Nov 21, 2022

Public workspacePAXgene Processing by RNA Extraction

DOI
dx.doi.org/10.17504/protocols.io.kxygxpnywl8j/v1
  • Clemens Scherzer1,2,
  • Bradley Hyman3,2,
  • Charles Jennings1,2
  • 1Brigham and Women's Hospital;
  • 2Harvard Medical School;
  • 3Massachusetts General Hospital
Daniel El Kodsi
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DOI: dx.doi.org/10.17504/protocols.io.kxygxpnywl8j/v1
Protocol Citation: Clemens Scherzer, Bradley Hyman, Charles Jennings 2022. PAXgene Processing by RNA Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxpnywl8j/v1
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: February 18, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 47408
Keywords: paxgene, processing, RNA, extraction, ASAPCRN
Abstract
This protocol explains the Standard Operating Protocol for performing Paxgene Processing by RNA extraction.
Guidelines
PROTOCOLS NO LONGER IN USE

PURIFICATION OF MIRNA FROM RNA EXTRACTION FLOW-THROUGH
MiRNA Extraction
MATERIALS:
  1. RNeasy MinElute Cleanup Kit (QIAgen, Cat #74204)
  2. 15 mL falcon tubes (BD, Cat #352097)
  3. 5 mL syringe reservoirs (Applied Biosystems, Cat #4344437)
  4. Freezerbondz labels (Fischer Scientific, Cat #22500521)
PROTOCOL:
  1. Keep the flow-through from steps 18 and 20 in RNA extraction protocol.
  2. The miRNA protocol should be performed during or after completion of RNA purification to ensure consistency with previous samples. Keep flow-through covered until beginning miRNA purification.
  3. Take RNeasy MinElute spin columns out from 4°C and equilibrate to room temperature (25°C) for several minutes.
  4. Prepare the Qiavac 24 Plus Vacuum Manifold with one RNeasy spin column per subject. Use disposable adaptors to prevent contamination.
  5. Attach a 5 mL syringe reservoir to RNeasy spin column to accommodate for the volume of miRNA flow through.
  6. Combine flow-through for each subject into a 15 ml tube.
  7. Add 700 µl of 100% ethanol for each 500 µl of flow through. Mix thoroughly by vortexing.
  8. Pour the entire flow-through into the 5 mL syringe reservoir and turn on vacuum.
  9. When all flow-through has been passed through the spin column, turn off the vacuum.
  10. Add 500 µl of Buffer RPE into the spin column and turn on the vacuum until the buffer has passed.
  11. Remove spin columns from the Quivac and place in a 2 ml processing tube.
  12. Add 500 µl of 80% ethanol to the spin column and centrifuge at 13,000 rpm for 2 min at 25°C.
  13. Place spin column in a new 2ml processing tube and discard old tube. (Remove spin column carefully so that the column does not touch the flow -through.)
  14. Open lid of the spin column and centrifuge at 13,000 rpm for 5 min at 25°C.
  15. Place the spin column in a low-retention 1.5 ml microcentrifuge tube.
  16. Pipette 15 µl of RNase-free water directly on the spin column membrane. Close lid gently and sit for 1 min, then to elute, centrifuge at 13,000 rpm for 1 min at 25°C.
  17. Label miRNA tube (miRNA).
  18. MiRNA is logged and stored the same way as RNA.


FREEZER STORAGE

Freezers are divided into 4 shelves, with 6 racks per shelf, and 24 boxes that can be held in each shelf. In total, 576 boxes, approximately 2,160 sample sets, can be stored in one -80°C freezer. The first three shelves are designated by visit number: Shelves A1-6 (top shelf) house samples from enrollment visits, shelves B1-6 (2nd shelf) house samples from the 1st year follow-up, and shelves C1-6 (3rd shelf) house samples from the 2nd year follow-up. Shelves D1-6 contain packed red blood cell tubes (PRBC), DNA, and RNA, extracted from blood as described in the protocols above. CSF is designated between two freezers in selected racks. Freezer storage and transactions of samples are recorded in the Freezerworks Inventory software.
Materials
MATERIALS:
  1. PAXgene tubes from BLOOD DRAW (Two 2.5 cc PAXgene™ Blood RNA Tubes (VWR Ref# 77776-026))
  2. PreAnalyix PAXgene kit (Qiagen/BD Company, Cat # 762164)
  3. Freezerbondz labels (Fischer Scientific, Cat # 22500521)
  4. 1.5 mL low-retention microcentrifuge tubes (Fisher Scientific, Cat #02-681-320)
Safety warnings
Please refer to Safety Data Sheets (SDS) for health and environmental hazards. Gain all required consent and experimental approvals before beginning any procedures.
Before start
***NOTE: Please see Appendix in guidelines for miRNA Extraction Protocol. MiRNA Extraction was discontinued 5/1/2019

RNA Q/C GOALS
1. Nanodrop Concentration Assay
a. 260/280 > 2.0
b. 94 µg/mL (30 µg total) of RNA/subject
2. Agilent 2100 Bioanalyzer Assay
a. 28S/18S peaks = 1.0 -2.0
b. RNA Integrity Number (RIN) > 7.3
PAXgene Processing BY RNA Extraction
PAXgene Processing BY RNA Extraction
1d 0h 16m 10s
1d 0h 16m 10s
Place all 2 PAXgene tubes in the Temperature4 °C fridge from blood draw if RNA extraction is NOT to be done the next day.

