TITLE: RNA extraction from 10µL mouse blood samples (KingFisher Flex 96-well)
AUTHORS: Petra Schneider, Charlotte Repton, Sarah Reece

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>RNA extraction from 5uL mouse blood samples, for subsequent cDNA production and qPCR to detect </span><span style = "font-style:italic;">Plasmodium </span><span> parasites - specifically gametocytes - within the blood. Using KingFisher Flex (96 well) system with </span><a href="https://www.thermofisher.com/order/catalog/product/AM1830" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">MagMAX-96 Total RNA Isolation Kit</span></a><span>. Protocol to import into BindIt Software attached.</span></div></div>

[STEPS]
?. [Sample Collection]
Dispense 20 µL of RNAlater  into 0.2 mL strip tubes or 96-well plates, one well per sample. Cover tubes with lids, or plate with a plastic film (the thick version, not the optical film for qPCRs – that one will NOT come off). Note: using more RNAlater ends up inhibiting the downstream process.
?. [Sample Collection]
Take a 10 µL blood sample (we use glass capillaries) and dispense the blood immediately into the RNAlater. Repeat for all samples.
?. [Sample Collection]
Mix with pipette and replace lid; or replace lid and mix by carefully tapping the tube.
?. [Sample Collection]
Freeze samples at -70°C or immediately extract RNA.
?. [Prepare for RNA extraction]
Defrost your samples.
?. [Prepare for RNA extraction]
Get required plastics. You will need 6 Kingfisher DeepWell (DW; Cat# 95040460) plates, 2 Kingfisher 96 (KF; Cat# 97002540) plates and 1 DW tip comb (Cat# 97002534).
?. [Prepare for RNA extraction]
Prepare reagents in kit only if opening a new MagMAX-96 Total RNA Isolation Kit (Cat#AM1830), Add the appropriate amount of ethanol or isopropanol to the wash buffers (see table below) and mark the bottles clearly by crossing out the word “concentrate”, ticking the small checkbox and writing your name/initials and the date.   ABC1  Reagent    Location    Action  2  Lysis/Binding Concentrate    Bench  Add 9 mL isopropanol3  Wash Solution 1 Concentrate    Bench  Add 6 mL isopropanol4  Wash Solution 2 Concentrate    Bench    Add 44 mL 100% ethanol  5  Elution Buffer    Bench    None  6  Lysis/Binding Enhancer    -20°C freezer    None  7  RNA Binding Beads     4°C fridge    None  8  Turbo DNase    -20°C freezer    None  9  Turbo DNase Buffer    Bench    None  10  RNA Rebinding Concentrate    Bench    Add 6 mL isopropanol  Note: some of the bench-top reagents crystallise when the ambient temperature is low (below ~18°C). To combat this, set a heat block to 25°C before beginning and place the room temperature reagents on it until ready to use, remembering to mix well!
ABC1  Reagent    Location    Action  2  Lysis/Binding Concentrate    Bench  Add 9 mL isopropanol3  Wash Solution 1 Concentrate    Bench  Add 6 mL isopropanol4  Wash Solution 2 Concentrate    Bench    Add 44 mL 100% ethanol  5  Elution Buffer    Bench    None  6  Lysis/Binding Enhancer    -20°C freezer    None  7  RNA Binding Beads     4°C fridge    None  8  Turbo DNase    -20°C freezer    None  9  Turbo DNase Buffer    Bench    None  10  RNA Rebinding Concentrate    Bench    Add 6 mL isopropanol
?. [Prepare for RNA extraction]
Prepare bead mix. Mix 10µL of RNA binding beads (vortex!)  and 10µL of lysis/binding enhancer (total = 20 µL) per sample, in a sterile DNase/RNase-free eppendorf tube. Vortex the mixture after preparation, and again immediately before use. Note: this stuff sinks to the bottom quickly, vortex regularly if dispensing for many samples.
?. [Prepare for RNA extraction]
Fill the plates as shown in the table:  AB1  Plate (type)    Contents /well  2Tip plate (KF) + DWTip Comb  N/A  3  Sample (DW)  filled at step 10: 380 µL sample+lysis/binding solution4  Wash 1 (DW)  150 µL wash buffer 15  Wash 2_1 (DW)  150 µL wash buffer 26DNAse (DW)50 µL DNAse mix7  Wash 2_2 (DW)  150 µL wash buffer 28Wash 2_3 (DW)150 µL wash buffer 29  Elution (KF)  75 µL RNA elution buffer
AB1  Plate (type)    Contents /well  2Tip plate (KF) + DWTip Comb  N/A  3  Sample (DW)  filled at step 10: 380 µL sample+lysis/binding solution4  Wash 1 (DW)  150 µL wash buffer 15  Wash 2_1 (DW)  150 µL wash buffer 26DNAse (DW)50 µL DNAse mix7  Wash 2_2 (DW)  150 µL wash buffer 28Wash 2_3 (DW)150 µL wash buffer 29  Elution (KF)  75 µL RNA elution buffer
?. [RNA extraction]
Connect the Kingfisher instrument to the computer using USB cable (note: this should already be there).
