TITLE: T-2 TICK PROCESSING
AUTHORS: REDI-NET Consortium

[DESCRIPTION]
This protocol details tick 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 1) to Thermo Scientific Screw Cap Micro 1.5 ml Tubes.
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 min (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 clearly document the correct process for effective standardized field collections of ticks, and recommended personal protection measures.

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 field tick collections in the vicinity of watering holes by dragging.

RESPONSIBLE PERSON

Principal Investigator, Study Coordinator, Entomology Component Lead, Managers
 

MAINTENANCE OF EQUIPMENT

BEFORE EACH COLLECTION
Clean forceps with 70%-ethanol.
Freeze and clean ice-packs.
Clean cool-boxes
Fully charge all equipment (e.g., GPS unit, tablets/phone). Make sure the tablet has enough free-space for field sampling pictures.

AFTER EACH COLLECTION
Clean all equipment thoroughly between sampling sites, including boots, cooler box (inside and outside), etc.
Tick drags should be sealed into trash bags for transport and cleaned without detergents (only use tap water and then air-dry) before the next collection.
Lint roller sheets should be disposed of in the waste bag after all ticks have been removed for testing.
Store sterile equipment separate from used equipment and samples.

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 MEASURING SPOON FOR 0.1 mm BEATING BEADS

The spoon (Next Advance, MSP01-RNA) is used for 0.1 mm beating beads measurement. The step is described on 6.6.4 the preparation before tick homogenization. One spoon equals to 100 uL.  
 

APPENDIX 4 EXPECTED OUTCOMES

 


[STEPS]
SECTION: 1. SORT TICK SAMPLES
1. 
 
Sort larvae (pooled and stored by the same genus), nymph (individual), and adults (individual) into separate vials in the lab.
 

SECTION: 2. PREPARING TICK e-ID (IDX): Device setup using Bluetooth
2. 
 
Plug in the device. On a phone or computer which has both Google Chrome and Bluetooth capabilities, visit https://mos-id.vectech.io (if on mobile, use the desktop site option) and log in. For first time use, click the “Sign Up” tab, enter in a username (email), and a password. We will approve your account, and then you can continue.
SECTION: 2. PREPARING TICK e-ID (IDX): Device setup using Bluetooth
3. If set-up with ethernet is desired, skip to Section 3. 
SECTION: 2. PREPARING TICK e-ID (IDX): Device setup using Bluetooth
4. Click the drop-down arrow at the top-center of the page (see figure). Click “Add New Device,” click on the bluetooth icon button. When a device with a name “Agamotto” appears, select the device and click “Pair”. When given the opportunity, select and enter your Wi-Fi credentials [user name, passcode] (the website may throw an error,but ignore this error). Return to the “Data” tab.
 

SECTION: 2. PREPARING TICK e-ID (IDX): Device setup using Bluetooth
5. Click the drop-down arrow at the top-center of the page, and you should see a device with status [booting] or [Idle]. If [booting], wait for status to change to [idle] and stay. Then select “Device.” Setup is complete. If your device soon changes to [Idle] under the device drop down menu, continue to 4. Imaging Protocol.
SECTION: 3. PREPARING TICK e-ID (IDX): Device setup using Ethernet
6. If setting up with ethernet instead of Bluetooth, follow these steps. Select the user menu at the bottom right corner of the screen. Select “Connect Device.”
 
SECTION: 3. PREPARING TICK e-ID (IDX): Device setup using Ethernet
7. Select “Device.”If the device name is not known, contact Vectech for support. Then select your Wi-Fi name from the “Wi-Fi SSD” drop down menu. If you do not see your Wi-Fi in the drop down, verify the network is functioning on other devices. Then enter your Wi-Fi password. Select connect.
SECTION: 3. PREPARING TICK e-ID (IDX): Device setup using Ethernet
8. If a successful Wi-Fi connection is established, disconnect the device from power and ethernet, then reconnect power. If your device soon changes to [Idle] under the device drop down menu, continue to Section 4. Imaging Using Tick e-Id.
SECTION: 3. PREPARING TICK e-ID (IDX): Device setup using Ethernet
9. If you have trouble connecting to the device via Wi-Fi, try to connect an Ethernet cable directly from your device to your modem. You should soon see your device status change to [Idle] under the device drop down menu.
 

