Dna master sybr green 1 kit

RNA was isolated from DCs (after 7 days of differentiation) using the NucleoSpin RNA XS kit (Macherey-Nagel, Hoerd, France) as recommended by the manufacturer and photometrically measured and quantified using the Qubit RNA Broad-Range (BR) Assay Kit and a corresponding 3.0 fluorometer (Invitrogen/Thermo Fisher Scientific, Waltham, USA). Subsequently, cDNA synthesis was performed using 200 ng of RNA and the SuperScript VILO Kit (Invitrogen/Thermo Fisher Scientific, Waltham, USA), as recommended by the manufacturer. To quantify mRNA expression levels, the real-time LightCycler System (Roche Diagnostics, Mannheim, Germany) was used. For the PCR reactions, DEPC-treated water, DNA Master SYBR Green I kit (Roche, Mannheim, Germany), and commercial primer kits (IL-12p35, IL-12p40, IL-12Rβ1, IL-12Rβ2, IFN-γ, TNF-α, IL-4, IL-8, IL-10, MIP-1α, MIP-1β, and G-CSF) from Search LC (Heidelberg, Germany) were used for 40 amplification cycles of the target DNA. The target gene transcript levels were normalized to those of the housekeeping gene GAPDH (Search LC, Heidelberg, Germany). Finally, the ratio of the β-TCP/Co group was set as 1 (control), and x-fold induction indices in relation to this control were calculated.

RNA was isolated from HUVECs using the NucleoSpin RNA kit (Macherey-Nagel, Hoerd, France) according to the manufacturer’s guidelines. After isolation, RNA was quantified using a Nanodrop micro-volume spectrophotometer (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA). Then, cDNA synthesis was performed using the SuperScript™ VILO™ cDNA synthesis kit according to the instructions of the manufacturer (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA). To quantify mRNA expression levels, the real-time LightCycler System (Roche Diagnostics, Mannheim, Germany) was used. For the PCR reactions, DEPC-treated water, DNA Master SYBR Green I kit (Roche, Mannheim, Germany), and commercial primer kits (FLT-1, KDR, HGF, MET, IL-8, HIF-1α, MMP-1, TIMP-1, IGFBP-1, IGFBP-2, VCAM-1, and ANGPT-1) from Search LC (Heidelberg, Germany) were used for 40 amplification cycles of the target cDNA following the manufacturer’s instructions. The target gene transcript levels were normalized to those of the housekeeping gene GAPDH. X-fold induction values were calculated related to the corresponding control. All cDNA samples (from 3 independent experiments) for each experimental group (+VEGF (positive control), 1 µM E7-QK, 1 µM QK, −VEGF (negative control)) were analyzed in triplicates.

To confirm microarray results, the relative abundance of gene transcripts were analysed by qPCR (S4 Table). One μg of total RNA from 8-day old fungal mycelia grown on cellophane placed on CM agar was reverse transcribed using PrimeScript RT reagent Kit (Takara). The average threshold cycle (Ct) was normalized against actin transcript and relative quantification of gene expression was calculated by the 2^ΔΔCt method[85 ]. Primer efficiency was tested using dilutions of cDNA samples. qPCR reactions were carried out with 1 μl of reverse transcribed products and fast-start DNA master SYBR green I kit (Roche Diagnostics) in a final reaction of 20 μl using the following program: one cycle of 95°C for 4 min and 40 cycles of 94°C for 30 s and 60°C for 30 s. The Ct (threshold cycle) provided a measure for the starting copy numbers of the target genes. Three technical repetitions from three independent biological experiments were used for each gene. For ROS detection in M. oryzae fungal structures, NBT staining[65 (link)] and quantification method of pixel intensities in hyphal tips[86 (link)] were carried out as previously described.

DNA extraction was performed using the DNeasy Blood & Tissue kit (Qiagen, Courtaboeuf) and quantified using a spectrophotometer (Nanodrop, Labtech, Palaiseau). qPCR was performed on 25 ng DNA in a total volume of 10 μl that contained 5 μl of DNA Master SYBR Green I kit (Roche Diagnostics, Meylan) and 0.05 μM primers for Alu sequence or 0.5 μM for mouse β-actin. The primer sequences for human Alu sequence and mouse β-actin sequences were listed: Alu: 5′-CAT​GGT​GAA​ACC​CCG​TCT​CTA-3' (F), 5′-GCC​TCA​GCC​TCC​CGA​GTA​G-3′ (R); β-actin: 5′-CCA​CCA​TGT​ACC​CAG​GCA​TT-3′ (F), 5′-AGG​GTG​TAA​AAC​GCA​GCT​CA-3′ (R). PCR conditions were as follow: 95°C for 15 min followed by 40 cycles at 95°C for 15 s and 64°C for 30 s and then 40°C for 30 s. Standard curves were generated by adding ten-fold serial dilutions of hHF-MSCs DNA in the DNA of mouse cells (the total DNA amount was kept constant to 25 ng). The results were expressed as the percentage of hHF-MSCs DNA in mouse DNA.