?. [RNA extraction]
Open the BindIt software. Select ‘Your Kingfisher Machine’ from the drop-down ‘connect’ list to connect to your Kingfisher instrument (note: connection has likely established automatically).
?. [RNA extraction]
Select the protocol (see step 22), and click on the ‘start’ (green arrow) button on the BindIt software.
?. [RNA extraction]
Load the plates into the machine. The machine will turn the turntable and prompt you to load the plates in the correct order. Press the ‘start’ button on the instrument to confirm you have loaded the current plate, this tells the machine to progress to the next plate. Make sure to line up the A1 well of the plate with the marked corner on the turntable position.
?. [RNA extraction]
Follow dispense step instructions. The run will begin once all plates are loaded. The instrument will prompt you to add the correct reagents at the dispense steps (grey cells in machine protocol, below). The first dispense step is roughly 2min after start-time. A guide to the approximate length of time until a step ends (so that you can work out how long between dispense steps) is also included in the machine protocol table below. Note: when adding beads - vortex first!
?. [RNA extraction]
Remove plates when the run has ended (as prompted by machine). Export AND SAVE the run details as a PDF (automatically opened in the software) –  it contains details of the times each steps were begun as well as temperatures at frequent intervals, if something has gone wrong this may give you the answer why. Do not yet discard plates. Stop and switch off machine.
?. [RNA extraction]
Check, seal and label RNA extracts. Check there is liquid in the elution plate (if not, something has gone wrong!). Seal the elution plate with a plastic film (make sure it is freezer-suitable and do not use the optical qPCR films – these do NOT come off) and label it carefully.
?. [RNA extraction]
Freeze at -20C (short term) or -70°C (long term) or use immediately for cDNA+qPCR. We recommend 1) Additional DNAse treatment (see step 22-30); 2) diluting at least 1 volume extracted RNA + 4 volumes water before cDNA production to remove the effects of inhibitors 3) if diluted before cDNA production, cDNA can go directly into qPCR without further dilution step.
?. [RNA extraction]
Discard all plates, except the elution plate. If you haven’t used every well, you can keep them to be reused (be sure to use different, clean wells to the ones you have already used). And, if you do keep them for a while – remove liquids from used wells before storage.
?. [Kingfisher Protocol]
Optimized machine ProtocolFile to import into your BindIt software:Summary: Plates (add to BindIt software under the ‘Layout’ tab):Tip plate (KF 96 plate with DW 96 tip comb inserted)Sample plate (DW microtiter plate)Wash 1 (DW microtiter plate)Wash 2_1 (DW microtiter plate)Wash 2_2 (DW microtiter plate)Wash 2_3 (DW microtiter plate)Turbo DNase (DW microtiter plate)Elution (KF 96 plate)Protocol (add to BindIt software under the ‘Protocol’ tab):  ABCD1Step name  Description    Plate  Step end (approx. time from start)2Pick up tip     Tip plate     3  Mix 1  Heating during mixing 20°C  1 min; medium mix  Sample  2 min 20 s4Add beadsDispense.  Message: “Add 20 µL beads.”  Sample  4 min 30s (~2 min)5Bind RNA of lysed sample&nbsp;Heating during mixing 20°C.  5 min; medium mix.  Collect beads count 5, time 5s  Sample  11 min6Release beads 1Heating during mixing 20°C. Release beads 10s, slow.  Wash 1  11 min 30s7Recollect beadsHeating during mixing 20°C . Collect beads count 5, time 5s.  Sample  13 min 30s8  Wash 1  Heating during mixing 20°C. Release beads 10s, slow  45s slow mix  Collect beads count 5, time 5s  Wash 1  16 min9Wash 2_1Heating during mixing 20°C. Release beads 10s, slow  45s slow mix  Collect beads count 5, 5sWash   2_118 min10Dry 1Dry outside well/tube, 30 s.Wash  2_118 min 30s11DNase 10 minHeating during mixing 25°C. Release beads 10s, slow  10 min slow mixTurbo Dnase29 min 30s12Add rebinding solutionDispense.  Message: “Add 100 µL rebinding solution.”Turbo DNase32 min 30s (~30 min)13Rebind RNAHeating during mixing 20°C. 3 min slow mix  Collect beads count 5, time 5s.Turbo DNase  36 min  142nd  Wash 2Heating during mixing 20°C. Release beads 10s slow, 45s slow mix  Collect beads count 3, time 5s.Wash 2_2  38 min  15  3rd  Wash 2  Heating during mixing 20°C. Release beads 10s slow  45s slow mix  Collect beads count 3, time 5sWash 2_3  40 min  16  Dry 2  Dry 1 min outside well/tubeWash 2_3  41 min  17  Elution  Heating during mixing 65°C. Release beads 10s slow. 7min 30s slow mix  Collect beads count 5, time 20s.Elution  51 min  18Release beads into 2_1Release beads 10s slowWash 2_1  52 min  19Recollect beads from eluCollect beads count 5, time 20s.Elution  53 min  20Leave tip     Wash 2_153 min