SECTION: 4. IMAGING USING TICK E-ID
10. On https://mos-id.vectech.io, navigate to the “Trays” tab. Verify that the device is plugged in. The device should flash lights when booting up.
SECTION: 4. IMAGING USING TICK E-ID
11. Organize the specimens to be multi-imaged in sample tubes laid out in tubes in a 96-well sample rack (8x12). MAINTAIN THE COLD CHAIN. Keep samples in dry ice or in polystyrene boxes filled with cool beans until ready to use. Place a glass tray on the chill table as you fill the rows before imaging and immediately return to storage tubes once imaged. Sequentially lay out the sequential sample across the row, with each row of 12 equaling one tray of multiplex photos.
 
 
SECTION: 4. IMAGING USING TICK E-ID
12. Wipe forceps using bleach wipes between each sample and rinse each RNA-later preserved sample in water before pulling tick sample on Kimwipes on chiller table to allow absorption of excess fluid before slotting into the imaging wells.
 

SECTION: 4. IMAGING USING TICK E-ID
13. Repeat step 12, until all 12 specimens are placed in the tray, dorsal side facing up.
SECTION: 4. IMAGING USING TICK E-ID
14. Close the tray by placing the lid on the well-side of the tray until you hear the gentle click as the magnets connect to keep the tray closed.
 
SECTION: 4. IMAGING USING TICK E-ID
15. Insert tray into device tray slot. Tray will “click” into place when fully inserted.
SECTION: 4. IMAGING USING TICK E-ID
16. To capture the dorsal view, go to the “Trays” tab in the website, click, “Capture New Tray.”
 
SECTION: 4. IMAGING USING TICK E-ID
17. Images auto upload.
 
 
SECTION: 4. IMAGING USING TICK E-ID
18. Enter the following information:
SECTION: 4. IMAGING USING TICK E-ID
18.1. Genus and species (if known). 
 

SECTION: 4. IMAGING USING TICK E-ID
18.2. Plate and well number. 
 

SECTION: 4. IMAGING USING TICK E-ID
19. Remove tray, flip upside down (i.e. dorsal to ventral), and insert tray again to capture the ventral image of the tick.
SECTION: 4. IMAGING USING TICK E-ID
20. Click “Capture Other Side”.
 
SECTION: 4. IMAGING USING TICK E-ID
21. Click “Finish,” and return specimens to original storage. Make a note of the tray number that corresponds to your sample row. 
SECTION: 5. PREPARING DINOSCOPE
22. Place a portable chill table on the adjustable stand.
SECTION: 5. PREPARING DINOSCOPE
23. Place white porcelain stage directly on top of the chill table surface. If a porcelain stage is not available, cut a 5cm x 5cm square from a paper note card and use color lab tape to attach to the bottom of a petri dish to make a stage.
SECTION: 5. PREPARING DINOSCOPE
24. Once in place, secure the stage by taping either porcelain or petri dish stands to sides of the chill table.
SECTION: 5. PREPARING DINOSCOPE
25. Adjust the DinoScope holster on the stand so it is 4.5 cm above the surface of the stage. 


SECTION: 5. PREPARING DINOSCOPE
26. Turn on the chill table (skip if not maintaining a cold chain) and wait until the surface temperature monitor reads -20 °C .
SECTION: 5. PREPARING DINOSCOPE
27. Ensure all specimens slated for imaging are arranged in individual Eppendorf tubes and stored in 96-well racks labeled with a plate number.
SECTION: 5. PREPARING DINOSCOPE
28. Use a Kimwipe and 75% ethanol to wipe clean the stage surface. If using a petri dish stage, replace paper cards.
SECTION: 6. IMAGING USING DINOSCOPE
29. Remove a specimen from tube with forceps and place in the center of the stage with the dorsal side facing up.
SECTION: 6. IMAGING USING DINOSCOPE
30. Using the DinoScope interface and stand fine adjustment, bring the specimen into focus (magnification requirements will differ between larvae/ nymphs and adults). Specimens should be centered in the field of view. Make sure all important characters are visible. 
SECTION: 6. IMAGING USING DINOSCOPE
31. Annotate with the file naming convention: Sample ID-View (D = Dorsal, V = Ventral) Example: NMNH2020-D.
SECTION: 6. IMAGING USING DINOSCOPE
32. Ensure the correct magnification is entered into the Magnification field and that all metadata is included in the image output (magnification, magnification profile, scale, username and annotation) but does not overlap with specimen in the image. 
SECTION: 6. IMAGING USING DINOSCOPE
33. Just before taking an image readjust the white balance for each individual image to standardize the background color for all images.
SECTION: 6. IMAGING USING DINOSCOPE
34. Take a single image and review the photo to ensure quality (focus, frame, taxonomic characters). If the image is not satisfactory delete and repeat steps 31 to 35.
SECTION: 6. IMAGING USING DINOSCOPE
35. Use forceps to flip the specimen so that the ventral side is facing up.
SECTION: 6. IMAGING USING DINOSCOPE
36. Repeat steps 31 to 35 for ventral view.
SECTION: 6. IMAGING USING DINOSCOPE
37. Return specimen to tube and repeat steps 30 to 38 until a dorsal and ventral image are captured for all specimens.
SECTION: 6. IMAGING USING DINOSCOPE
38. When the imaging session is completed, copy photos from the dated folder and save. 
SECTION: 7. BEFORE TICK HOMOGENIZATION
39. 
 
