Pcr thermal cycler dice touch

Total RNA (100 or 200 ng) was transcribed at 42 °C for 60 min using the SuperScript VILO kit (Thermo Fischer Scientific). Aliquots of cDNA (1/10 of RT reaction) were amplified using Hot Start Taq DNA Polymerase (New England Biolabs). Amplification was conducted using a PCR Thermal Cycler Dice Touch (TaKaRa). PCR was carried out with initial activation at 95 °C for 30 s followed by 20 (β-actin) or 27 cycles (REST, sREST) of amplification (95 °C for 15 s, 58 °C for 15 s, and 68 °C for 30 s) and 68 °C for 2 min. Forward and reverse primers (5 pmol each) were used in 50 μL reactions. PCR products were analyzed by electrophoresis using 5% Mini-PROTEAN TBE Precast Gels (Bio-Rad), followed by SYBR Gold or EtBr staining (Thermo Fisher Scientific). The primer sequences were as follows: REST forward: 5ʹ- gaacgcccatataaatgtgaa-3ʹ; REST reverse: 5ʹ-tttgaagttgcttctatctgctgt-3ʹ; actin forward: 5ʹ-ggccgtcttcccctccatcg-3ʹ; actin reverse: 5ʹ-ccagttggtgacgatgccgtgc-3ʹ. The number of % exclusion was calculated as the intensities of REST band against total band intensities of REST and sREST.

Maxillary samples were fixed in RNAlater (Sigma-Aldrich) for 12 h at 4 °C and separated into the GT, PRT, and BT (Supplementary Fig. 3a; sampling of single ligature model was only performed in the tissues around the second molar) in RNAlater. Total RNA was isolated using TRI Reagent (MRC, Cincinnati, US) and a Direct-zol-96 RNA Kit (Zymo Research, Irvine, US) according to the manufacturer’s instructions, separately for the three tissues. For cell samples in macrophage polarization experiments, total RNA was isolated using TRI Reagent and purified as the manufacturer suggested. First-strand cDNAs were synthesized with PCR Thermal Cycler Dice Touch (TaKaRa Bio, Shiga, JP) using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO). RT-qPCR analysis was performed with the CFX384 Touch Real-Time PCR Detection System and Bio-Rad CDX Manager software (v3.11517.0823; Bio-Rad Laboratories, Hercules, US) using THUNDERBIRD Next SYBR qPCR/UNG Set (TOYOBO). All genes were measured using the primers listed in Supplementary Table 4, 5. The relative expression levels normalized to internal reference control (Eef2 for tissue samples; Stx5a for macrophage samples) were calculated using the standard curve method.

Blood samples were collected from the dogs by veterinarians. Genomic DNA was isolated using the QIAamp® DNA Mini Kit (QIAGEN, Hilden, Germany). The numbers of dogs of each breed are shown in Table S1. Polymorphisms were searched using the Ensembl and National Centre for Biotechnology Information databases. The fragments including the selected polymorphisms were amplified by PCR using the primer pairs shown in Table S2. The PCR reactions were performed with KOD-FX Neo DNA polymerase (TOYOBO, Osaka, Japan) in a PCR Thermal Cycler Dice Touch (Takara, Shiga, Japan). The samples were initially denatured at 94 °C for 2 min, followed by 40 cycles of 98 °C for 10 s and 65 °C for 30 s, and a final extension at 72 °C for 7 min. The PCR products were purified using Nucleo Spin® Gel and a PCR Clean-up Kit (Takara) after being confirmed using 1.5% agarose gel electrophoresis. The samples were shipped to the Takara CDM Centre (Mie, Japan) and subjected to Premix Sequence Analysis (Takara).

RNA (250 ng) was reverse transcribed into cDNA using a Prime Script II 1st strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan). Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was conducted using Ex Taq (Takara Bio, Shiga, Japan) on a PCR Thermal Cycler Dice Touch (Takara Bio, Shiga, Japan). The PCR products were visualized by 2% agarose gel electrophoresis stained with ethidium bromide. The primer sequences were as follows: GAPDH forward primer, 5′-agccacatcgctcagacac-3′; GAPDH reverse primer, 5′-gcccaatacgaccaaatcc-3′; TIPARP forward primer, 5′-gtccctgtttctgcagagga-3′; TIPARP reverse primer, 5′-atcctgtcacggccaaacat-3′.

Total RNA was extracted using TRIZOL regent (Life Technologies) and treated with a TURBO DNA-free kit (Life Technologies), and 500 ng of RNA was reverse transcribed into cDNA using the Prime Script^TM II 1st strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan). Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed with Ex Taq (Takara Bio) on a PCR Thermal Cycler Dice Touch (Takara Bio). The PCR products were run on a 2% (w/v) agarose gel and visualized with ethidium bromide. Real-time quantitative RT-PCR was performed with TB green Premix Ex Taq II (Takara Bio) on a LightCycler 480 (Roche, Basel, Switzerland). GAPDH was used as an internal control. Total RNA of human bladder tissue (BioChain Institute Inc., Newark, CA, USA) was used as a positive control. The sequences of the primers used in these studies are described in Table S1.