Abi quantstudio 7

The relative expression of mRNA was examined by real‐time PCR using SYBR Green Master Mix (Takara) on an ABI QuantStudio™ 7 real‐time PCR system (Thermo Fisher Scientific, Wlathm, MA, USA). The thermal cycling conditions included an initial hold period at 95 °C for 30 s followed by a two‐step PCR program, comprising 95 °C for 5 s and 60 °C for 30 s with 40 cycle repeats. To evaluate the relative expression, the Ct value of the examined sample gene was first normalized with the Ct value of endogenous Gapdh and then with the Ct values of the respective control sample gene. All experiments were performed with three biological repeats and three technique repeats. Student's t‐test was used for statistical analysis. The primer sequences for real‐time PCR are provided in Table 1.

RNA was extracted from cells using Isolate II RNA mini kit (Bioline, Alexandria, NSW, Australia) according to manufacturer's instructions. RNA (up to 2 μg) was treated to remove genomic DNA with 1 unit of DNase I (New England Biolabs, Beverly, MA) in 10 μL, incubated at 37°C for 10 minutes then DNase Inactivated at 75°C. cDNA was synthesized from DNase-treated RNA using a Tetro cDNA synthesis kit (Bioline) according to manufacturer's instructions. The oligonucleotide primers employed in these analyses are listed in Supplementary Table 1. Samples were analyzed in duplicate using a qPCR mix containing 25-50 ng cDNA, 1x iTAQ Sybr Green PCR master mix (Biorad) and 0.2 μM of each primer in a total volume of 10 μL. qPCR reactions were run on ABI Quantstudio7 (Thermo Fisher Scientific) with the following conditions: 10 mins of denaturation at 95°C; 40 cycles of: 95°C (15s), 60°C (60s). To discriminate primer specific amplicon from primer dimers or unspecific PCR products, we also performed melt curve analysis with the following conditions: 95°C (15s), 60°C (60s), 95°C (15s). Comparative (delta-CT) values were calculated using Gus as a housekeeping gene, and expressed as relative to the vehicle control.

Total RNA was extracted from the cortex of the treated kidney with RNeasy Plus Universal Mini Kit (Qiagen) according to the manufacturer’s directions. Aliquots (10 μl) of 500 ng of RNA were reverse transcripted to complementary DNA using PrimeScript RT reagent kit (Takara Bio) according to manufacturing protocol. Quantitative real-time reverse-transcriptase polymerase chain reaction was performed using ABI PowerUp SYBR Green Master Mix on an ABI Quant Studio 7 (Thermo Fisher Scientific). Primers amplifying the rat mRNA regions designed using the Primer Express software package v3.0.1 (Thermo Fisher Scientific, No4363991) or referred as previously published^{43 (link)}. The PCR primer sets used for cDNA amplification were as follows: caspase-3 sense 5- AAT TCA AGG GAC GGG TCA TG -3, antisense 5- GCT TGT GCG CGT ACA GTT TC -3; CCL2 sense 5-CTA TGC AGG TCT CTG TCA CGC TTC-3, antisense 5-CAG CCG ACT CAT TGG GAT CA-3; IL-6 sense 5-ATT GTA TGA ACA GCG ATG ATG CAC-3, antisense 5-CCA GGT AGA AAC GGA ACT CCA GA-3; TIMP-1 sense 5-CGA GAC CAC CTT ATA CCA GCG TTA-3, antisense 5-TGATGTGCAAATTTCCGTTCC-3; β-actin 5-GGA GAT TAC TGC CCT GGC TCC TA-3, antisense 5-GAC TCA TCG TAC TCC TGC TTG CTG-3. Data are expressed as the comparative cycle threshold. The normalized cycle threshold value of each gene was obtained by subtracting the cycle threshold value of β-actin RNA.

For gene expression analysis mice were sacrificed in ad libitum conditions 2 h post injection with PBS, AMPH or PEGyAMPH, tissues were collected and immediately frozen. RNA from SCG and BAT was extracted using RNeasy Plus Micro Kit (Qiagen) and from all other tissues the RNA was extracted using PureLink RNA Mini Kit (Life Technologies) according to manufacturer’s instructions. From total tissue RNA of all samples complementary DNA was reverse-transcribed by using SuperScript II (Invitrogen) and random primers (Invitrogen). Quantitative PCR was performed with SYBR Green (Invitrogen) in ABI QuantStudio 7 (Applied Biosystems). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as housekeeping gene to normalize liver and gastrocnemius muscle tissue samples. Acidic ribosomal phosphoprotein P0 (Arbp0) was used as housekeeping gene to normalize adipose tissues samples. The list of primers used is shown in Table S1.

Circulating miRNAs that were significantly up-regulated in individuals affected by FSHD1 were examined in a separate set of non-overlapping CINRG patients used as a validation group. For this group, FSHD patients had a confirmed diagnosis of FSHD1 (n = 12; 9 females, 3 males) and were compared to healthy volunteer control samples (n = 7; 4 females, 3 males). RNA was isolated from 150 µL of plasma using Trizol LS liquid extraction. Total RNA was converted to cDNA using a High Capacity Reverse Transcription Kit with multiplexed RT primers, pre-amplified using PreAmp MasterMix with multiplexed TM primers, and quantified with individual TaqMan assays on an ABI QuantStudio 7 real time PCR machine (Applied Biosystems; Foster City, CA, USA). Assay IDs used are: miR-32-002109, miR-103-000439, miR-505-002089, miR-146b-001097, miR-29b-000413, miR-34a-000426, miR-141-000463, miR-98-000577, miR-576-3p-002351, miR-9-000583, and miR-142-3p-000464. Expression levels of all miRNAs were normalized to the geometric mean of multiple control genes (miR-150 and miR-342-3p) determined previously to be stable circulating miRNA controls [35 (link),51 (link)]. Expression was analyzed in FSHD1 versus healthy control patients via t-test analysis, including assessment of directionality. A p-value of ≤ 0.05 was considered significant. Data are presented as mean ± SEM unless otherwise noted.