TITLE: PAXgene Processing by RNA Extraction
AUTHORS: Clemens Scherzer, Bradley Hyman, Charles Jennings

[DESCRIPTION]
This protocol explains the Standard Operating Protocol for performing Paxgene Processing by RNA extraction.

[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

[GUIDELINES]
PROTOCOLS NO LONGER IN USE

PURIFICATION OF MIRNA FROM RNA EXTRACTION FLOW-THROUGH 
MiRNA Extraction 
MATERIALS: 
RNeasy MinElute Cleanup Kit (QIAgen, Cat #74204) 
15 mL falcon tubes (BD, Cat #352097) 
5 mL syringe reservoirs (Applied Biosystems, Cat #4344437) 
Freezerbondz labels (Fischer Scientific, Cat #22500521) 
PROTOCOL:
Keep the flow-through from steps 18 and 20 in RNA extraction protocol. 
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. 
Take RNeasy MinElute spin columns out from 4°C and equilibrate to room temperature (25°C) for several minutes. 
Prepare the Qiavac 24 Plus Vacuum Manifold with one RNeasy spin column per subject. Use disposable adaptors to prevent contamination. 
Attach a 5 mL syringe reservoir to RNeasy spin column to accommodate for the volume of miRNA flow through. 
Combine flow-through for each subject into a 15 ml tube. 
Add 700 µl of 100% ethanol for each 500 µl of flow through. Mix thoroughly by vortexing. 
Pour the entire flow-through into the 5 mL syringe reservoir and turn on vacuum. 
When all flow-through has been passed through the spin column, turn off the vacuum. 
Add 500 µl of Buffer RPE into the spin column and turn on the vacuum until the buffer has passed. 
Remove spin columns from the Quivac and place in a 2 ml processing tube. 
Add 500 µl of 80% ethanol to the spin column and centrifuge at 13,000 rpm for 2 min at 25°C. 
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.) 
Open lid of the spin column and centrifuge at 13,000 rpm for 5 min at 25°C. 
Place the spin column in a low-retention 1.5 ml microcentrifuge tube. 
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. 
Label miRNA tube (miRNA). 
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.

[STEPS]
SECTION: PAXgene Processing BY RNA Extraction
1. Place all 2 PAXgene tubes in the 4 °C fridge from blood draw if RNA extraction is NOT to be done the next day.

SECTION: PAXgene Processing BY RNA Extraction
2. Incubate PAXgene Blood RNA Tubes for 1440 min at 4 Room temperature (25°C) after blood collection or removal from 4°C before processing.

SECTION: PAXgene Processing BY RNA Extraction
3. Prepare 55°C and 65°C heating blocks.
SECTION: PAXgene Processing BY RNA Extraction
4. Centrifuge tubes at 4000 rpm, 12 min, 25 °C.

SECTION: PAXgene Processing BY RNA Extraction
5. Remove supernatant and add 4 mL to each tube.

SECTION: PAXgene Processing BY RNA Extraction
6. Close tube using a fresh secondary Hemogard closure.
SECTION: PAXgene Processing BY RNA Extraction
7. Vortex 10 s until pellet is dissolved.

SECTION: PAXgene Processing BY RNA Extraction
8. Centrifuge at 4000 rpm, 10 min, 25 °C. Discard supernatant.
SECTION: PAXgene Processing BY RNA Extraction
9. Add 350 µL, cap and vortex until the pellet is visibly dissolved.

SECTION: PAXgene Processing BY RNA Extraction
10. Pipette sample into a 1.5 ml microcentrifuge tube.
SECTION: PAXgene Processing BY RNA Extraction
11. Add 300 µL and 40 µL.

SECTION: PAXgene Processing BY RNA Extraction
12. Mix by votexing briefly, then incubate for a total of 10 minutes on a heating block at 55 °C as follows: incubate for 5 min, briefly vortex, incubate 5 min. 
 


SECTION: PAXgene Processing BY RNA Extraction
13. Pipette lysate directly onto the membrane of a PAXgene Shredder spin column (lilac-colored) placed in a 2 ml processing tube.
SECTION: PAXgene Processing BY RNA Extraction
14. Centrifuge at 13000 rpm, 3 min, 25 °C.

