RNA extraction from 10µL mouse blood samples (KingFisher Flex 96-well)

Petra Schneider; Charlotte Repton; Sarah Reece

Nov 12, 2019

RNA extraction from 10µL mouse blood samples (KingFisher Flex 96-well)

DOI

dx.doi.org/10.17504/protocols.io.88fhztn

Petra Schneider¹,
Charlotte Repton¹,
Sarah Reece¹

¹University of Edinburgh

Petra Schneider

DOI: dx.doi.org/10.17504/protocols.io.88fhztn

Protocol Citation: Petra Schneider, Charlotte Repton, Sarah Reece 2019. RNA extraction from 10µL mouse blood samples (KingFisher Flex 96-well). protocols.io https://dx.doi.org/10.17504/protocols.io.88fhztn

Manuscript citation:

Protocol originates from previous method described in: P. Schneider et al, Mol Biochem Parasit 2015, 199: 29-33, DOI: 10.1016/j.molbiopara.2015.03.006. Protocol previously shortly described in P. Birget et al, Proc Roy Soc Lond B, 2017, 284 (1860): 20171229, DOI: 10.1098/rspb.2017.1229; and in P. Schneider et al, PLoS Pathogens 2018, 14(11): e1007371, DOI: 10.1371/journal.ppat.1007371.

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: November 12, 2019

Last Modified: November 12, 2019

Protocol Integer ID: 29671

Keywords: RNA extraction, mouse, blood, Plasmodium

Abstract

RNA extraction from 5uL mouse blood samples, for subsequent cDNA production and qPCR to detect Plasmodium  parasites - specifically gametocytes - within the blood. Using KingFisher Flex (96 well) system with MagMAX-96 Total RNA Isolation Kit. Protocol to import into BindIt Software attached.

Guidelines

Please note that RNA was extracted using the semi-automatic Kingfisher Flex Magnetic Particle Processor and the MagMAX-96 Total RNA Isolation Kit (AM1830 Thermo Fisher Scientific) but with adjustments to the manufacturer's protocols (standard protocols AM1830DW ) to improve recovery. Note: the adapted protocol requires MORE lysis buffer than is provided in the kit: order extra (AM8500)

Materials

MATERIALS
ReagentRQ1 RNase-Free DNase, 1,000uPromegaCatalog #M6101 
ReagentKingFisher&trade; Flex&trade; Systems Consumables, KingFisher Flex Microtiter Deepwell 96 plate, sterileThermo FisherCatalog #95040460 
ReagentKingFisher&trade; Flex&trade; Systems Consumables, KingFisher 96 tip comb for DW magnetsThermo FisherCatalog #97002534 
ReagentKingFisher&trade; Flex&trade; Systems Consumables, KingFisher 96 KF microplate (200&micro;L)Thermo FisherCatalog #97002540 
ReagentMagMAX&trade;-96 Total RNA Isolation KitThermo FisherCatalog #AM1830 
ReagentMagMAX&trade; Lysis/Binding Solution ConcentrateThermo FisherCatalog #AM8500 
In addition you will need: RNAlater, isopropanol, absolute ethanol, and general lab materials (plastics, pipettes, pipette tips)

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.

Take a 10 µL blood sample (we use glass capillaries) and dispense the blood immediately into the RNAlater. Repeat for all samples.

Mix with pipette and replace lid; or replace lid and mix by carefully tapping the tube.

Freeze samples at -70°C or immediately extract RNA.

Prepare for RNA extraction

Defrost your samples.

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 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. 

  
  Reagent    Location    Action  
  Lysis/Binding Concentrate    Bench  Add 9 mL isopropanol
  Wash Solution 1 Concentrate    Bench  Add 6 mL isopropanol
  Wash Solution 2 Concentrate    Bench    Add 44 mL 100% ethanol  
  Elution Buffer    Bench    None  
  Lysis/Binding Enhancer    -20°C freezer    None  
  RNA Binding Beads     4°C fridge    None  
  Turbo DNase    -20°C freezer    None  
  Turbo DNase Buffer    Bench    None  
  RNA Rebinding Concentrate    Bench    Add 6 mL isopropanol  
New MagMAX-96 Total RNA Isolation Kit - Preparation of reagents (Thermofisher Cat#AM1830)
 
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!

