm to 37°C to dissolve.
Lysate Preparation
Centrifuge microcentrifuge tube containing 1 mL of whole blood for 10 minutes at 3,000 x g.
Discard supernatant in biohazardous waste container by pipetting. Add 425 µL of RNase-Free Water to the pellet and resuspend pellet by pipetting up and down several times. Vortex until the pellet is completely dissolved.
Centrifuge the tube for 10 minutes at 3,000 x g. During this spin, heat shaking heat block to 55°C. Discard the entire supernatant in biohazardous waste container.
Add 350 µL of Buffer BR1 to the pellet and resuspend pellet by pipetting up and down several times. Vortex to completely dissolve the pellet.
Add 300 µL Buffer BR2 and 40 µL Proteinase K to the sample.
Note: Buffer BR2 contains guanidine thiocyanate, which is not compatible with bleach.
Mix by vortexing for 5 seconds and incubate for 10 min. at 55°C using a shaker heat block at 1300 rpm.
Pipette the lysate directly into a PAXgene Shredder spin column (lilac) placed in a 2 mL processing tube and centrifuge for 3 minutes at 20,000 x g.
Transfer the entire supernatant of the flowthrough fraction to a fresh 1.5 mL microfuge tube without disturbing the pellet.
Add 350 µL ethanol (96-100%). Mix by vortexing and spin briefly to remove any drops of liquid from the lid.
Note: The length of the spin should not exceed 1-2 seconds, as this may result in pelleting of the nucleic acids and reduce yields of the RNA
Binding RNA to Column
Pipette 700 µL of the sample into the PAXgene RNA spin column (red) and centrifuge for 1 minute at 20,000 x g. Place the spin column into a new 2 mL processing tube and discard flowthrough in a chemical waste container. From this point on, discard all waste in this chemical waste container.
Pipette the remaining sample into the PAXgene RNA spin column (red) and centrifuge for 1 minute at 20,000 x g. Place the spin column into a new 2 mL processing tube and discard flowthrough.
Pipette 350 µL Buffer BR3 into the spin column. Centrifuge for 1 minute at 20,000 x g. Place the spin column into a new 2 mL processing tube and discard flowthrough.
Note: Buffer BR3 contains guanidine thiocyanate, which is not compatible with bleach.
For every on-column DNase I reaction to be performed, prepare a mix of 10 µL of DNase I and 70 µL of Buffer RDD in a 1.7 mL microfuge tube. Mix gently by pipetting up and down a few times. DO NOT VORTEX.
Apply 80 µL of DNase I solution mix to each column and incubate the column assembly at 20 – 30°C for 15 minutes.
Apply 350 µL of Buffer BR3 to the column and centrifuge for 1 minute at 20,000 x g. Place the spin column into a new 2 mL processing tube and discard flowthrough.
Note: Buffer BR3 contains guanidine thiocyanate, which is not compatible with bleach.
Apply 500 µL of Buffer BR4 to the column and centrifuge for 1 minute at 20,000 x g. Place the spin column into a new 2 mL processing tube and discard flowthrough.
Apply another 500 µL of Buffer BR4 to the column and centrifuge for 3 minutes at 20,000 x g.
Discard the processing tube and flowthrough and place column in a new 2 mL processing tube. Centrifuge for 1 minute at 20,000 x g.
Discard the processing tube and flowthrough and place column in a 1.7 mL microfuge tube.
RNA Elution
Pipette 40 µL Buffer BR5 directly onto the column membrane. Centrifuge for 1 minute at 20,000 x g to elute the RNA.
Repeat the dilution step above, using another 40 µL Buffer BR5 and the same microfuge tube.
Quality Control
Determine RNA concentration using Qubit RNA assay kit.
The purified RNA sample should be stored at –80°C.