TITLE: B2- BLOOD PROCESSING
AUTHORS: REDI-NET Consortium

[DESCRIPTION]
This protocol details blood processing.

[BEFORE_START]
BEFORE START
 

Pre-cool the Bullet Blender by adding dry ice into the cooling compartment and running the cooling program. 
Clean the work surfaces with RNaseZap, then wipe the surfaces with 70% molecular biology grade ethanol to remove additional contaminants.
Transfer 0.1 mm zirconium oxide beads (2 spoons, Appendix 2) to Thermo Scientific 1.5 ml Screw Cap Micro Tubes.
Buffer ATL may form precipitates upon storage. If necessary, warm to 56 °C until the precipitates have fully dissolved. Prepare buffer ATL-DX: add 100 μl Reagent DX to 15 ml Buffer ATL. If smaller amounts are needed, transfer 1.5 mL of Buffer ATL into a sterile 2 ml vial and add 10 µL Reagent DX. Mix well, after addition of Reagent DX. After preparation, the mixture is stable for 6 months at Room temperature (15-25°C).
For the first time use of IndiMag pathogen kit, add 100% ethanol to Buffer AW1 and AW2, and add 100% isopropanol to ACB as indicated on the bottles.
MagAttract Suspension G from IndiMag pathogen kit needs to be vortexed thoroughly for 3 min (before first use) or 1 minute (before subsequent uses) to ensure that the magnetic silica particles are fully resuspended. 
Aliquot nuclease-free water in big bottle into a few 15 ml tubes for preparing TNA elution in KingFisher Flex or KingFisher Duo Prime to avoid cross-contamination.

 







[GUIDELINES]
OBJECTIVE 

To outline procedures for total nucleic acid extraction from blood samples in various formats (whole blood, plasma, serum, buffy coat and dried blood spot on FTA cards).

SUMMARY/SCOPE

The overarching aim of the REDI-NET is to develop a collaborative laboratory network between domestic and international partnering institutions to address disease surveillance needs in order to effectively detect, predict and contain potentially emergent zoonosis. This SOP provides guidance on procedures for total nucleic acid extraction from blood samples to provide materials for downstream library preparation and sequencing for pathogen detection.

MAINTENANCE OF EQUIPMENT

Caution on RNA handling

RNases are very stable and difficult to inactivate, and only minute amounts are sufficient to destroy RNA. 
Care should be taken to avoid inadvertently introducing RNases into the samples during or after the purification procedure. 
Sample handling and extraction should be performed under an extraction hood and respecting Good Laboratory Practices. 
Use filter tips all the time.

Storage of the buffers from IndiMag Pathogen Kit

Proteinase K is stable for at least 1 year after delivery when stored at Room temperature (15-25°C). To store for more than 1 year or if ambient temperature often exceeds 25°C, storage at 2-8 °C is recommended. Do not add Proteinase K directly to the Buffer VXL mixture! This can cause clogs or precipitates.
Precipitation may form after storage at low temperature or prolonged storage. To dissolve precipitate, incubate Buffer VXL or ACB for 30 min at 37 °C, with occasional shaking.
Reconstituted Buffer AW1 can be stored at Room temperature (15-25°C) for up to 1 year. Mix well after adding Ethanol.
Buffer AVE is RNase-free upon delivery. It contains sodium azide, an antimicrobial agent that prevents growth of RNase-producing organisms. However, as this buffer does not contain any RNase degrading chemicals, it will not actively inhibit RNases introduced by inappropriate handling. When handling Buffer AVE, take extreme care to avoid contamination with RNases. Follow general precautions for working with RNA, such as frequent change of gloves and keeping tubes closed whenever possible.

QUALITY CONTROL

This SOP is reviewed by the applicable supervisor annually or as required in order to maintain its relevance.

APPENDICES


APPENDIX 1. WORKFLOW OF BLOOD SAMPLE PROCESSING
 
APPENDIX 2. MEASURING SPOON FOR 0.1 MM BEATING BEADS 

The spoon (Next Advance, MSP01-RNA) is used for 0.1 mm beating beads measurement. One scoop equals to 100 µL.
 
