TITLE: Mouse Tissue mtDNA Copy Number Protocol
AUTHORS: Livia Hecke Morais, Jack Devine

[DESCRIPTION]
 Mouse Tissue mtDNA Copy Number Protocol developed in the Picard lab at Departments of Psychiatry and Neurology, Robert N Butler Columbia Aging Center, Columbia
University Irving Medical Center, New York, NY, USA

[STEPS]
SECTION: Consumables, with catalog numbers:
1. 
BD Vacutainer Tubes (BD #363083)
Tris HCl (Sigma #T3253)
Tween 20, 10% (Sigma #P1379)
Nuclease free water (Thermofisher Scientific #AM9939)
Proteinase K (20 mg/mL) (Thermofisher Scientific #AM2548)
Stericup Quick Release-GV Sterile Vacuum Filtration System – 500 mL (Millipore Sigma S2GVU05RE)
BrandTech 96-well semi-skirted plate (BrandTech #781375/VWR #10141-434)
BrandTech 8-strip domed tube caps (BrandTech #781340/VWR #80087-132)
2x TaqMan Universal MasterMix Fast (LifeTech #4444965)
MicroAmp‱ Optical 384-Well Reaction Plate with Barcode (ThermoFisher
#4309849) MicroAmp‱
Optical Adhesive Film (ThermoFisher #4311971) Validated
mtDNA/nDNA Primer & Probe sequences (idtDNA.com)
Additional consumables not listed here: 1.5 mL Eppendorf tubes, pipette tips as needed.
SECTION: Lysis buffer for mtDNA measurement
3. Keep everything sterile. The below calculations provide sufficient lysis buffer for
1 L of complete lysis buffer, or about 5,000 reactions.
SECTION: Lysis buffer for mtDNA measurement
4. Tris-HCl (0.38M, pH 8.5)                                                                                     (Sigma #T3253)
-        18.168 g of Tris HCl (MW = 157.60 g/mol) in 200 mL of nuclease-free dH2O
-        pH to 8.5 with 5M NaOH (about 12.5 mL, but measure pH continuously)
-        Note: Accurate pH is critical for proper extraction. Make sure your pH meter has been recently calibrated.
-       Make up to 300 mL with nuclease-free dH2O
-       Sterile filter Tris-HCl buffer using a SteriCup  (Make 6 mL or 12 mL aliquots, depending on if you want to run 1 or 2 plates at once)
SECTION: Lysis buffer for mtDNA measurement
5. Tween 20, 10%                                                                                                   (Sigma #P1379)
-       540 mL of ultrapure dH2O  
-       60mL of Tween 20 (wash tip thoroughly or leave in solution)  
-       Sterile filter Tween 10% using a SteriCup  (Make 12 mL or 24 mL aliquots, depending on if you want to run 1 or 2 plates at once)  
NB: Thawing these 10% Tween aliquots can be slow. I recommend using a 37ºC bead bath.
SECTION: Lysis buffer for mtDNA measurement
8.  
 
 
SECTION: Lysis buffer for mtDNA measurement
9.  
 
 
SECTION: Lysis buffer for mtDNA measurement
6.     ddH2O (Sterile, nuclease-free)                                            (Thermofisher Scientific #AM9939)
(Make 5 mL aliquots or use direct from bottle)
SECTION: Lysis buffer for mtDNA measurement
7. Proteinase K (20 mg/mL)                                                     (Thermofisher Scientific #AM2548)
-       To be put in fresh from 20 mg/mL                        (1:100 dilution, final 200 µg/mL)
 
Store aliquots at -30°C
SECTION: Lysis buffer for mtDNA measurement
10.     
Lysis for Mouse Tissue
       Thaw all reagents and prepare lysis buffer as described above.
       Add 180 μL of lysis buffer in each well of a 96-well semi-skirted plate. We use BrandTech #781375. Supplied in US by VWR #10141-434.
       Add 20 μL of sample into each well.
       We recommend one extra well with 200 μL lysis buffer only to use as a “no template control”.
       Seal wells with 8-strip domed caps. We use BrandTech #781340. Supplied in US by VWR #80087-132.
      To ensure proper mixing of the sample and buffer, vortex vigorously. 
       To pull the sample/buffer mixture to the bottom of the wells, quickly centrifuge
at 1,000g for 10 sec before lysis.
       Proceed to heat-activated lysis of the samples in a Thermocycler for 16 hours at 55oC,
followed by heat inactivation for 10 minutes at 95oC. 
SECTION: Lysis buffer for mtDNA measurement
11. o   
 
This digested sample can be used directly as template DNA in qPCR for cf-DNA measurements. 
       Before performing qPCR, vortex and centrifuge again the template DNA-containing tube(s)/plate(s) at 1,000g for 10 sec at room temperature.  If not using within 24 - 48h, freeze the DNA samples at -80ºC or colder.

2. 
SECTION: 6.	qPCR data analysis and error checking
12. Compute the mean, standard deviation, and coefficient of variation (CV) for the cycle thresholds (Ct) across each triplicate for each sample. 
SECTION: 6.	qPCR data analysis and error checking
15. The average Ct for each sample should be used compute copies/mL.
SECTION: 6.	qPCR data analysis and error checking
13.  If the CV across the 3 triplicates is >2%, check the triplicates to see if any of the wells has a Ct that is >1 unit different from the other two, which could indicate a qPCR failure in that well. 
SECTION: 6.	qPCR data analysis and error checking
14. In the case where amplification has failed in a well, remove the outlier. 
SECTION: Appendix A: qPCR Reagents and MasterMix Recipe
16.  
 
 
SECTION: Appendix A: qPCR Reagents and MasterMix Recipe
17. 2x TaqMan Universal MasterMix Fast                                            (Life Tech #4444965)
MicroAmp‱ Optical 384-Well Reaction Plate with Barcode       (ThermoFisher #4309849)
MicroAmp‱ Optical Adhesive Film                                               (ThermoFisher #4311971)
Validated mtDNA/nDNA Primer & Probe sequences                    (idtDNA.com)
SECTION: Appendix A: qPCR Reagents and MasterMix Recipe
18.  
 
 
SECTION: Appendix A: qPCR Reagents and MasterMix Recipe
19. -       Reconstitute primers in appropriate volume of nuclease free water to achieve
100 μM stock concentration.
-       Combine 120 μL each of 100 μM forward and reverse primers with 960 μL
nuclease free water to achieve 10 μM working concentration.
-       Store primers at -30ºC or -80ºC until use.
-       Reconstitute probe in appropriate volume of nuclease free water to
achieve 100 μM stock concentration.
-       Dilute 100 μM stock probe 20x to achieve 5 μM working concentration.
-       Store probes at -30oC or -80oC until use (avoid
freeze-thaw) and protect probes from light.