Incubate PAXgene Blood RNA Tubes for Duration24:00:00 at TemperatureRoom temperature (25°C) after blood collection or removal from 4°C before processing.

1d
Incubation
Prepare 55°C and 65°C heating blocks.
Centrifuge tubes at Centrifigation4000 rpm, 25°C, 00:12:00 .

Centrifigation
Remove supernatant and add Amount4 mL RNase-free water to each tube.

Close tube using a fresh secondary Hemogard closure.
Vortex Duration00:00:10 until pellet is dissolved.

10s
Centrifuge at Centrifigation4000 rpm, 25°C, 00:10:00 . Discard supernatant.
Centrifigation
Add Amount350 µL Buffer BR 1 , cap and vortex until the pellet is visibly dissolved.

Pipette sample into a 1.5 ml microcentrifuge tube.
Pipetting
Add Amount300 µL Buffer BR2 and Amount40 µL proteinase K .

Pipetting
Mix by votexing briefly, then incubate for a total of 10 minutes on a heating block at Temperature55 °C as follows: incubate for Duration00:05:00 , briefly vortex, incubate Duration00:05:00 .
Note
Do not mix Buffer BR2 and proteinase K together before adding to samples!


10m
Incubation
Mix
Pipette lysate directly onto the membrane of a PAXgene Shredder spin column (lilac-colored) placed in a 2 ml processing tube.
Pipetting
Centrifuge at Centrifigation13000 rpm, 25°C, 00:03:00 .

Centrifigation
Carefully pipette supernatant of the flow-through to a 1.5 microcentrifuge tube without disturbing pellet.
Pipetting
Add Amount350 µL 100% ethanol , mix by votexing, and centrifuge Centrifigation1000 x g, 00:00:02 , 1-2sec at 500-1000 x g to remove drops from inside of the tube lid.
Note
Do not centrifuge any longer to avoid pelleting of nucleic acids.

Centrifigation
Mix
Pipette Amount700 µL sample into the PAXgene RNA spin column (pink) placed in a 2 ml processing tube, and centrifuge at Centrifigation10000 rpm, 25°C, 00:01:00 .

Centrifigation
Pipetting
Place the spin column in a new 2 ml processing tube.
Pipette the remaining sample from step 16 into the spin column and centrifuge at Centrifigation10000 rpm, 25°C, 00:01:00 .
Centrifigation
Pipetting
Place spin column in a new 2 ml processing tube.
Add Amount350 µL Buffer BR3 into the spin column and centrifuge at Centrifigation10000 rpm, 25°C, 00:01:00 .

Centrifigation
Pipetting
Place spin column in a new 2 ml processing tube and discard old tube.
Add Amount10 µL DNase I stock solution to Amount70 µL Buffer RDD in a 1.5 ml microcentrifuge tube and mix by gently flicking the tube (do not vortex). Centrifuge briefly to collect residual liquid on sides.

Centrifigation
Mix
Add Amount80 µL DNase I incubation mix directly onto the membrane of the spin column, and place on benchtop (20°C - 30°C, TemperatureRoom temperature ) for Duration00:05:00 .

5m
Incubation
Pipette Amount350 µL Buffer BR3 into the spin column and centrifuge at Centrifigation10000 rpm, 25°C, 00:01:00 .

Centrifigation
Pipetting
Place spin column in a new 2 ml processing tube and discard old tube.
Add Amount500 µL Buffer BR4 (diluted in 1:4 in 100% ethanol) into the spin column, and centrifuge at Centrifigation10000 rpm, 25°C, 00:01:00 .

Centrifigation
Pipetting
Place spin column in a new 2 ml processing tube and discard old tube.
Add another Amount500 µL Buffer BR4 to the spin column and centrifuge at Centrifigation10000 rpm, 25°C, 00:01:00 .
Centrifigation
Pipetting
Place spin column into a new 2 ml processing tube and discard old tube. Centrifuge at Centrifigation13000 rpm, 25°C, 00:03:00 .
Centrifigation
Place spin column into a new 1.5 ml microcentrifuge tube and discard old tube.
Add Amount41 µL Buffer BR5 directly onto spin column membrane and sit for Duration00:01:00 .

1m
Incubation
Pipetting
Centrifuge for Centrifigation10000 rpm, 25°C to elute the RNA.

Centrifigation
Repeat step 32 with Amount41 µL Buffer BR5 and the same microcentrifuge tube: sit for Duration00:01:00 . Centrifuge for Centrifigation10000 rpm, 25°C to elute the RNA.
1m
Incubation
Centrifigation
Pipetting
Combine all two elutes into one tube. Split volume in half between two tubes and label (RNA-01 and 02).
Place the aliquots in the Temperature65 °C heat block for Duration00:05:00 without shaking. After incubation, chill immediately TemperatureOn ice .

5m
Aliquot Amount3 µL RNA into a 1.5 mL tube for Nanodrop concentration assay.

Pipetting
Aliquot Amount2 µL RNA into a PCR tube for Agilent 2100 Bioanalyzer assay. (Store in Temperature-20 °C if not being assay immediately.)

Pipetting
Pause
RNA Sample Storage
RNA Sample Storage
Scan and position RNA in the Freezerworks Inventory Program.
Store in corresponding -80°C freezer.
Note
Split and store RNA-01 and RNA-02 aliquots in separate freezers in case of freezer failure.