Sybr premix ex taq rt pcr kit

Total RNA was isolated from rice leaf samples (100 mg tissue per sample) using TRIzol reagent (TIANGEN Biotech, Beijing, China). The concentration of total RNA in each sample was determined using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). cDNA was synthesized using 1 µg total RNA per 20 µL reaction using the PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara, Dalian, China). Quantitative RT-PCRs were performed on an ABI PRISM 7500 device using a SYBR Premix ExTaq RT−PCR Kit (Takara). Relative transcript levels were calculated by the 2^-ΔΔCT method as previously described (Rao et al., 2013 (link)), and the rice ubiquitin gene (Os03g0234350) was used as an internal control. The primers for quantitative RT−PCR analysis are listed in Table S1.

Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instruction. PrimeScript RT Reagent kit (Takara) and SYBR Premix Ex Taq RT-PCR kit (Takara) were used to detect the quantities of mRNA expression. The fold-change of each gene was normalized to GAPDH as the internal control using 2–ΔΔCt method. TaqMan MicroRNA Assay kit (Applied Biosystems) and specific TaqMan probes (Applied Biosystems) were used to detect the expression of miRNAs. RNU6 was used as the internal control for humans, and sno202 was used as the internal control for mice.

Total RNA from epididymal WAT was extracted with Trizol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. The cDNA was synthesized from total RNA (3 µg) in a 25 μL reaction volume using the TIANScript RT Kit (Tiangen Biotech, Beijing, China). Real-time PCR was performed using 1 μL cDNA with the SYBR Premix Ex Taq RT-PCR kit (Takara, Otsu, Shiga, Japan) on Techne Quantica real-time PCR detection system (Techne, Staffordshire, UK). The thermal profile settings were starting with a denaturation at 95 °C for 180 s followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s. The SYBR green fluorescence was read at the end of each extension step (72 °C). Relative expression levels of the mRNA of the target genes were normalized to 18S mRNA levels. PCR sequences of various genes are presented in Table 1.

Muscle tissues were obtained from the left biceps of IIMs patients and from HC who underwent joint replacement. Total RNA was extracted from muscle tissue using TRIzol reagent according to the manufacturer’s instructions. To detect the expression of IL18R1, RT-qPCR was performed. RNA concentration and quality were determined using a QS3000 spectrophotometer. RT-qPCR was performed using gene-specific primers with SYBR Green (SYBR Premix Ex Taq RT-PCR kit; Takara) on a 7,500 Real-Time PCR System (Applied Biosystems, Waltham, MA, United States ). The sequences of primers used in the present study were as follows: IL18R1, forward: 5′-GGA​GGC​ACA​GAC​ACC​AAA​AGC​T-3′ and; IL18R1, reverse: 5′-AGG​CAC​ACT​ACT​GCC​ACC​AAG​A-3′. β-actin was used as an internal control and the relative expression levels were determined by the 2 ˗∆∆ CT method.

The spikes of S-Cp1-1 and its WT counterpart were collected at GS24, GS29, and GS32 (Zadoks et al. 1974 ). There were three biological replicates for each stage, with at least 10 spikes for each replicate. Root, stem, and leaf samples of S-Cp1-1 and its WT at GS24 stage were collected as well. Three biological replicates were done for each tissue, with 10 plants used per replicate. The harvested samples were ground in liquid nitrogen, and RNA was extracted using the Plant RNA extraction kit V1.5 (Biofit, Chengdu, China).
qRT-PCR reactions were performed using a SYBR premix Ex Taq RT-PCR kit (Takara, Dalian, China). RNA clean up, cDNA synthesis, qRT-PCR analyses were performed as described in Wang et al. (2010) . Two housekeeping genes (Long et al. 2010 (link)), i.e., Scaffold-associated regions (SAR) DNA binding protein (NCBI UniGene Ta.14126) and methionine aminopeptidase 1 (Ta.7894), were amplified as reference genes for normalization of data. The primers used for qRT-PCR are listed in Table S2 in File S1.