ABCD1Step name  Description    Plate  Step end (approx. time from start)2Pick up tip     Tip plate     3  Mix 1  Heating during mixing 20°C  1 min; medium mix  Sample  2 min 20 s4Add beadsDispense.  Message: “Add 20 µL beads.”  Sample  4 min 30s (~2 min)5Bind RNA of lysed sample&nbsp;Heating during mixing 20°C.  5 min; medium mix.  Collect beads count 5, time 5s  Sample  11 min6Release beads 1Heating during mixing 20°C. Release beads 10s, slow.  Wash 1  11 min 30s7Recollect beadsHeating during mixing 20°C . Collect beads count 5, time 5s.  Sample  13 min 30s8  Wash 1  Heating during mixing 20°C. Release beads 10s, slow  45s slow mix  Collect beads count 5, time 5s  Wash 1  16 min9Wash 2_1Heating during mixing 20°C. Release beads 10s, slow  45s slow mix  Collect beads count 5, 5sWash   2_118 min10Dry 1Dry outside well/tube, 30 s.Wash  2_118 min 30s11DNase 10 minHeating during mixing 25°C. Release beads 10s, slow  10 min slow mixTurbo Dnase29 min 30s12Add rebinding solutionDispense.  Message: “Add 100 µL rebinding solution.”Turbo DNase32 min 30s (~30 min)13Rebind RNAHeating during mixing 20°C. 3 min slow mix  Collect beads count 5, time 5s.Turbo DNase  36 min  142nd  Wash 2Heating during mixing 20°C. Release beads 10s slow, 45s slow mix  Collect beads count 3, time 5s.Wash 2_2  38 min  15  3rd  Wash 2  Heating during mixing 20°C. Release beads 10s slow  45s slow mix  Collect beads count 3, time 5sWash 2_3  40 min  16  Dry 2  Dry 1 min outside well/tubeWash 2_3  41 min  17  Elution  Heating during mixing 65°C. Release beads 10s slow. 7min 30s slow mix  Collect beads count 5, time 20s.Elution  51 min  18Release beads into 2_1Release beads 10s slowWash 2_1  52 min  19Recollect beads from eluCollect beads count 5, time 20s.Elution  53 min  20Leave tip     Wash 2_153 min
?. [Prepare for RNA extraction]
Prepare the DNAse mix. Mix 49 µL Turbo DNase buffer (mix well before use) and 1 µL Turbo DNase enzyme per sample, in a sterile DNase/RNase-free eppendorf tube. Mix thoroughly with a pipette, and leave at room temperature until needed.
?. [Prepare for RNA extraction]
Mix samples with lysis/binding solution. Label a Deep Well (DW) plate with the word ‘Sample’. Add 175 μL Lysis/Binding solution to the samples; pipette up and down (multichannel comes in handy), then move to the Deep Well 'Sample' plate. Add a further 175 μL of Lysis/Binding solution to each - now almost empty - sample tube to collect any remaining blood, mix, and transfer to the corresponding wells of the Deep Well 'Sample' plate. Note: we add a total of  350μL Lysis/Binding solution to each sample. There’s only 20mL per 100 samples available in the kit. Order AM8500 (100mL Lysis/Binding solution to make up the difference)
?. [DNAse treatment]
We currently use RQ1 RNase-Free DNaseDefrost. Take RQ1 RNase-Free DNase kit and samples (if frozen) from freezer. Thaw on ice. Vortex RQ1 buffer once defrosted.
?. [DNAse treatment]
Program PCR machine.  You will need 37ºC for 30min and 65ºC for 10min.
?. [DNAse treatment]
Prepare RQ1 reaction mix. Per sample you will need 2 µL RQ1 10x reaction buffer and 4 µL RQ1 enzyme. Prepare RQ1 reaction mix for all samples and aliquot 6 µL per sample into PCR tubes/plate.
?. [DNAse treatment]
Add RNA extract. Add 14 µL of RNA extract to each tube/well with RQ1 reaction mix (total volume 20 µL). Mix gently, Seal tubes/plate.
?. [DNAse treatment]
37C 30 min. Place tubes/plate in PCR machine. Run at 37ºC for 30 min.
?. [DNAse treatment]
Spin down. Briefly centrifuge to spin down contents.
?. [DNAse treatment]
Add stop solution. Add 2 µL RQ1 stop solution to each sample.
?. [DNAse treatment]
65C 10 min. Place tubes/plate in PCR machine. Run at 65ºC for 10 min.
?. [DNAse treatment]
Cool to 4 °C for 5 mins before using cDNA production, or store samples at -20ºC (short term) or -70ºC (longer term)