Qpcr rt kit

Total RNA from cells or tissues was extracted using TRIzol reagent (Invitrogen, 15596026, USA). Primer mix, miR-302a and U6 primers were designed for cDNA synthesis using a qPCR RT Kit (TOYOBO, FSQ-101, Japan). The PCR reaction solution (20 μL) was prepared according to the instructions for SYBR Premix Ex Taq (TaKaRa, DRR081A, Japan) under the following conditions: 95 °C for 1 min; followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. U6 and β-actin were used as internal controls. Three biological replicates were conducted. The data were analyzed using SPSS 20.0 software, and the 2^−ΔΔCT method was used according to the following formula: $$\mathrm{\Delta }\mathrm{\Delta }\mathrm{Ct}=\{\mathrm{Ct}(\mathrm{positive})-\mathrm{Ct}(\mathrm{reference})\}-\{\mathrm{Ct}(\mathrm{control})-\mathrm{Ct}(\mathrm{reference})\}.$$ Here, 2−ΔΔCt refers to the relative expression ratio and relative expression levels were calculated using the 2 −ΔΔCt method.

hBMSCs were harvested at assigned time points post osteogenic induction. Total RNA was extracted using TRIzol Reagent (Life Technologies, Gaithersburg, MD, USA) according to manufacturer’s instruction. Reverse transcription was performed using the qPCR RT Kit (TOYOBO, Co. Ltd., Osaka, Japan) according to manufacturer’s instructions. mRNA levels of target genes were measured using quantitative PCR (qPCR) analysis with SYBR^® Green qPCR Mix (TOYOBO Co. Ltd., Osaka, Japan). The quantitative assay was performed in a 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and the following PCR cycling protocol was used: 95 °C for 1 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The relative expression of gene-specific products was analyzed using the 2^−△△CT method and normalized to the corresponding GAPDH values. The primer sequences used are shown in Table 2.

Total RNA was isolated using the RNeasy Mini Kit (Qiagen). cDNA was synthesized using qPCR RT Kit (Toyobo). For the detection of Ccl2 (Mm00441242), Il6 (Mm00446190), and Il1b (Mm00434228) mRNA, TaqMan Gene Expression Assays on the AB 7500 Real Time PCR System (Applied Biosystems) was used. Other PCR primers are described in the Supplementary Table 2. Densitometric quantification was done using Image J software (http://rsb.info.nih.gov/ij/).

The following reagents were purchased from Roche: FastStart Universal SYBR Green Master ROX (2×) kit (Roche Cat# 4913914001), Tripure isolation reagent (Cat# 11667165001), FastStart Universal SYBR Master (Cat# 04913850001), and In Situ Cell Death Detection Kit (Cat# 11684817910). The ReverTra Ace quantitative real-time polymerase chain reaction (qPCR RT) kit was obtained from TOYOBO (Cat# FSQ-101). The Animal Total RNA Rapid Extraction Kit was from JieRui (Cat# GK3016). The RevertAid First Strand cDNA Synthesis Kit (K1622) was from Thermo Scientific (Cat# 1622). The following measurement kits were purchased from Nanjing Jiancheng Bioengineering Institute: superoxide dismutase (SOD) (Cat# A001-1), catalase (CAT) (Cat# A007-1), malondialdehyde (MDA) (Cat# A003-1), nitric oxide synthase (NOS) (Cat# A014-2), and NO (Cat# A012). Rabbit-anti-mouse NF-κB antibody was from Abcam (Cat# Ab32536). The immunohistochemistry (IHC) and enhanced DAB chromogenic kits were obtained from Mai Xin.

Total RNA was extracted from PBLs. The cDNA synthesized by using a qPCR-RT kit (Toyobo, Japan) was subjected to quantitative real-time PCR (qPCR) to measure the mRNA levels of cytokines and transcription factors with SYBR green PCR master mix (Applied Biosystems) on a StepOnePlus real-time PCR system (Applied Biosystems). The primers specific for the mouse or sheep TGF-β, IFN-γ, IL-2, IL-12, IL-4, IL-6, IL-10, T-bet, and GATA-3 genes are listed in Table S1 in the supplemental material.

Total RNA was performed according to the instructions of Novogene (Beijing) RNA extraction kit, which was extracted from all tissues, including roots, stems, leaves, flowers and ovaries, at each fruit stage. The total RNA was detected by 1% (w/v) agarose gel electrophoresis. In addition, the non-degraded RNA was selected as OD260: OD280 > 1.80 for cDNA synthesis. cDNA synthesis was performed according to the qPCR RT Kit (Code No. Fsq-101, TOYOBO, Tokyo, Japan). The first step was to denaturate RNA, that is, the qualified RNA was placed in a 65 °C metal bath for 5 min and then immediately put in an ice box for cooling, so as to improve the reverse transcription efficiency. The cDNA after reverse transcription was stored at −20 °C for qRT-PCR and coding region cloning.