Sybr green master mix

ChIP assay was performed as described previously40 (link). Crosslinked, sonicated chromatin was precleared before being incubated with 2.5 μg of the indicated antibodies and rotated at 4 °C overnight. Normal mouse or rabbit IgG (Millipore) was used for the mock immunoprecipitation. After extensive washes, immunocomplexes were treated with Proteinase K and decrosslinked. Bound DNA in the precipitates, as well as input DNA (1/10 fragmented chromatin), was extracted, purified, and subjected to real-time PCR analysis using primers corresponding to a region of the repo promoter (−1,337 to −1,138; forward primer, 5′-ATCTGGCCAAAAGGTCACAC-3′; reverse primer, 5′-CTCATGAGGGTGGTTCCATT-3′). Real-time PCRs were conducted on the Bio-Rad iQ5 Gradient Real-Time PCR system, using the 2 × SYBR-Green Master mix (Bio-Rad, USA). Results were corrected for non-specific binding to IgG (whose values were considered as 1 and not shown in the bar graphs). Triplicate PCRs for each sample were carried out.

Gene-specific primers (Table 1) were used in PCR reactions (20 µl) containing 7µl of ddH₂O, 10 µl of 2×SYBR Green MasterMix (Bio-Rad), 1 µl of each specific primer (10 µM), and 1 µl of first-strand cDNA template. The qPCR program included an initial denaturation for 3 min at 95°C followed by 40 cycles of denaturation at 95°C for 10 s, annealing for 30 s at 55°C, and extension for 30 s at 72°C. For melting curve analysis, a dissociation step cycle (55°C for 10 s, and then 0.5°C for 10 s until 95°C) was added. The reactions were set up in 96-well format Microseal PCR plates (Bio-Rad) in triplicates. All experiments were replicated in triplicate.
Reactions were performed in a MyiQ single Color Real-Time PCR Detection System (Bio-Rad). Existence of single peaks in melting curve analysis was used to confirm gene-specific amplification and rule out non-specific amplification and primer-dimer generation. The qRT-PCR was determined for each gene using slope analysis with a linear regression model. Relative standard curves for the transcripts were generated with serial dilutions of cDNA (1/5, 1/25, 1/125, 1/625, and 1/3125). The corresponding qRT-PCR efficiencies (E) were calculated according to the equation: E = (10^[-1/slope] -1)×100.

Total RNA was isolated using Trizol reagent from control and shRNA treated cells following the instructions of the manufacturer. RNA was transcribed using the RT-PCR kit (Promega, Madison, WI, USA). Gene expression was detected by quantitative real-time RT-PCR (qRT-PCR) using 2 × SYBR Green master mix (BioRad, Hercules, CA, USA). The primer sequences are presented in Supplementary Table S3. Each qPCR experiment was carried out three independent times in triplicate.

The RNA was extracted using Qiagen miRNeasy Micro kit (catalog 1071023). Next, 100–500 ng of RNA was used for cDNA synthesis (iScript cDNA synthesis, Bio-Rad, 1708890). No-RT and no-RNA controls were used to ensure DNase digestion and no residual contamination. The cDNA was diluted to different concentrations and eventually were diluted by 5-fold to obtain qPCR values in the linear range. qPCR primers (Supplemental Table 6) were from PrimerBank. A dilution curve that included primers and qPCR Master Mix were utilized to ensure that PCR primers did not self-anneal and to reduce background amplification. The qPCR was performed with 2× SYBR Green Master Mix (Bio-Rad, 170-8880), 0.5 μM primer (forward and reverse), and 5 μL of 1:5 diluted cDNA and rest water. All qPCR reactions had 3 technical replicates and at least 3 biological replicates per genotype. All values were normalized to the reference gene Gapdh.

Details regarding RT-qPCR amplifications and programs were provided in Yang et al. (2014) (link); briefly, PCR reactions (20 μl) contained 7.0 μl of ddH₂O, 10.0 μl of 2× SYBR Green Master Mix (Bio-Rad), 1.0 μl of each specific primer (10 μM), and 1.0 μl of first-strand cDNA template. Reactions occurred in 96-well format Microseal PCR plates (Bio-Rad) in triplicate. Reactions were performed in a MyiQ single Color Real-Time PCR Detection System (Bio-Rad). The standard curve for each candidate was generated from cDNA serial dilutions (1/5, 1/25, 1/125, 1/625, and 1/3125). The corresponding RT-qPCR efficiencies (E) were expressed in percentage according to the equation: E = (10^[-1/slope]-1) × 100. The coefficients of determination (R²) for a linear regression model with one independent variable was obtained according to the method in excel.

Total DNA from cultured cells was isolated using a NucleoSpin Tissue Purification Kit (Macherey-Nagel; 740952) according to the manufacturer's protocol. The mtDNA content was quantified and normalized to nuclear DNA (nucDNA). Measurements were performed using quantitative real-time PCR (qPCR) with primers for cytochrome b (CytB, mtDNA) and amyloid precursor protein (APP, nucDNA): CytB-Fw 5′-GCCTGCCTGATCCTCCAAAT-3′, CytB-Rv 5′-AAGGTAGCGGATGATTCAGCC-3′; APP-Fw 5′-TTTTTGTGTGCTCTCCCAGGTCT-3′, APP-Rv 5′-TGGTCACTGGTTGGTTGGC-3′. Each qPCR reaction contained 25 ng of purified total DNA, 2.5 mM of forward and reverse primers, 10 μl of 2× SYBR Green Master Mix (Bio-Rad; #4309155) and was measured in triplicate in Hard-Shell 96-Well PCR plates (Bio-Rad; #HSP9635). The amplification program was as follows: 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. The fluorescent signal was acquired by a CFX96 Real-Time System (Bio-Rad). The absence of non-specific amplicons was confirmed using melting curve analysis. Fold changes in the relative mtDNA copy number after C17orf80 depletion or ddC treatment were calculated using the 2^−ΔΔCT method.