APPENDIX 4. EXPECTED OUTCOMES 

 








[STEPS]
SECTION: 1. WHOLE BLOOD AND BUFFY COAT LYSIS
1. 
 
 

SECTION: 1. WHOLE BLOOD AND BUFFY COAT LYSIS
2. Add 200 µL sterile 1x PBS to a 200 µL whole blood or buffy coat sample. 
 


SECTION: 1. WHOLE BLOOD AND BUFFY COAT LYSIS
4. Include a positive control for each batch of samples: transfer 37.5 µL ZymoBIOMICS Microbial Community Standard Material, 100 µL EBV, and 100 µL ZIKV standard into a tube from step 3 of Before Start section. Add 100 µL ATL-DX buffer and 162.5 µL sterile 1x PBS.

SECTION: 1. WHOLE BLOOD AND BUFFY COAT LYSIS
5. Include a negative control for each batch of samples: a bead tube from step 3 of Before Start section with 100 µL ATL-DX buffer and 400 µL sterile 1x PBS.
SECTION: 1. WHOLE BLOOD AND BUFFY COAT LYSIS
6. Load all samples into the Bullet Blender (refill dry ice if necessary).
SECTION: 1. WHOLE BLOOD AND BUFFY COAT LYSIS
7. Set the speed at 12 and the time at 3. Press Start.
SECTION: 1. WHOLE BLOOD AND BUFFY COAT LYSIS
8. Let the samples settle for 1 min in the Bullet Blender and then repeat step 7. 
 


SECTION: 2. CELL-FREE FLUID LYSIS (SERUM/PLASMA)
9. Add up to 1.5 mL fluid sample to a 2 ml microcentrifuge tube (depending on available volume, no less than 0.5 mL) and centrifuge the tube for 5 min at maximum speed (12000- 14000 xg). 

SECTION: 2. CELL-FREE FLUID LYSIS (SERUM/PLASMA)
10. Use a pipet to remove the supernatant.
SECTION: 2. CELL-FREE FLUID LYSIS (SERUM/PLASMA)
11. Add 500 µL ATL-DX buffer and resuspend the pellet in by vortexing.

SECTION: 2. CELL-FREE FLUID LYSIS (SERUM/PLASMA)
12. Transfer the entire pellet resuspension to the bead tubes prepared in step 3 of Before Start section.
SECTION: 2. CELL-FREE FLUID LYSIS (SERUM/PLASMA)
13. Include a positive control for each batch of samples: transfer 37.5 µL ZymoBIOMICS Microbial Community Standard Material, 100 µL EBV, and 100 µL ZIKV standard into a tube from step 3 of Before Start section. Add 250 µL ATL-DX buffer.

SECTION: 2. CELL-FREE FLUID LYSIS (SERUM/PLASMA)
14. Include a negative control for each batch of samples: a bead tube from step 3 of Before Start section with 500 µL ATL-DX buffer.

SECTION: 2. CELL-FREE FLUID LYSIS (SERUM/PLASMA)
15. Load all samples into the Bullet Blender (refill dry ice if necessary).
SECTION: 2. CELL-FREE FLUID LYSIS (SERUM/PLASMA)
16. Set the speed at 12 and the time at 3. Press Start.
SECTION: 2. CELL-FREE FLUID LYSIS (SERUM/PLASMA)
17. Let the samples settle for 1 min in the Bullet Blender and then repeat step 16. 
 


SECTION: 3. FTA CARD LYSIS
18. Punch FTA card with dried blood spots with a 3.0mm single-hole puncher. 
SECTION: 3. FTA CARD LYSIS
19. Use a forceps to place three card punches of the same sample into a bead tube prepared in step 3 of Before Start section.
SECTION: 3. FTA CARD LYSIS
20. Add 20 µL Proteinase K and 200 µL QIAcard FTA Wash Buffer and 200 µL ATL-DX buffer to the sample. Mix immediately by vortexing for 15 s.

SECTION: 3. FTA CARD LYSIS
21. Include a positive control for each batch of samples: transfer 37.5 µL ZymoBIOMICS Microbial Community Standard Material, 100 µL EBV, and 100 µL ZIKV standard into a tube from step 3 of Before Start section. Add 125 µL QIAcard FTA Wash Buffer and  125 µL ATL-DX buffer and 20 µL proteinase K.

