Apr 28, 2024

Public workspaceMHV Tissue Titering Protocol V.1

This protocol is a draft, published without a DOI.
  • 1CU Anschutz
Siddharth Krishnamurthy
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Protocol Citation: Siddharth Krishnamurthy 2024. MHV Tissue Titering Protocol. protocols.io https://protocols.io/view/mhv-tissue-titering-protocol-dcuh2wt6
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: April 28, 2024
Last Modified: April 28, 2024
Protocol Integer ID: 98921
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Abstract
This protocol is for isolating total nucleic acid from soft tissues in mice, for subsequent analysis of Viral RNA levels
Protocol materials
ReagentRPMI 0%Merck MilliporeSigma (Sigma-Aldrich)Catalog #R7755
Reagent2.0 mm Zirconia beadsBioSpec ProductsCatalog #11079124ZX
Reagent1.1 mL Polypropylene Cluster Tubes, 12-Tube Strip Format, NonsterileVWR International (Avantor)Catalog #89005-574
Reagent12-Well Cluster Tube CapsVWR International (Avantor)Catalog #89005-728
ReagentRPMI 0%Merck MilliporeSigma (Sigma-Aldrich)Catalog #R7755
ReagentSterile 24 Well PlateVWR International (Avantor)Catalog #103348-844
ReagentDTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0632
ReagentBuffer RLT PlusQiagenCatalog #1053393
ReagentDeep 96 Well PlateVWR International (Avantor)Catalog #10011-940
ReagentEconoSpin 96 Well DNA & RNA Binding PlateEpoch Life ScienceCatalog #2020-001
ReagentEthyl alcohol, PureMerck MilliporeSigma (Sigma-Aldrich)Catalog #E7023
ReagentDeep 96 Well PlateVWR International (Avantor)Catalog #10011-940
ReagentBuffer RW1QiagenCatalog #1053394
Day -1 (Or earlier): Design Well Layout for Tissue Collection tubes
Day -1 (Or earlier): Design Well Layout for Tissue Collection tubes
Design the 96-well plate layout in which you will process (and eventually store) your samples
15m
Day -1: Prepare tissue collection tubes and plate
Day -1: Prepare tissue collection tubes and plate
Label a sufficient number of 1.1 mL 12-well cluster tubes, and then place them in a new rack. Reagent1.1 mL Polypropylene Cluster Tubes, 12-Tube Strip Format, NonsterileVWR InternationalCatalog #89005-574
10m
Retrieve enough 12-well cluster tube caps for your cluster tubes Reagent12-Well Cluster Tube CapsVWR InternationalCatalog #89005-728

Add 5-10 2.0 mm Zirconia beads to each tube that will have tissue in it Reagent2.0 mm Zirconia beadsBioSpec ProductsCatalog #11079124ZX
5m
Add Amount650 µL RPMI 0% to each bead containing tube
ReagentRPMI 0%Merck MilliporeSigma (Sigma-Aldrich)Catalog #R7755
5m
Pipetting
Cover rack and tubes with resealable plate mat and leave in Temperature4 °C until ready to use

5m
Pause
Temperature
Add Amount6 mL (Approx) of RPMI 0% to a 24 well deep well plateReagentRPMI 0%Merck MilliporeSigma (Sigma-Aldrich)Catalog #R7755 ReagentSterile 24 Well PlateVWR InternationalCatalog #103348-844



Day 0
Day 0
1h 30m
1h 30m
Dissect the mouse and open the abdominal cavity without disturbing the adipose tissue
Critical
Collect mesenteric lymph nodes, Peyer's patches, and proximal colon
Orient mesenteric adipose tissue so mesenteric lymph nodes (mLN) are easily identifiable and place all mesenteric lymph nodes (with capsule) in the 1.1 cluster tube
Extract all the Peyer's patches and place them in the cluster tube
Cut Amount0.5 cm of proximal colon (the part of the colon that connects to the cecum)

Seal tube tightly with cap. Put whole weight on it, if necessary; it must be sealed by any means necessary
1m
Bead beat plate in SPEX MiniG Tissue Homogenizer for 5 min at 1500 rpmShaker1500 rpm, Room temperature , 00:05:00
Equipment
new equipment
NAME
SPEX
BRAND
SP 1600
SKU
MiniG 1600 Automated Tissue Homogenizer and Cell Lyser
SPECIFICATIONS


5m
Seal the bead beater with the 96 well plate mat and a paper towel to examine if there is serious leakage
Thaw Concentration2 Molarity (M) DTT to make a sufficient amount of RLT + 50 mM DTT
ABC
Number of SamplesAmount of RLT Plus (mL)Amount of 2 M DTT (uL)
3012.5250
Spreadsheet to calculate how much RLT Plus and DTT will be needed
ReagentDTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0632 ReagentBuffer RLT PlusQiagenCatalog #1053393

Aliquot 350 uL of RLT+DTT buffer to appropriate wells in a new 96 well deep well plate
ReagentDeep 96 Well PlateVWR InternationalCatalog #10011-940

Pipetting
Once the plate is done beating:Centrifigation3000 x g, Room temperature, 00:02:00

2m
Centrifigation
While the plate is spinning, set up the Multi-well Plate Manifold with a 96-well silica plate on top
ReagentEconoSpin 96 Well DNA & RNA Binding PlateEpoch Life ScienceCatalog #2020-001

Equipment
Multi-well plate manifold
NAME
Vacuum Manifold
TYPE
Pall
BRAND
5017
SKU
https://www.cytivalifesciences.com/en/us/shop/lab-filtration/manifolds-and-accessories/microbiology-manifold/vacuum-manifold-and-accessories-p-36407
LINK

Once the plate is done spinning, add 100 uL of tissue supernatant to theRLT-DTT plate
Pipetting
Critical
Use the pipet that you transfer the organ homogenate to pipet up and down to mix the RLT with the homogenate
Mix
Critical
Once the 100 uL has been transferred to the RLT-DTT plate, the RNA is stable, and you can transfer the 200 uL of the remaining homogenate to another deep 96-well plate for FFU titersReagentDeep 96 Well PlateVWR InternationalCatalog #10011-940
Add 350 uL of pure ethanol to the RLT-DTT well plate and do not mix hereReagentEthyl alcohol, PureMerck MilliporeSigma (Sigma-Aldrich)Catalog #E7023
Pipetting
Turn the vacuum on to prepare the silica plate
With a new pipet tip, mix the RLT+Ethanol solution and transfer to the silica plate
Pipetting
Mix
Transfer the solution with the same pipet you mix the ethanol with the pipet
The solution should take ~1 minute, usually less to suck through
Wash the silica plate with 350 uL RW1 Buffer
ReagentBuffer RW1QiagenCatalog #1053394
Pipetting
Wash
Wash the silica plate with 800 uL RPE Buffer (10 mM Tris-Cl + 80% Ethanol)
Pipetting
Wash
Dry the plate Centrifigation3000 x g, Room temperature, 00:02:00

2m
Centrifigation
Place plate onto an elution skirted plate.
Elute by adding 75 uL of DEPC treated water to the wells and Centrifigation3000 x g, Room temperature, 00:02:00

2m
Centrifigation
Pipetting