Pre-cool the Bullet Blender by adding dry ice into the cooling compartment and running the cooling program. 
SECTION: 7. BEFORE TICK HOMOGENIZATION
40. Clean the work surfaces with RNaseZap, then wipe the surfaces with 70% molecular biology grade ethanol to remove additional contaminants.
SECTION: 7. BEFORE TICK HOMOGENIZATION
41. Transfer 0.1 mm zirconium oxide beads (two spoons, Appendix 1) to Clear RINO brand 1.5 ml screw-cap microcentrifuge tubes.
SECTION: 7. BEFORE TICK HOMOGENIZATION
42. For the 1st time use of the IndiMag pathogen kit, add 100% ethanol to Buffer AW1 and AW2, and add 100% Isopropanol to ACB as indicated on the bottles.
SECTION: 7. BEFORE TICK HOMOGENIZATION
43. 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).

SECTION: 7. BEFORE TICK HOMOGENIZATION
44. MagAttract Suspension G from the IndiMag pathogen kit needs to be vortexed thoroughly for 3 mins (before first use) or 1 minute (before subsequent uses) to ensure that the magnetic silica particles are fully resuspended.
SECTION: 7. BEFORE TICK HOMOGENIZATION
45. Prepare a few 15 ml or 50 ml conical centrifuge tubes with nuclease-free water for preparing TNA elution in KingFisher Flex or KingFisher Duo Prime to avoid cross-contamination.
SECTION: 8. TICK HOMOGENIZATION
46. Clean forceps with 70% ethanol and Kimwipes before use and between samples.
SECTION: 8. TICK HOMOGENIZATION
47. Label orange RINO RNA lysis tubes on the cap. 
 

SECTION: 8. TICK HOMOGENIZATION
48. Wash tick using ice-cold 1x PBS:
SECTION: 8. TICK HOMOGENIZATION
48.1. For adult ticks: 3 times sequentially in Petri dishes to wash away any external pathogens.
SECTION: 8. TICK HOMOGENIZATION
48.2. For nymph ticks: in a clean microcentrifuge tube 1.5mL, add 250 µL of PBS and pipette off the supernatant without pipetting the nymph. Repeat sequentially 3 times to wash away any external pathogens. 



SECTION: 8. TICK HOMOGENIZATION
48.3. 
 

For larval ticks: 
Transfer 60 tick from lint roller to an orange RINO tube containing five 3.2 mm beads or use pooled larval ticks stored at -80 °C in orange RINO tubes (see step 1). 
Add 200 µL 1x PBS to wash the ticks. The ticks will be floating at the top of the 1x PBS. 
To remove the PBS, using a pipette with 200 µL tip, directly insert the tip to the bottom of the tube and aspirate the PBS until all PBS is retrieved. Discard the PBS and repeat the wash twice. The minimum 1x PBS residuals retaining on the beads will not affect the result.


SECTION: 8. TICK HOMOGENIZATION
49. Tick transfer:
SECTION: 8. TICK HOMOGENIZATION
49.1. For adult ticks: Transfer individual ticks into labeled orange RINO tubes and set on ice.

SECTION: 8. TICK HOMOGENIZATION
49.2. For nymph ticks: For a good amount of material used in downstream processing, transfer a pool of ticks (approximately 10-15 nymph, according to their size) until reaching the size of one bead (see Appendix 2), into labeled orange RINO tubes and set on ice.
SECTION: 8. TICK HOMOGENIZATION
49.3.  
For larval ticks: See step 51.3. A pool of ticks reaching the size of one 3.2 mm bead will be sufficient (approximately 60 larvae, according to their size, see Appendix 2). Label the orange RINO tubes from step 51.3 and set on ice. 
 
