Quantifying ATP and mtDNA Levels

ATP was measured according to manufactures protocol using the ATP Determination Kit (Molecular Probes). COS-7 cells were transfected with siRNAs as previously described and homogenized in ATP assay buffer at 48 hours post transfection, or 5 whole zebrafish embryos were homogenized in ATP assay buffer at 24 hours post fertilization. Homogenates were spun at 12,000 g for 5 min to pellet tissues debris. 10 ul supernatant were added to a 97 well dish containing 90 ul of the luciferase reaction mix from the ATP Determination Kit (Molecular Probes). Luminescence was immediately read on a luminometer. ATP values were calculated by comparison to a standard curve generated from a series of ATP concentrations. Values were then normalized by the total amount of protein present in each sample. Samples were run in triplicate. mtDNA levels were quantified by quantitative PCR as described^{38 (link)}.

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Abrams A.J., Hufnagel R.B., Rebelo A., Zanna C., Patel N., Gonzalez M.A., Campeanu I.J., Griffin L.B., Groenewald S., Strickland A.V., Tao F., Speziani F., Abreu L., Schüle R., Caporali L., La Morgia C., Maresca A., Liguori R., Lodi R., Ahmed Z.M., Sund K.L., Wang X., Krueger L.A., Peng Y., Prada C.E., Prows C.A., Bove K., Schorry E.K., Antonellis A., Zimmerman H.H., Abdul-Rahman O.A., Yang Y., Downes S.M., Prince J., Fontanesi F., Barrientos A., Nemeth A.H., Carelli V., Huang T., Zuchner S, & Dallman J.E. (2015). Mutations in the UGO1-like protein SLC25A46 cause an optic atrophy spectrum disorder. Nature genetics, 47(8), 926-932.

Publication 2015

ATP Determination Kit

Assay Buffer Cos 7 cells Dish Embryos Fertilization Luciferase Luminescence Molecular probes Mtdna Protein Sirnas Tissues Transfection Zebrafish

Corresponding Organization :

Other organizations : Dr. John T. Macdonald Foundation, University of Miami, Cincinnati Children's Hospital Medical Center, Ospedale Bellaria, Istituti di Ricovero e Cura a Carattere Scientifico, Istituto delle Scienze Neurologiche di Bologna, University of Michigan–Ann Arbor, Policlinico S.Orsola-Malpighi, University of Bologna, University of Maryland, Baltimore, University of Mississippi Medical Center, Baylor College of Medicine, John Radcliffe Hospital, University of Oxford

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Variable analysis

independent variables

SiRNA transfection in COS-7 cells
Time post-transfection (48 hours)
Time post-fertilization for zebrafish embryos (24 hours)

dependent variables

ATP levels
MtDNA levels

control variables

Homogenization of cells/embryos in ATP assay buffer
Centrifugation at 12,000 g for 5 minutes to pellet tissue debris
Addition of 10 μL supernatant to 97-well plate containing 90 μL luciferase reaction mix
Immediate luminescence reading on a luminometer
ATP standard curve for quantification
Normalization of ATP values by total protein amount
Triplicate samples

positive controls

ATP standard curve

negative controls

Not explicitly mentioned

Annotations

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