Sybr green real time pcr master mix

Total RNA was isolated from liver (10 µg) or cells using miRNeasy (QIAGEN, Hilden, Germany) following manufacturer’s instructions. cDNA was synthesized with a High-Capacity cDNA Reverse transcription Kit (Applied Biosystems, Foster City, CA, USA). RNA quality was assessed via agarose gel and nanodrop prior to library preparation and transcriptome sequencing was conducted by Novogene Co., LTD (Beijing, China). Genes with adjusted p-value < 0.05 and |log2(Fold-Change)| > 0 were considered as differentially expressed. Relative mRNA expression was also determined by quantitative PCR using SYBR Green Real-Time PCR Master Mix (QIAGEN, Hilden, Germany). Equal amounts of cDNA samples were subjected to qPCR using the StepOnePlus (Applied Biosystems, Foster City, CA, USA) in a 96-well plate. mRNA isolated from cells were analyzed relative to Thyroxine-binding globulin (TBG), whereas, arrays were analyzed per manufactures instruction⁴³ . Qiagen RT2 custom arrays (CLAM28936C) layout and information is presented in Supplemental Table 2. The nucleotide sequences of oligonucleotides used for qPCR are presented in Supplemental Table 3.

RAW 264.7 cells were attained from Pasteur Institute of Iran; Cell culture reagents including RPMI, HEPES, bovine serum albumin (BSA), penicillin and streptomycin media supplement, DMSO, elaidic acid (EA) and fetal bovine serum (FBS) were obtained from Sigma-Aldrich (Sigma, Deisenhofen, Germany) and Gibco (Life Technologies GmbH, Karlsruhe, Germany). SYBR Green Real-Time PCR Master Mix, Kits and reagents for RNA extraction (RNeasy plus Mini Kit), reverse transcription and primers (ABCA1, PPAR-γ, β-Actin) were purchased from Qiagen (Hilden, Germany).

The relative-expression of selected gene was carried out with real-time PCR System (Rotor-Gene Q-QIAGEN). The reaction mixture contained 5 μL SYBR Green real-time PCR Master Mix (QIAGEN) which enclosed Taq DNA polymerase, dNTP, MgCl₂ and SYBR Green I dye, 0.2 μL of a 10 mM solution of sense/anti-sense primer, 0.5 μL of template cDNA added with H₂O to a total of 10 μL. The negative controls were also designed as above excluded cDNA. Thermal cycling conditions were carried out by an initial denaturation stage at 95 °C for 2 min, followed by 40 cycles at 95 °C for 15 s, 60 °C for 20 s and 72 °C for 20 s. At the completion of each run, melting curves for the amplicons were measured by raising the temperature by 0.3 °C from 57 to 95 °C while monitoring fluorescence. To determine the specificity of the PCR amplification, the melting curve for Tm, its symmetry, and the lack of non-specific peaks were checked. All tests were conducted in triplicate. The expression ratio was recorded as the fold difference in quantity of real-time PCR product from samples. Each mRNA expression value was normalized against a housekeeping gene expression (GAPDH).

RNA from skin tissues was extracted using TRIzol (#15596026, Invitrogen, USA). cDNA was synthesized with 1 mg of total RNA via reverse transcription. Then, the synthesized DNA (10 ng) was subjected to CFX96 cycler (Bio-Rad, Hercules, CA, USA) using SYBR Green Realtime PCR Master Mix (#204243, Qiagen, USA). The quantification of gene expression was normalized utilizing the expression of GAPDH as a reference gene. The relative quantification of mRNA expression was assessed using ΔΔCt method.

RNA samples were isolated from GC tissues and cells using Trizol reagent (Takara, Dalian, China) in accordance with the manufacturer’s instructions. The complementary DNA was synthesized using 2 μg of RNA template as template and reverse transcription kit (Takara) or microRNA Reverse Transcription Kit (Qiagen, Hilden, Germany). All complementary DNA products were quantitatively analyzed using SYBR Green Real-Time PCR Master Mix (Qiagen) under ABI Prism 7900 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The transcription levels of circHIPK3, HIPK3, miR-876-5p, and PIK3R1 were computed based on the 2^−ΔΔCt method. Notable, phosphate dehydrogenase (GAPDH) and small nuclear RNA U6 were used to as housekeeping genes.
The sequences of primers were listed:
circHIPK3 (F-5′-GGGTCGGCCAGTCATGTATC-3′; R-5′-ACACAACTGCTTGGCTCTACT-3′);
HIPK3 (F-5′-TCACAAGTCTTGGTCTACCCA-3′; R-5′-CACATAGGTCCGTGGATAGTTTC-3′);
miR-876-5p (F-5′-GCCGAGTGGATTTCTTTGTG-3′; R-5′-CTCAACTGGTGTCGTGGA-3′);
PIK3R1 (F-5′-ACCACTACCGGAATGAATCTCT-3′; R-5′-GGGATGTGCGGGTATATTCTTC-3′);
GAPDH (F-5′-TCCCATCACCATCTTCCAGG-3′; R-5′-GATGACCCTTTTGGCTCCC-3′);
U6 (F-5′-AACGCTTCACGAATTTGCGT-3′; R-5′-CTCGCTTCGGCAGCACA-3′).

Cells were lysed and total RNA was prepared using the Monarch Total RNA Miniprep Kit (NEB #T2010) according to the manufacturer’s protocol. cDNA was synthesized using LunaScrip RT SuperMix Kit (NEB# E3010). Quantitative real-time reverse transcription PCR (qRT-PCR) was carried out with SYBR Green Real-Time PCR Master Mix (Qiagen) in a CFX-96 C1000 platform (Rio-Rad). The transcript levels were normalized by the readings for GAPDH (see Additional file 2: Table S8 for primer sequences). RNA-seq library was prepared using a standardized rRNA depletion and dUTP protocol (NEB) followed by sequencing on either a HiSeq4000 or NextSeq500 platform (Illumina) at the Oxford Genomics Centre (Wellcome Centre for Human Genetics, Oxford, UK).

RNA was isolated using the RNeasy plus kit (Qiagen) according to the manufacturer’s instructions. RNA integrity was confirmed by agarose gel prior to cDNA synthesis. cDNA was synthesized using SuperScript II reverse transcriptase (Thermo Fisher) and real-time quantitative PCR (qPCR) was carried out using SYBR Green Real Time PCR master mix (Qiagen). Normalization of target genes was done against TATA-box-binding protein (TBP) using the 2^−ΔΔCt method. The sequences of the qPCR primers used in this study have been included in Supplementary Table 1.