 
 
 

SECTION: 8. TICK HOMOGENIZATION
50. Ensure the Bullet Blender is fully cooled down. Add more dry ice into the cooling compartment of Bullet Blender, if necessary, then load the RINO tubes with ticks.
SECTION: 8. TICK HOMOGENIZATION
51. Set the controls for Speed 10 and Time 3. Press Start. 
 

SECTION: 8. TICK HOMOGENIZATION
52. After the run, remove the tubes from the instrument, briefly spin down and set on ice.

SECTION: 8. TICK HOMOGENIZATION
53. Add 400 µL cold sterile 1xPBS into each orange RINO tube with ticks.

SECTION: 8. TICK HOMOGENIZATION
54. Return RINO tubes to the Bullet Blender.
SECTION: 8. TICK HOMOGENIZATION
55. Set the controls for Speed 10 and Time 3. Press Start.
SECTION: 8. TICK HOMOGENIZATION
56. After the run, remove the tubes from the instrument and visually inspect the samples. If homogenization is incomplete, repeat the homogenization at speed 10 and Time 3 (for larvae, one time beating should be sufficient for homogenization).
SECTION: 8. TICK HOMOGENIZATION
57. Centrifuge the suspension at 100 x g, 1 min to pellet debris.

SECTION: 8. TICK HOMOGENIZATION
58. Without disturbing the tubes, carefully transfer the top 320 µL supernatant into 1.5 mL tubes.
SECTION: 9. MICROBE LYSIS
59. 
 
Add 80 µL of ATL-DX Lysis Buffer to the 1.5 mL tubes containing 0.1mm beads from step 52.

SECTION: 9. MICROBE LYSIS
60. Transfer 320 µL homogenate to each of the pre-labeled bead tubes.
SECTION: 9. MICROBE LYSIS
61. Include a positive control for each batch of samples: transfer 37.5 µL ZymoBIOMICS Microbial Community Standard Material and 100 µL EBV, and 100 µL HIV standard into a tube from step 62. Add 82.5 µL 1x PBS.

SECTION: 9. MICROBE LYSIS
62. Include a negative control for each batch of samples: a bead tube with 320 µL cold sterile 1xPBS only.
SECTION: 9. MICROBE LYSIS
63. Add more dry ice into the cooling compartment of Bullet Blender, if necessary and then load the all bead tubes (samples and controls).
SECTION: 9. MICROBE LYSIS
64. Set the speed at 12 and time at 3. Press Start.
SECTION: 9. MICROBE LYSIS
65. Let the samples settle for 1 min and then repeat step 64.

SECTION: 9. MICROBE LYSIS
66. Centrifuge the tube at 100 x g, 1 min.

SECTION: 9. MICROBE LYSIS
67. Carefully transfer the 350 µL supernatant from the RINO tube to a new snap-cap 1.5 ml microcentrifuge tube, avoiding bead carryover (slight bead contamination is tolerated). 
 


SECTION: 10. INSTRUMENT SET UP
68. 
 

Confirm 96 deep-well magnetic heads and 96 well deep-well heat blocks are being used.
SECTION: 10. INSTRUMENT SET UP
69. Ensure the program IndiMag_Pathogen_KF_Flex_4wash has been downloaded and loaded onto the KingFisher Flex instrument.
SECTION: 10.1 SET UP THE PROCESSING PLATES
70. Set up the Wash, Elution, and Tip Comb 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: 10.2 EXTRACTION
71. Centrifuge the 1.5 mL tubes with lysate from step 70 for 12000 x g, 5 min. 

SECTION: 10.2 EXTRACTION
72. Add 20 µL of Proteinase K into wells (based on number of samples) of a new Deep-well plate.

SECTION: 10.2 EXTRACTION
73. Transfer 270 µL supernatant of step 71 without any particle carryover to the wells of the Deep-well plate containing proteinase K. This plate becomes the Sample Plate.

SECTION: 10.2 EXTRACTION
74. Add 135 µL Buffer VXL, 540 µLBuffer 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 (can be mixed on Hula mixer for 2 min). Add 695 µL mixture to each sample.

SECTION: 10.2 EXTRACTION
75. Select the program IndiMag_Pathogen_KF_Flex_4wash on the instrument.
SECTION: 10.2 EXTRACTION
76. Start the run, then load the prepared plates into position when prompted by the instrument.
SECTION: 10.3. QUANTIFICATION AND STORAGE
77. After the running protocol is completed (~35 minutes), 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: 10.3. QUANTIFICATION AND STORAGE
78. In a 0.6 mL microcentrifuge tube, use 1 µL total nucleic acid for DNA and RNA concentration measurement using Qubit 4 Fluorometer following manufacturer instructions.
 