All or part of the organs were mechanically dissociated and DNA extraction was performed using the DNeasy Blood & Tissue kit (Qiagen, Courtaboeuf) [9 (link)]. Real time PCR (qPCR) was performed on 25ng DNA in 10μl that contained 5μl of DNA Master SYBR Green I kit (Roche Diagnostics, Meylan) and 0.05μM primers. Sequences of primers and PCR conditions were as described [9 (link)]. Standard curves were generated by adding ten-fold serial dilutions of hASCs in murine MSCs (10⁶ total cell number). Results were expressed as the percentage of hASCs per organ or blood (total blood evaluated at 1.5ml/mouse) and limit of detection was estimated to be 0.005% hASCs of total cells [9 (link)].

Total RNA was extracted from cells using TRIzol reagent (Invitrogen). After total RNA was reverse transcribed into cDNA by using a Prime-Script™ One Step RT-qPCR kit (Takara Biotechnology Co., Ltd., Dalian, China), PCR reactions for SIRT1, PGC-1α, PPAR-γ, IL-1β, IL-6 and TNF-α mRNA were performed on a LightCycler 480 (Roche, Mannheim, Germany) system with GAPDH used as an internal control. The light cycler DNA master SYBR green I kit (Roche Molecular Biochemicals, Mannheim, Germany) was then used for PCR experiments. The following primers were used: SIRT1 Forward: 5′-GGTGTTAAATACCAAACTGC-3′ and reverse: 5′-AGGAGTGATGTTCAAAATG-3′; PGC-1α Forward: 5’-AATTCACAATCACAGGATCAGAACA3’ and reverse: 5’-ACTTAAGGTGCGTTCAATAGTCTT-3’; PPAR-γ Forward: 5’-TTGGCCATATTTATAGCTGTCATTATT-3′ and reverse: 5’- TGTCCTCGATGGGCTTCA-3’; TNF-α Forward: 5’-GAGCTGTGGGGAGAACAAAAGGA-3′ and reverse: 5’- TTGGCCCTTGAAGAGGACCTG-3’; IL-1β Forward: 5’-GAC CTT CCA GGA TGA GGA CA-3′ and reverse: 5’-AGC TCATATGGGTCCGACAG-3’; IL-6 Forward: 5’-TCC AGT TGC CTTCTT GGG AC-3′ and reverse: 5’-GTGTAATTAAGCCTCCGACTTG-3’; GAPDH Forward: 5’-AGAAGGCTGGGGCTCATTTG-3’ and reverse: 5’-AGGGGCCATCCACAGTCTTC-3. The relative expression of the target genes was calculated using the 2^−ΔΔCt method. All experiments were repeated in triplicate.

RNA isolation from JPCs, iPSCs and iMSCs (1 × 10⁵ cells per sample) was carried out using the NucleoSpin RNA kit (Macherey-Nagel, Düren, Germany) following the manufacturer’s instructions. The amount of isolated RNA was quantified with a Qubit 3.0 fluorometer and the corresponding RNA BR Assay Kit (Thermo Fisher Scientific). The first-strand cDNA synthesis was performed with 0.5 μg of RNA using the SuperScript Vilo Kit (Thermo Fisher Scientific). The quantification of mRNA expression levels was performed using the real-time LightCycler System (Roche Diagnostics, Mannheim, Germany). For the PCR reactions, commercial OCT4, NANOG, ALP, CD44, and CD73 primer kits (Search LC, Heidelberg, Germany) and DNA Master SYBR Green I kit (Roche, Basel, Switzerland) were used. The amplification was performed with a touchdown PCR protocol of 40 cycles (annealing temperature between 68–58 °C), following the manufacturer’s instructions. GOI (gene of interest) transcript levels of each sample were normalized to those of the housekeeping gene GAPDH, divided by the corresponding control samples and displayed as x-fold induction indices.