Superscript first strand synthesis kit

RT-PCR was used to qualify the mRNA expression levels of osteogenesis-related genes including alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OCN), type I collagen (Col-I), and runt-related transcription factor 2 (Runx2). After variable time intervals (1, 7, and 14 days) of cultivation, total RNA was extracted from the cells by using the TRIzol reagent (Invitrogen, USA). Then, total RNA was subjected to reverse transcription to cDNA using the SuperScript First Strand Synthesis Kit (Fermentas, Japan) following the manufacturer's protocol. The real-time PCR analysis was conducted with SYBR Green PCR Kit (Thermo Fisher Scientific, USA) in a volume of 25 μL reaction mixture, with 12.5 μL of the SYBR Green Master Mix, 0.5 μL of both forward and reverse primers, 2 μL of specimen cDNA, and 9.5 μL of ddH₂O. All reactions were carried out for 10 minutes at 95°C, 40 cycles of 15 seconds at 95°C and 45 seconds at 60°C, 15 seconds at 95°C, 1 minute at 60°C, 15 seconds at 95°C, and 15 seconds at 60°C. β-Actin was selected as the internal control. Relative expressions of mRNA levels were calculated by the 2^−ΔΔCt method. The genes and primers used are listed in Table 1.

Total RNA was extracted using TRIzol reagent (Invitrogen). The purity of the RNA (A260/A280 >1.8) was examined using a spectrophotometer, and the integrity of the RNA was confirmed by visualization of the 28S and 18S bands (2:1) on 1% agarose gels. The RNA (1 µg) was used to synthesize cDNA using the SuperScript First-Strand Synthesis kit (Fermentas, Pittsburgh, PA, USA) following the manufacturer’s instructions.
Quantitative PCR was then performed using the SYBR-Green qPCR Super Mixture (Takara, Shiga, Japan) and an ABI Prism 7500 Sequence Detection system (Life Technologies, Grand Island, NY, USA). The following primer sequences were used: rat VEGF (forward, 5′-AAAGCC AGCACATAGGAGAG-3′ and reverse, 5′-AGGATTTAAACCGGGATTTC-3′); COX-2 (forward, 5′-TACAACAACTCCATCCTCCTTG-3′ and reverse, 5′-TTCATCTCTCTGCTCTGGTCAA-3′) and rat β-actin (forward: 5′-CACCCGCGAGTACAACCTTC-3′ and reverse, 5′-CCCATACCCACCATCACACC-3′). The amplification conditions were as follows: 95°C for 10 min followed by 40 cycles of 95°C for 15 sec, 60°C for 60 sec and 72°C for 60 sec. The specificity of the detected signals was confirmed by a dissociation curve, which consisted of a single peak. All samples were run in triplicate in each experiment. Data were analyzed using the 2^−ΔΔCT method.

Total RNA was extracted from liver tissue using Trizol reagent (Invitrogen). RNA concentrations were determined by SmartSpecTM Plus (BIO-RAD, USA). 1 μg of total RNA was transcribed to cDNA using the superscript first-strand synthesis kit (Thermo) following instructions. Real-time PCR analysis was performed with a LightCycler instrument (Roche Applied Science) and SYBR green detection of amplified products. Primers for GCK, PK, PEPCK, G6Pase, IL-6, TNF-α, and MCP-1 [46 (link)] were previously described. PCR reactions were performed in triplicate in 96-well plates and normalized to house-keeping genes (GAPDH) using the 2^−ΔΔCt method.

Spleens from line 6 and 7 chicks uninfected or infected with 2000 pfu Md5 strain MDV at day of hatch, collected in the course of a previous study [50 (link)] at 7, 14 and 21 days post-infection, were spectratyped for TCRβ length. cDNA was prepared from spleen samples preserved at −20 °C in RNAlater, using the Absolutely RNA Miniprep kit (product #400800, Agilent Technologies) followed by the Invitrogen™ Superscript™ First Strand Synthesis kit (product #11904018, Thermo Fisher Scientific) or Applied Biosystems™ High Capacity cDNA kit (product #4368814, Thermo Fisher Scientific) and oligo-dT primers. Nested PCR was performed for TCR Vβ1 and TCR Vβ2 using previously described primers and methods [51 (link)]. PCR products were diluted at 1:200 and fragment analysis was performed on an ABI (Applied Biosystems™,., Thermo Fisher Scientific) 3730xL machine. Fragment data were analyzed in Peak Scanner v.2 (Thermo Fisher Scientific).

Pre-B cells were treated with 5 Gy γ-irradiation or with 10 µM Nutlin3a (Nut3a), in the presence of 25 µM QVD-OPH (Sigma-Aldrich, St. Louis, MO, USA) in culture. WT and Zmat3^−/− mice, 2–5 months old, were either exposed to 8 Gy γ-irradiation or left untreated and lung tissue was harvested after 6 h. RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and converted to cDNA using the SuperScript First Strand Synthesis Kit (Thermo Fisher Scientific). Quantitative RT-PCR was performed using TaqMan probes (Thermo Fisher Scientific) for Zmat3 (Mm01292424_m1), Puma (Mm00519268_m1), p21 (Mm00432448_m1) and analyzed by the ΔΔCT method relative to HMBS (Mm01143545_m1).

Total RNA was extracted from liver tissue using TRIzol reagent (Invitrogen). RNA concentrations were determined by SmartSpec Plus (BIO-RAD, USA). 1 μg of total RNA was transcribed to cDNA using the superscript first-strand synthesis kit (Thermo) following instructions. UCP1 and PGC-1α expression were analyzed using semiquantitative RT-PCR. The cDNA was served as a template in a 50 μL reaction mixture and was processed using an initial step at 94°C for 5 min, followed by 32 amplification cycles (94°C for 30 s, 55°C for 30 s, and 72°C for 35 s), and 72°C for 5 min. Primers were as follows: UCP1 (forward, 5′-GGTTTTGCACCACACTCCTG-3′; reverse, 5′-ACATGGACATCGCACAGCTT-3′), PGC-1α (forward, 5′-TAAATCTGCGGGATGATGGA-3′; reverse, 5′-GTTTCGTTCGACCTGCGTAA-3′), and GAPDH (forward, 5′-AGAACATCATCCCTGCATCC-3′; reverse, 5′-TCCACCACCCTGTTGCTGTA-3′). Quantitative RT-PCR analysis was performed with a LightCycler instrument (Roche Applied Science) and SYBR green detection of amplified products. Primers for SREBP-1c, ACC, FAS, CPT-1α, and CD36 were previously described [15 (link)]. PCR reactions were performed in triplicate and normalized to cyclophilin using the 2^−ΔΔCt method.