SECTION: 3. FTA CARD LYSIS
22. Include a negative control for each batch of samples: a bead tube from step 3 of Before Start section with 200 µL QIAcard FTA Wash Buffer and  200 µL  ATL-DX buffer and  20 µL proteinase K.

SECTION: 3. FTA CARD LYSIS
23. Incubate in a thermomixer at  .

SECTION: 3. FTA CARD LYSIS
24. Refill the dry ice compartment of the Bullet Blender if necessary. After incubation, load all samples into the Bullet Blender.
SECTION: 3. FTA CARD LYSIS
25. Set the speed at 12 and the time at 3. Press Start.
SECTION: 3. FTA CARD LYSIS
26. Let the samples settle for 1 minute in the Bullet Blender and then repeat step 25. 
 

SECTION: 4. INSTRUMENT SET UP
27. 
 

Confirm 96 deep-well magnetic heads and 96 well deep-well heat blocks are being used.
SECTION: 4. INSTRUMENT SET UP
28. Ensure the program IndiMag_Pathogen_KF_Flex_4washor the program has been downloaded and loaded onto the KingFisher Flex instrument.
SECTION: 5. SET UP THE PROCESSING PLATES
29. Set up the Tip Comb, Wash, and Elution Plates outside the instrument according to the following table. 
 
 
     Plate ID      Plate position      Plate type      Reagent      Volume per well        Tip   comb      7      Place   a 96 Deep-well Tip comb in a deep-well plate        Elution      6      Deep-Well      Nuclease-free   water      75   µL        Wash   4      5      Deep-Well      100   % ethanol      750   µL        Wash   3      4      Deep-Well      80%   ethanol      750   µL        Wash   2      3      Deep-Well      Buffer   AW2      700   µL        Wash   1      2      Deep-Well      Buffer   AW1      700   µL        Sample         1      Sample   Lysate      Lysate   and lysis buffer      985   µL   
 
SECTION: 6. EXTRACTION
30. Centrifuge the bead tubes with lysate from the sample lysis step for 12000 x g, 5 min.

SECTION: 6. EXTRACTION
31. Add 20 µL of Proteinase K into wells of a Deep-well plate (based on the number of samples). 
 


SECTION: 6. EXTRACTION
32. Transfer 270 µL supernatant (for FTA card samples of section 3, transfer 290 µL supernatant) without any particle carryover to the wells of the Deep-well plate. This plate becomes the Sample Plate.
SECTION: 6. EXTRACTION
33. Add 135 µL Buffer VXL, 540 µL Buffer ACB, and 20 µL MagAttract Suspension G to each sample in the sample plate. For multiple samples, make a master mix with 10% overage. Invert slowly to mix the master mix; avoid foaming (it can be mixed on a Hula mixer for 2 min). Add 695 µL mixture to each sample. Each well including sample lysate should be 985 µL.

SECTION: 6. EXTRACTION
34. Select the program IndiMag_Pathogen_KF_Flex_4wash on the instrument.
SECTION: 6. EXTRACTION
35. Start the run, then load the prepared plates into position when prompted by the instrument.
SECTION: 7. QUANTIFICATION AND STORAGE
36. After the running protocol is completed (~35 min), immediately remove the elution plate from the instrument and cover the plate or transfer the eluate to the final tube or plate of choice for final storage. 

SECTION: 7. QUANTIFICATION AND STORAGE
37. In  0.6 mL microcentrifuge tubes, use 1 µL  total nucleic acid from whole blood/buffy coat samples, or  3 µL  total nucleic acid from FTA card/cell-free fluid samples, for DNA and RNA concentration measurements using Qubit 4 Fluorometer following manufacturer instructions. 
 




SECTION: 7. QUANTIFICATION AND STORAGE
38. Proceed with sample testing following the REDI-NET SOP B-4 Blood Testing or store at -20 °C for less than 2 weeks. 
 

SECTION: 8. INSTRUMENT SET UP
39. 
 

