Determination of Mitochondrial DNA Upon Drug Treatment

Authors

Michel Perron and Joy Y. Feng

Summary

This protocol describes the method to quantify mitochondrial DNA level against nuclear DNA level, by amplification of a fragment of the mitochondrial specific cytochrome b gene and amplification of a fragment of  the β-actin gene, and is analysed using 2^-ΔΔCT formula.

Introduction

Drug-induced mitochondrial injury can be caused by many different mechanisms including inhibition of mitochondrial DNA replication, transcription, translation, and altered protein function. Determination of the level of mitochondrial DNA relative to the nuclear DNA levels provides important information on potential mitochondrial toxicity.

Materials

1. HepG2 cells (ATCC, catalog number: HB 8065)
2. DMSO (cell culture grade) (Sigma-Aldrich, catalog number: D2650)
3. Phosphor-buffered saline (PBS) (Life Sciences, catalog number: 10010049)
4. QIAamp DNA mini kit (QIAGEN, catalog number: 51304)
5. TaqMan universal mastermix (Life Technologies, Applied Biosystems®, catalog number:4352042)
6. β-actin Assay-on-Demand kit (Life Technologies, Applied Biosystems®, catalog number:4331182)
7. Eagle’s minimum essential medium (Life Technologies, Gibco®, catalog number: 41090)
8. GlutaMAXTM
9. Fetal bovine serum (FBS) (HyClone, catalog number: SH30071.03)
10. 100 units/ml penicillin, 100 units/ml streptomycin (Life Technologies, Gibco®, catalog
number: 15140)
11. Sodium pyruvate (Life Technologies, Gibco®, catalog number: 11360)
12. Cells were cultured in Eagle’s minimum essential medium (see Recipes)
Note: Cells were cultured in Eagle’s minimum essential medium.

Equipment
1. 12-well plates (Corning, catalog number: 3513)
2. ABI Prism 7900HT Fast Real-Time PCR system (Life Technologies, Applied Biosystems®)

Recipes

Eagle’s minimum essential medium

GlutaMAXTM
10% fetal bovine serum
100 units/ml penicillin
100 units/ml streptomycin
1 mM sodium pyruvate

Procedure

1. HepG2 cells were seeded into 12-well plates at a density of 2 x 105
cells per well and allowed to attach overnight. The volume of medium was 1.0 ml in each well.
2. After the overnight incubation, the media in each well was replaced with 1.0 ml of fresh
media containing tested compounds and controls, and incubated for 10 more days. The
media was replaced with fresh media and compounds every 3 to 4 days. The DMSO
concentration was kept at 1.0% for all treatments including control samples (no drug,
DMSO only).
3. Following the incubation, the cells were washed once with PBS and the total DNA was
extracted from the cells using the QIAamp DNA Mini Kit according to the manufacturer’s
protocol.
4. Real-time PCR reactions were performed using TaqMan universal mastermix in an ABI
Prism 7900HT Fast Real-Time PCR System.
5. Quantification of mtDNA was achieved by amplification of a fragment of the mitochondrial specific cytochrome b gene using the primers and probe described in table 1.Chromosomal DNA was quantified by the amplification of a fragment of the β-actin gene using a β-actin Assay-on-Demand kit.
6. Amplification reactions for the quantification of mitochondrial and chromosomal DNA
were performed independently using approximately 25 ng of total DNA in a volume of 20
μl.
7. Data analysis
a. The relative amount of mtDNA in treated samples was determined using a relative
quantification method based upon the 2^-ΔΔCT formula (Livak and Schmittgen, 2001).
b. The amount of mtDNA (% mtDNA) in compound treated samples relative to the
DMSO treated controls was calculated based upon the following formula:

% mtDNA = 100 x 2^-ΔΔCT
ΔΔCT = ΔC_T, _treated – ΔC_T, _control
ΔC_{T, treated} = (C_T, cyt b – C_T, β-actin) _treated
ΔC_{T, control} = (C_T, cyt b – C_T, β-actin) _control
C_T, cyt b and C_T, β-actin represent the cycle threshold values for the amplification of cytochrome b and β-actin, respectively, as determined by the computational analysis of amplification curves using the ABI Prism software. The final results are presented as the mean % mtDNA ± SD from 3 independent experiments, each performed in triplicate.
The 2-ΔΔCT method was validated for cytochrome b and β-actin genes by determining the ΔCT values for amplification reactions containing various amounts of total cellular DNA. Minimal differences were observed in the ΔCT values in samples containing 5 to 40 ng of total cellular DNA; indicating that neither the amplification nor detection efficiencies of cytochrome b and β-actin were affected by the amount of DNA template within the dilution range relevant for the quantitative analysis performed in this study Table 2.
The effect of a positive control compound ddC (dideoxy cytidine) is shown in Table 3.

References

1. Feng, J. Y., Cheng, G., Perry, J., Barauskas, O., Xu, Y., Fenaux, M., Eng, S., Tirunagari,
N., Peng, B., Yu, M., Tian, Y., Lee, Y. J., Stepan, G., Lagpacan, L. L., Jin, D., Hung, M.,
Ku, K. S., Han, B., Kitrinos, K., Perron, M., Birkus, G., Wong, K. A., Zhong, W., Kim, C. U.,
Carey, A., Cho, A. and Ray, A. S. (2014). Inhibition of hepatitis C virus replication by GS-
6620, a potent C-nucleoside monophosphate prodrug. Antimicrob Agents Chemother
58(4): 1930-1942.
2. Livak, K. J. and Schmittgen, T. D. (2001). Analysis of relative gene expression data using
real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4): 402-408.

Documents for download

• 1377 Determination of Mitochondrial DNA Upon Drug Treatment.pdf

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