TITLE: MHV Tissue Titering Protocol
AUTHORS: Siddharth Krishnamurthy

[DESCRIPTION]
This protocol is for isolating total nucleic acid from soft tissues in mice, for subsequent analysis of Viral RNA levels

[STEPS]
SECTION: Day -1: Prepare tissue collection tubes and plate
5. Add 650 µLto each bead containing tube
  
SECTION: Day -1: Prepare tissue collection tubes and plate
2. Label a sufficient number of 1.1 mL 12-well cluster tubes, and then place them in a new rack.     
SECTION: Day -1: Prepare tissue collection tubes and plate
4. Add 5-10 2.0 mm Zirconia beads to each tube that will have tissue in it   
SECTION: Day -1: Prepare tissue collection tubes and plate
6. Cover rack and tubes with resealable plate mat and leave in 4 °C until ready to use

SECTION: Day -1 (Or earlier): Design Well Layout for Tissue Collection tubes
1. Design the 96-well plate layout in which you will process (and eventually store) your samples
SECTION: Day 0
8. Dissect the mouse and open the abdominal cavity without disturbing the adipose tissue
SECTION: Day 0
9. Collect mesenteric lymph nodes, Peyer's patches, and proximal colon
SECTION: Day 0
9.1. 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
SECTION: Day 0
9.2. Extract all the Peyer's patches and place them in the cluster tube
SECTION: Day 0
9.3. Cut 0.5 cmof proximal colon (the part of the colon that connects to the cecum)

SECTION: Day 0
10. Seal tube tightly with cap. Put whole weight on it, if necessary; it must be sealed by any means necessary
SECTION: Day -1: Prepare tissue collection tubes and plate
7. Add 6 mLof RPMI 0% to a 24 well deep well plate    



SECTION: Day -1: Prepare tissue collection tubes and plate
3. Retrieve enough 12-well cluster tube caps for your cluster tubes   

SECTION: Day 0
11. Bead beat plate in SPEX MiniG Tissue Homogenizer for 5 min at 1500 rpm  
 


SECTION: Day 0
11.1. Seal the bead beater with the 96 well plate mat and a paper towel to examine if there is serious leakage
SECTION: Day 0
14.      Once the plate is done beating:3000 x g, 2 min 

SECTION: Day 0
15.      While the plate is spinning, set up the Multi-well Plate Manifold with a 96-well silica plate on top
  

 

SECTION: Day 0
16. Once the plate is done spinning, add 100 uL of tissue supernatant to theRLT-DTT plate 
SECTION: Day 0
16.1. Use the pipet that you transfer the organ homogenate to pipet up and down to mix the RLT with the homogenate
SECTION: Day 0
16.2. 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 titers  
SECTION: Day 0
17. Add 350 uL of pure ethanol to the RLT-DTT well plate and do not mix here  
SECTION: Day 0
18. Turn the vacuum on to prepare the silica plate
SECTION: Day 0
19. With a new pipet tip, mix the RLT+Ethanol solution and transfer to the silica plate
SECTION: Day 0
19.1. Transfer the solution with the same pipet you mix the ethanol with the pipet 
SECTION: Day 0
19.2. The solution should take ~1 minute, usually less to suck through
SECTION: Day 0
20. Wash the silica plate with 350 uL RW1 Buffer
  
SECTION: Day 0
21. Wash the silica plate with 800 uL RPE Buffer (10 mM Tris-Cl + 80% Ethanol)
SECTION: Day 0
22.  Dry the plate 3000 x g, 2 min 

SECTION: Day 0
23.  Place plate onto an elution skirted plate.
SECTION: Day 0
23.1. Elute by adding 75 uL of DEPC treated water to the wells and 3000 x g, 2 min 

SECTION: Day 0
12. Thaw 2 Molarity (M)DTT to make a sufficient amount of RLT + 50 mM DTT  
 
 
 
   

SECTION: Day 0
13. Aliquot 350 uL of RLT+DTT buffer to appropriate wells in a new 96 well deep well plate