SECTION: PAXgene Processing BY RNA Extraction
15. Carefully pipette supernatant of the flow-through to a 1.5 microcentrifuge tube without disturbing pellet.
SECTION: PAXgene Processing BY RNA Extraction
16. Add 350 µL, mix by votexing, and centrifuge 1000 x g, 2 s to remove drops from inside of the tube lid. 
 

SECTION: PAXgene Processing BY RNA Extraction
17. Pipette 700 µL into the PAXgene RNA spin column (pink) placed in a 2 ml processing tube, and centrifuge at 10000 rpm, 1 min, 25 °C. 

SECTION: PAXgene Processing BY RNA Extraction
18. Place the spin column in a new 2 ml processing tube.
SECTION: PAXgene Processing BY RNA Extraction
19. Pipette the remaining sample from step 16 into the spin column and centrifuge at 10000 rpm, 1 min, 25 °C.
SECTION: PAXgene Processing BY RNA Extraction
20. Place spin column in a new 2 ml processing tube.
SECTION: PAXgene Processing BY RNA Extraction
21. Add 350 µL into the spin column and centrifuge at 10000 rpm, 1 min, 25 °C.

SECTION: PAXgene Processing BY RNA Extraction
22. Place spin column in a new 2 ml processing tube and discard old tube.
SECTION: PAXgene Processing BY RNA Extraction
23. Add 10 µL to 70 µL 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.

SECTION: PAXgene Processing BY RNA Extraction
24. Add 80 µL directly onto the membrane of the spin column, and place on benchtop (20°C - 30°C, 55 Room temperature) for 5 min.

SECTION: PAXgene Processing BY RNA Extraction
25. Pipette 350 µL into the spin column and centrifuge at 10000 rpm, 1 min, 25 °C.

SECTION: PAXgene Processing BY RNA Extraction
26. Place spin column in a new 2 ml processing tube and discard old tube.
SECTION: PAXgene Processing BY RNA Extraction
27. Add 500 µL (diluted in 1:4 in 100% ethanol) into the spin column, and centrifuge at 10000 rpm, 1 min, 25 °C.

SECTION: PAXgene Processing BY RNA Extraction
28. Place spin column in a new 2 ml processing tube and discard old tube.
SECTION: PAXgene Processing BY RNA Extraction
29. Add another 500 µL to the spin column and centrifuge at 10000 rpm, 1 min, 25 °C.
SECTION: PAXgene Processing BY RNA Extraction
30. Place spin column into a new 2 ml processing tube and discard old tube. Centrifuge at 13000 rpm, 3 min, 25 °C.
SECTION: PAXgene Processing BY RNA Extraction
31. Place spin column into a new 1.5 ml microcentrifuge tube and discard old tube.
SECTION: PAXgene Processing BY RNA Extraction
32. Add 41 µL directly onto spin column membrane and sit for 1 min.

SECTION: PAXgene Processing BY RNA Extraction
33. Centrifuge for 10000 rpm, 25 °C to elute the RNA.

SECTION: PAXgene Processing BY RNA Extraction
34. Repeat step 32 with 41 µL and the same microcentrifuge tube: sit for 1 min. Centrifuge for 10000 rpm, 25 °C to elute the RNA.
SECTION: PAXgene Processing BY RNA Extraction
35. Combine all two elutes into one tube. Split volume in half between two tubes and label (RNA-01 and 02).
SECTION: PAXgene Processing BY RNA Extraction
36. Place the aliquots in the 65 °C heat block for 5 min without shaking. After incubation, chill immediately on ice.

SECTION: PAXgene Processing BY RNA Extraction
37. Aliquot 3 µL into a 1.5 mL tube for Nanodrop concentration assay.

SECTION: PAXgene Processing BY RNA Extraction
38. Aliquot 2 µL into a PCR tube for Agilent 2100 Bioanalyzer assay. (Store in -20 °C if not being assay immediately.)

SECTION: RNA Sample Storage
39. Scan and position RNA in the Freezerworks Inventory Program.
SECTION: RNA Sample Storage
40. Store in corresponding -80°C freezer.