TITLE: Protocol for Extraction of Viral RNA with added DNase treatment
AUTHORS: Megan Folkerts, Ashlyn Pfeiffer, mnguyen , Danielle Vazquez,  Amber Jones

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is modified from the MagMAX MVP II protocol from Thermo Fisher, and the MagMAX mirVana protocol from Thermo Fisher. Modifications were necessary to include a DNAse treatment step, which improves viral recovery in low-burden samples.</div></div>

[STEPS]
?. [RNA extraction using MVP II kit and Kingfisher]
Aliquot 5 ul of proteinase k to each sample well of a 96-well deep-well plate to be used for extraction.
?. [RNA extraction using MVP II kit and Kingfisher]
Add 50ul sample to wells
This can be increased to as much as 200ul if sufficient sample volume is available
?. [RNA extraction using MVP II kit and Kingfisher]
Bring sample volume up to 200ul with 150ul molecular biology grade H20
If you increased sample volume in step 3, decrease H20 here for a final volume of 200ul.
?. [RNA extraction using MVP II kit and Kingfisher]
Prepare Binding Bead Mix according to the following:  AB1ComponentVolume/well (ul)2Binding Solution2653Magnetic Beads104Total Volume275
AB1ComponentVolume/well (ul)2Binding Solution2653Magnetic Beads104Total Volume275
Binding solution is very viscous. Pipette slowly. Make enough binding solution to allow for ~15-20% overage to account for volume lost in pipetting.
?. [RNA extraction using MVP II kit and Kingfisher]
Add 275ul Binding Bead mix to each sample well and pipette to mix.
Mix slowly! Binding solution can become very frothy, and well-to-well contamination is possible if care is not taken at this step.
?. [RNA extraction using MVP II kit and Kingfisher]
Prepare Dnase solution on ice as follows. Keep on ice until needed (step 10).  AB1ComponentVolume/well (ul)2Turbo Dnase buffer483Turbo Dnase24Total Volume50
AB1ComponentVolume/well (ul)2Turbo Dnase buffer483Turbo Dnase24Total Volume50
?. [RNA extraction using MVP II kit and Kingfisher]
Prepare remaining reagents as follows:   ABCD1Plate
  IDPlate
  typeReagentVolume/well
  (ul)2Wash
  1Deep
  WellWash
  Solution10003Wash
  2Deep
  Well80%
  Ethanol10004DnaseDeep
  WellDnase
  solution505Wash
  3Deep
  Well80%
  Ethanol5006Wash
  4Deep
  Well80%
  Ethanol5007ElutionStandard
  200ulElution
  Solution508Tip
  CombDeep
  Wellnonenone
ABCD1Plate
  IDPlate
  typeReagentVolume/well
  (ul)2Wash
  1Deep
  WellWash
  Solution10003Wash
  2Deep
  Well80%
  Ethanol10004DnaseDeep
  WellDnase
  solution505Wash
  3Deep
  Well80%
  Ethanol5006Wash
  4Deep
  Well80%
  Ethanol5007ElutionStandard
  200ulElution
  Solution508Tip
  CombDeep
  Wellnonenone
Wash and elution plates can be prepared ahead of time. Be sure to seal plates tightly to avoid evaporation, and centrifuge briefly prior to use.
?. [RNA extraction using MVP II kit and Kingfisher]
Run the following program on the Kingfisher flex. Add the plates from steps 1-8 to the machine when prompted to do so.
?. [RNA extraction using MVP II kit and Kingfisher]
Return Dnase plate to Kingisher and press start to continue protocol.
?. [RNA extraction using MVP II kit and Kingfisher]
When run has finished, remove elution plate, seal, and store at -80C.
?. [RNA extraction using MVP II kit and Kingfisher]
Discard remaing plates into appropriate biohazard waste container.
?. [RNA extraction using MVP II kit and Kingfisher]
When prompted, ~30minutes into protocol, remove DNAse plate from Kingfisher and add 50ul binding solution and 150ul isopropanol (molecular biology grade) to each sample well
?. [RNA extraction with MVP II kit and Kingrisher ]
Gather all plastic ware needed for the Kingfisher program. This will include 6 deep well plates, one tip comb, and one standard 200ul plate. Some plates can be prepared in advance to save time the day of extraction. See step 8 for more details.
?. [Optional QC]
Optional QCRun a sub-sample of extracted RNA on a Bioanalyzer or Tape Station to check RNA quality and DNA removal success.