SECTION: 10.3. QUANTIFICATION AND STORAGE
79. Proceed with sample testing following the REDI-NET SOP T-4 Tick Testing or store at -20 °C for less than 2 weeks.
 

SECTION: 11. INSTRUMENT SET UP
80. 
 

Confirm 12-tip magnetic head and 12 well deep-well heat blocks are being used.
SECTION: 11. INSTRUMENT SET UP
81. Ensure the program IndiMag_Pathogen_KF_Duo_4wash has been downloaded and loaded onto the KingFisher Duo Prime instrument.
SECTION: 11.1 SET UP THE SAMPLE PLATE AND ELUTION STRIP
82. 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       700 µL               G         Empty               H   
 
SECTION: 11.1 SET UP THE SAMPLE PLATE AND ELUTION STRIP
83. Set up the Elution Strip according to the table below:
 

 
     Row ID      Plate Row      Reagent      Volume per well        Elution       A      Nuclease-free water      75 µL   
 
SECTION: 11.2. EXTRACTION
84. Centrifuge the bead tubes with lysate from step 68 for 12000 x g, 5 min. 

SECTION: 11.2. EXTRACTION
85. Add 20 µL of Proteinase K into wells (based on number of samples) of a sample row.

SECTION: 11.2. EXTRACTION
86. Transfer 270 µL supernatant without any particle carryover to the wells of the sample row containing proteinase K.

SECTION: 11.2. EXTRACTION
87. Add 135 µL Buffer VXL, 540 µL Buffer ACB, and 20 µL MagAttract Suspension G to each sample in the sample row. For multiple samples, make a master mix with 10% overage. Invert slowly to mix the master mix, avoid foaming (can be mixed on Hula mixer for 2 min). Add 695 µL mixture to each sample.
SECTION: 11.2. EXTRACTION
88. Select program IndiMag_Pathogen_KF_Duo_4wash on the instrument.
SECTION: 11.2. EXTRACTION
89. Start the run, then load the prepared plate/strip into position when prompted by the instrument.

 

SECTION: 11.3 QUANTIFICATION AND STORAGE
90. After the protocol is completed (~35 minutes), immediately remove the elution strip from the instrument and transfer the eluate to the final tube or plate of choice for final storage.
SECTION: 11.3 QUANTIFICATION AND STORAGE
91. Use 1 µL total nucleic acid for DNA and RNA concentration measurement using Qubit 4 Fluorometer.
 


SECTION: 11.3 QUANTIFICATION AND STORAGE
92. Proceed with sample testing following the REDI-NET SOP T-4 Tick Testing or store at -20 °C for less than 2 weeks. 
 

SECTION: APPENDIX 2. SIZING CRITERIA FOR TICK NYMPH AND LARVA POOLING
93. NOTE: The size of nymphs from different tick species could vary which may influence the number of nymphs needed for pooling. 

The picture below provides an “actual” size reference for 10 nymphs of Amblyomma americanum pooled in a 1.5 ml microcentrifuge tube compared to a 3.2 mm diameter stainless steel bead in an identical tube (used for tick sample processing). 

 
 Number of nymphs pooled: 10 Amblyomma americanumThe diameter of the stainless steel bead: 3.2 mm


 
 
The picture below provides an “actual” size reference for 60 larvae of Amblyomma americanum pooled in a 1.5 ml microcentrifuge tube compared to a 3.2 mm diameter stainless steel bead in an identical tube (used for tick sample processing). 

 Number of larvae pooled: 60 Amblyomma americanumThe diameter of the stainless steel bead: 3.2 mm
 
SECTION: APPENDIX 3. DNA and RNA Measurement Using QUBIT FLUOROMETER 4.0
94. 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. 

SECTION: APPENDIX 3. DNA and RNA Measurement Using QUBIT FLUOROMETER 4.0
95. RNA Quantification:
In a new microcentrifuge tube/falcon tube (depending on the number of samples processed), prepare a working solution of the Qubit HS RNA Assay: 
 
 
   ReagentsVolume/sampleVolume for n+1 sampleQubit RNA HS Assay buffer199 µL…. µLQubit RNA HS Assay Dye1 µL…. µL

SECTION: APPENDIX 3. DNA and RNA Measurement Using QUBIT FLUOROMETER 4.0
95.1. In a new 0.6 ml tube, mix 199 µL of Qubit HS RNA Assay working solution and 1 µL of the sample. Incubate for 1 minute at room temperature before reading.