Confirm 12-tip magnetic heads and 12 well deep-well heat blocks are being used.
SECTION: 8. INSTRUMENT SET UP
40. Ensure the program IndiMag_Pathogen_KF_Duo_4wash has been downloaded and loaded onto the KingFisher Duo Prime instrument. 
SECTION: 9. SET UP THE SAMPLE PLATE AND ELUTION STRIP
41. Set up the Sample Plate according to the table below: 
     Row ID      Plate Row      Reagent      Volume per well        Sample row      A      Lysate   and lysis buffer      985   µL        Wash 1      B      Buffer   AW1      700   µL        Wash 2      C      Buffer   AW2      700   µL        Wash 3      D      80   % ethanol      750   µL        Wash 4      E      100   % ethanol      750   µL        Tip Comb       F      Tip   comb                       G      Empty               H   
 
SECTION: 9. SET UP THE SAMPLE PLATE AND ELUTION STRIP
42. Set up the Elution Strip according to the table below: 
 
 
     Strip ID      Row       Reagent      Volume per well        Elution         A      Nuclease-free   water      75   µL   
 
SECTION: 10. EXTRACTION
43. Centrifuge the bead tubes with lysate from the sample lysis step for 12000 x g, 5 min.

SECTION: 10. EXTRACTION
44. Add 20 µL of Proteinase K into wells of a Deep-well plate (based on the number of samples).
 


SECTION: 10. EXTRACTION
45. Transfer 270 µL supernatant (for FTA card samples of section 3, transfer 290 µL supernatant) without any particle carryover to the wells of the Deep-well Row A. This row becomes the Sample Row.
SECTION: 10. EXTRACTION
46. Add 135 µL Buffer VXL, 540 µL Buffer ACB, and 20 µL MagAttract Suspension G to each sample in the Sample Plate. For multiple samples, make a master mix with 10% overage. Invert slowly to mix the master mix and avoid foaming. Add 695 µL mixture to each sample. Each well including sample lysate should be 985 uL.

SECTION: 10. EXTRACTION
47. Select the program IndiMag_Pathogen_KF_Duo_4wash on the instrument.
SECTION: 10. EXTRACTION
48. Start the run, then load the prepared plate and Elution Strip into position when prompted by the instrument.

 

SECTION: 11. QUANTIFICATION AND STORAGE
49. After the running protocol is completed (~35 min), immediately remove the elution plate from the instrument and cover the plate or transfer the eluate to the final tube or plate of choice for final storage.

SECTION: 11. QUANTIFICATION AND STORAGE
50. In  0.6 mL microcentrifuge tubes, use 1 µL  total nucleic acid from whole blood/buffy coat samples, or  3 µL  total nucleic acid from FTA card/cell-free fluid samples, for DNA and RNA concentration measurements using Qubit 4 Fluorometer following manufacturer instructions. 
 

SECTION: 11. QUANTIFICATION AND STORAGE
51. Proceed with sample testing following the REDI-NET SOP B-4 Blood Testing or store at -20 °C for less than 2 weeks.
 


SECTION: APPENDIX 3. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
52. DNA quantification: 
According to the volume of sample used, add the 1xHS dsDNA Qubit Assay for a final volume of 200 µL (i.e., if using 1 µL of sample, add 199 µL of 1x HS dsDNA Qubit Assay). Vortex for 5 - 10 seconds, then incubate for 2 min at Room temperature before reading.
SECTION: APPENDIX 3. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
53. RNA Quantification:
SECTION: APPENDIX 3. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
53.1. In a new microcentrifuge tube/falcon tube (depending on the number of samples processed), prepare a working solution of the Qubit HS RNA Assay:
     Reagents      Volume/sample      Volume   for n+1 sample        Qubit RNA HS Assay buffer      199   µL      ….   µL        Qubit RNA HS Assay Dye      1   µL      ….   µL   
 
SECTION: APPENDIX 3. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
53.2. In a new 0.6 ml tube, mix 197 µL of Qubit HS RNA Assay working solution and 3 µL of the sample. Incubate for 2 min at Room temperature before reading.
SECTION: 1. WHOLE BLOOD AND BUFFY COAT LYSIS
3. Transfer 400 µL blood and 1x PBS mixture to the bead tubes prepared in step 3 of Before Start section under the Guidelines & Warnings tab. Add 100 µL of ATL-DX buffer.