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 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.

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)

Fill the plates as shown in the table:
 
  Plate (type)    Contents /well  
Tip plate (KF) + DWTip Comb  N/A  
  Sample (DW)  filled at step 10: 380 µL sample+lysis/binding solution
  Wash 1 (DW)  150 µL wash buffer 1
  Wash 2_1 (DW)  150 µL wash buffer 2
DNAse (DW)50 µL DNAse mix
  Wash 2_2 (DW)  150 µL wash buffer 2
Wash 2_3 (DW)150 µL wash buffer 2
  Elution (KF)  75 µL RNA elution buffer
Plate filling instructions.

 
 

RNA extraction

Connect the Kingfisher instrument to the computer using USB cable (note: this should already be there). 

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). 

Select the protocol (see step 22), and click on the ‘start’ (green arrow) button on the BindIt software.

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. 

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!

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.

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. 

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.

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 Protocol

File to import into your BindIt software:RNA10uLMouseBlood.bdz  

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):
 
 
 
Step name  Description    Plate  Step end (approx. time from start)
Pick up tip     Tip plate     
  Mix 1  Heating during mixing 20°C  1 min; medium mix  Sample  2 min 20 s
Add beadsDispense.  Message: “Add 20 µL beads.”  Sample  4 min 30s (~2 min)
Bind RNA of lysed sample Heating during mixing 20°C.  5 min; medium mix.  Collect beads count 5, time 5s  Sample  11 min
Release beads 1Heating during mixing 20°C. Release beads 10s, slow.  Wash 1  11 min 30s
Recollect beadsHeating during mixing 20°C . Collect beads count 5, time 5s.  Sample  13 min 30s
  Wash 1  Heating during mixing 20°C. Release beads 10s, slow  45s slow mix  Collect beads count 5, time 5s  Wash 1  16 min
Wash 2_1Heating during mixing 20°C. Release beads 10s, slow  45s slow mix  Collect beads count 5, 5sWash   2_118 min
Dry 1Dry outside well/tube, 30 s.Wash  2_118 min 30s
DNase 10 minHeating during mixing 25°C. Release beads 10s, slow  10 min slow mixTurbo Dnase29 min 30s
Add rebinding solutionDispense.  Message: “Add 100 µL rebinding solution.”Turbo DNase32 min 30s (~30 min)
Rebind RNAHeating during mixing 20°C. 3 min slow mix  Collect beads count 5, time 5s.Turbo DNase  36 min  
2nd  Wash 2Heating during mixing 20°C. Release beads 10s slow, 45s slow mix  Collect beads count 3, time 5s.Wash 2_2  38 min  
  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  
  Dry 2  Dry 1 min outside well/tubeWash 2_3  41 min  
  Elution  Heating during mixing 65°C. Release beads 10s slow. 7min 30s slow mix  Collect beads count 5, time 20s.Elution  51 min  
Release beads into 2_1Release beads 10s slowWash 2_1  52 min  
Recollect beads from eluCollect beads count 5, time 20s.Elution  53 min  
Leave tip     Wash 2_153 min
KingFisher Protocol for RNA extraction from 10µL mouse blood samples (BindIt software) with added time frame for extraction run.
 


 

 
 

DNAse treatment

We currently use RQ1 RNase-Free DNase

Defrost. Take RQ1 RNase-Free DNase kit and samples (if frozen) from freezer. Thaw on ice. Vortex RQ1 buffer once defrosted.

Program PCR machine.  You will need 37ºC for 30min and 65ºC for 10min.

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.

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.

Spin down. Briefly centrifuge to spin down contents.

