Kapa sybr fast qpcr master mix 2x kit

We isolated RNA from cells using TRIzol (ThermoFisher Scientific, Inc.) followed by chloroform and isopropanol extraction. The RNA samples were treated with Turbo DNA-free Kit (Invitrogen) to eliminate any residual DNA from the preparation. Total RNA (2 μg) was reverse transcribed using the M-MLV reverse transcriptase (PROMEGA) and 0.25 μg of Anchored Oligo(dT)20 Primer (Invitrogen; 12,577–011). We performed qPCR reactions in triplicate using KAPA SYBR FAST qPCR Master Mix (2X) Kit (Kapa Biosciences) and primer concentrations of 0.4 μM (Additional file 10: Table S1). Cycling conditions were as follows: initial denaturation at 95 °C for 3 min, then 40 cycles with 95 °C for 5 s (denaturation) and 60 °C for 20 s (annealing/extension). To control specificity of the amplified product, a melting-curve analysis was carried out. No amplification of unspecific product was observed. Expression of each gene was relative to Polr2a gene (RNA pol II) and plotted as fold change compared to control in each case.

The total RNA from the muscle tissues was extracted by using the Trizol (Invitrogen Carlsbad, CA, USA) method. The same amount of RNA of each sample was reverse-transcribed according to the instructions of the reverse transcription kit (TaKaRa Biotech Co., Ltd., San Jose, CA, USA). Primers for qPCR analysis are shown in Table S8, and the mRNA expression was detected using the KAPA SYBR^® FAST qPCR Master Mix (2X) Kit (KAPA Biosystem, Wilmington, MA, USA.) in the QuantStudio 7 Flex Real-Time PCR System (life, Carlsbad, CA, USA). The relative abundance of target mRNAs was calculated using the 2^−ΔΔCt method.

Total ribonucleic acid (RNA) was isolated from the excised pinna from one ear per mice at 24-h post-challenge using a Total RNA Extraction Kit (RBC Bioscience, New Taipei City, Taiwan). Complementary deoxyribonucleic acid (cDNA) was then synthesized from 1 μg of total RNA using an iScript™ Select cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Real-time polymerase chain reaction (real-time PCR) analysis was performed using a LightCycler® 480 Instrument II (Roche Applied Science, Penzberg, Germany). The final reaction mixture in each reaction tube consisted of 0.5 μL of cDNA; 0.4 μL of sense and antisense primer solutions, including IFN-γ (forward: 5′-AACGCTACACACTGCATCT-3′, reverse: 5′-TGCTCATTGTAATGCTTGG-3′) and β-actin (ACTB, forward: 5′-ATGGATGACGATATCGCT-3′, reverse: 5′- ATGAGGTAGTCTGTCAGGT-3′) (Integrated DNA Technologies, Coralville, IA, USA); 10 μL of KAPA SYBR® Fast qPCR Master Mix (2x) Kit (Kapa Biosystems, Wilmington, MA, USA); and, 8.7 μL of sterile Milli-Q® water (Merck). The conditions for reverse transcription and the PCR steps were according to the manufacturer’s instructions. The normalization and quantification of mRNA expression was performed using LightCycler® 480 software (Roche Applied Science, Rotkreuz, Switzerland).

To evaluate the effect of drought stress on OsS1Fa1 expression, the mock- and drought-treated rice leaf samples were ground thoroughly to obtain a fine powder. Total RNA was isolated from the ground tissue using the FavorPrep^TM Plant Total RNA Mini Kit (Favorgen, Ping-Tung, Taiwan) and then reverse transcribed to produce cDNA using ReverTra Ace^® qPCR RT Master Mix, with gDNA Remover (TOYOBO, Osaka, Japan). Then, the cDNA template was amplified by RT-qPCR on LightCycler^®480 using the KAPA SYBR^® FAST qPCR Master Mix (2X) Kit (Kapa Biosystems, Wilmington, NC, USA) and OsS1Fa1-specific primers. The Ubiquitin 5 (UBQ5) gene was used as an internal reference.
To examine the level of OsS1Fa1 expression in rice seedlings during vegetative growth, total RNA was isolated from the shoot, leaf, culm, and root tissues of rice seedlings harvested at five different vegetative growth stages and amplified by RT-qPCR.
To examine the transcript levels of drought-responsive genes in Arabidopsis, total RNA was isolated from the leaves of 14-d-old WT and OsS1Fa1-overexpressing plants, and RT-qPCR was carried out using gene-specific primers. Primers for Actin (internal control) were added to the RT-qPCR reaction together with other gene-specific primers.
All experiments were repeated three times, with three replicates per sample. Primers used for all RT-qPCR assays are listed in Table S2.

Total RNA was prepared using Trizol reagent (Invitrogen, Waltham, MA, USA) according to the manufacture’s protocol. Total RNA (1 μg) was used in the reverse transcription (RT) reaction by iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). The quantitative real time PCR was performed using KAPA SYBR^® FAST qPCR Master Mix (2X) Kit (KAPA Biosystem, Wilmington, DE, USA) according to the manufacturer’s protocol. Quantitative PCR reactions were performed using a QIAGEN Rotor Gene Q Real-Time PCR. β-actin was used as the endogenous reference gene since it has the highest stability across pig tissues compared to the other reference genes [31 (link)]. The primer sequences (5′–3′; forward, reverse) were showed in Table 1. The amplification steps were set for 3 min at 95 °C, followed by 40 cycles of denaturation at 95 °C for 3 s, and annealing at 60 °C for 20 s. The data were calculated using the standardized mRNA level comparative methods 2^−ΔΔCt. The 2^−ΔΔCt method is a convenient way to analyze the relative changes in gene expression with a high efficiency qPCR assay [32 (link)].

Total RNA was extracted from half of the mouse kidneys homogenized in Sepasol-RNA1 Super G (Nacalai Tesque). After quantification with the NanoPhoto meter NP80 (Implen), extracted RNA was reverse transcribed into cDNA using a PrimeScript RT reagent Kit (Takara). The obtained templates were used for qPCR with the KAPA SYBR FAST qPCR master mix (2x) Kit (Kapa Biosystems) or Thunderbird qPCR Mix (Toyobo). Data were acquired by using a QuantStudio 6 Flex or a StepOnePlus Real-Time PCR system (Thermo Fisher Scientific) and normalized to the expression of the Hprt gene. The primer sets are described in Table S1.