Power sybr green polymerase chain reaction master mix

ρ0 cells have mtDNA-CN close to zero (0.1 is typically reported in HeLa ρ0) (10 ). We aimed to have a similarly low number in our BET-1A and BEAS-2B ρ0 cells.
The qPCR reaction was performed in triplicate for each sample at a volume of 20 μl per well. Each qPCR reaction contained 2 μl diluted sample DNA (varying concentrations of DNA tested as described below), 10 μl Power-SYBR Green Polymerase Chain Reaction (PCR) Master Mix (Applied Biosystems), 6 μl PCR water, and 2 μl primer mix containing 10 nM forward and reverse primers. qPCR reactions were performed on Applied Biosystems QuantStudio5 using MicroAmp Optical 384-Well Reaction Plates. The real-time PCR conditions were the following: initial denaturation 2 minutes at 50°C ramp to 95°C for 10 minutes followed by 40 amplification cycles of (95°C for 15 seconds denaturation ramp down to 60°C for 60 secs for annealing and extension) followed by a final 95°C for 15 second hold. The cycle threshold values (Ct values) were determined automatically via QuantStudio Design & Analysis software.
To optimize the qPCR methods, we tested several primer sets detecting both mtDNA and nDNA. Primer sets tested are reported in Table 1. For this purpose, master mix was prepared by mixing DNA, SYBR-Green and PCR water, and then added to the PCR plates which contain the 2 μL of primer sets.

Total RNA from kidney tissues was extracted using TRIzol reagent (Invitrogen, Middlesex, MA, USA). Complementary DNA was produced from messenger RNA in a reverse transcription reaction by using Reverse Transcriptase Premix (Elpis Biotech, Daejeon, Republic of Korea) and the primers (GenoTech Corporation, Daejeon, Republic of Korea) were then amplified using a Power SYBR^® Green polymerase chain reaction (PCR) Master Mix (Applied Biosystems, Foster City, CA, USA) with gene-specific primer pairs (Table 2). Relative amplitude of gene expression was quantified using an ABI 7500 FAST instrument and 7500 Software v2.0.6 (Applied Biosystems, Foster City, CA, USA). GAPDH was used to normalize the quantitative gene expression data.

Total RNA was isolated from kidney tissues and HK-2 cells using a NucleoSpin^® RNA II kit (Macherey-Nagel, Düren, Germany). cDNA was synthesized using Reverse Transcriptase Premix (Elpis Biotech, Daejeon, Korea) and amplified in a Power SYBR® Green polymerase chain reaction (PCR) Master Mix (Applied Biosystems, Warrington, UK) with gene-specific primer pairs (HIF-1α: F; 5’-TGCCCCAGATTCAAGATCAGC-3’, R; 5’-GGCTGGGAAAAGT TAGGAGTGT-3’) Quantitative real-time PCR was performed on an ABI 7500 FAST instrument (Applied Biosystems, Warrington, UK). The expression levels of mRNAs were normalized to the expression of GAPDH.