37C 30 min. Place tubes/plate in PCR machine. Run at 37ºC for 30 min.

Add stop solution. Add 2 µL RQ1 stop solution to each sample.

65C 10 min. Place tubes/plate in PCR machine. Run at 65ºC for 10 min.

Cool to 4 °C for 5 mins before using cDNA production, or store samples at -20ºC (short term) or -70ºC (longer term)

Reagent	Location	Action
Lysis/Binding Concentrate	Bench	Add 9 mL isopropanol
Wash Solution 1 Concentrate	Bench	Add 6 mL isopropanol
Wash Solution 2 Concentrate	Bench	Add 44 mL 100% ethanol
Elution Buffer	Bench	None
Lysis/Binding Enhancer	-20°C freezer	None
RNA Binding Beads	4°C fridge	None
Turbo DNase	-20°C freezer	None
Turbo DNase Buffer	Bench	None
RNA Rebinding Concentrate	Bench	Add 6 mL isopropanol

	Plate (type)	Contents /well
	Tip plate (KF) + DWTip Comb	N/A
	Sample (DW)	filled at step 10: 380 µL sample+lysis/binding solution
	Wash 1 (DW)	150 µL wash buffer 1
	Wash 2_1 (DW)	150 µL wash buffer 2
	DNAse (DW)	50 µL DNAse mix
	Wash 2_2 (DW)	150 µL wash buffer 2
	Wash 2_3 (DW)	150 µL wash buffer 2
	Elution (KF)	75 µL RNA elution buffer

Step name	Description	Plate	Step end (approx. time from start)
Pick up tip		Tip plate
Mix 1	Heating during mixing 20°C 1 min; medium mix	Sample	2 min 20 s
Add beads	Dispense. Message: “Add 20 µL beads.”	Sample	4 min 30s (~2 min)
Bind RNA of lysed sample	Heating during mixing 20°C. 5 min; medium mix. Collect beads count 5, time 5s	Sample	11 min
Release beads 1	Heating during mixing 20°C. Release beads 10s, slow.	Wash 1	11 min 30s
Recollect beads	Heating during mixing 20°C . Collect beads count 5, time 5s.	Sample	13 min 30s
Wash 1	Heating during mixing 20°C. Release beads 10s, slow 45s slow mix Collect beads count 5, time 5s	Wash 1	16 min
Wash 2_1	Heating during mixing 20°C. Release beads 10s, slow 45s slow mix Collect beads count 5, 5s	Wash 2_1	18 min
Dry 1	Dry outside well/tube, 30 s.	Wash 2_1	18 min 30s
DNase 10 min	Heating during mixing 25°C. Release beads 10s, slow 10 min slow mix	Turbo Dnase	29 min 30s
Add rebinding solution	Dispense. Message: “Add 100 µL rebinding solution.”	Turbo DNase	32 min 30s (~30 min)
Rebind RNA	Heating during mixing 20°C. 3 min slow mix Collect beads count 5, time 5s.	Turbo DNase	36 min
2nd Wash 2	Heating during mixing 20°C. Release beads 10s slow, 45s slow mix Collect beads count 3, time 5s.	Wash 2_2	38 min
3rd Wash 2	Heating during mixing 20°C. Release beads 10s slow 45s slow mix Collect beads count 3, time 5s	Wash 2_3	40 min
Dry 2	Dry 1 min outside well/tube	Wash 2_3	41 min
Elution	Heating during mixing 65°C. Release beads 10s slow. 7min 30s slow mix Collect beads count 5, time 20s.	Elution	51 min
Release beads into 2_1	Release beads 10s slow	Wash 2_1	52 min
Recollect beads from elu	Collect beads count 5, time 20s.	Elution	53 min
Leave tip		Wash 2_1	53 min

Public workspaceRNA extraction from 10µL mouse blood samples (KingFisher Flex 96-well)

We currently use RQ1 RNase-Free DNase

RNA extraction from 10µL mouse blood samples (KingFisher Flex 96-well)