Applied biosystems sybr green pcr master mix

Total RNA was extracted from the mice’s cerebral cortex using an RNA extraction kit (Servicebio, Wuhan, China, G3640-50T). Reverse-transcription of RNA to cDNA was performed using ReverTra Ace qPCR RT Master Mix (TOYOBO, FSQ-201) according to the manufacturer’s instructions. Real-time qPCR was conducted on an ABI 7500 system using Applied Biosystems SYBR-Green PCR Master mix (Thermo Fisher Scientific, Inc., USA). The total reaction volume was 10 μL and primer sequences are shown in Table 1. The following amplification steps were used: initial denaturation at 95°C for 10 min, followed by 40 denaturation cycles at 95°C for 15 s, and elongation at 60°C for 60 s. Gapdh was used as a housekeeping gene. Relative mRNA expression was calculated using the 2^–ΔΔCt method. Three replicates were performed for each group.

RNA was extracted from whole blood samples using the TRIzol™ reagent and the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The RNA was quantified using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and the samples were stored at −80 °C until further use. Complementary DNA (cDNA) was synthesized using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) following the manufacturer’s recommendations and stored at −20 °C for subsequent use in real-time PCR reactions. The real-time PCR amplification reaction was conducted in the Applied Biosystems 7500 Real-Time PCR Systems (Thermo Fisher Scientific, Waltham, MA, USA), with the Applied Biosystems SYBR Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). We used 240 ng of cDNA and 1 µM of primers for the following markers: IFNγ, IFNλ, IL-13, matrix metallopeptidase (MMP)9, retinoic acid-related orphan receptor (ROR)γt, AXL, CD209, transforming growth factor (TGF)-β, FoxP3, IL-10, and Interferon-stimulated gene (ISG)15 (Table 2). The mean of the constitutively expressed genes β-Actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used for normalization. Normalized expression was calculated as previously described using the 2^−ΔCt formula [19 (link)], and reaction specificity was evaluated by the dissociation curve.

Total RNA from rice and Arabidopsis leaf tissue or rice protoplast was extracted using TRIzol™ Reagent (Thermo Fisher Scientific) following the manufacturer’s instructions with slight modifications. DNA was then removed with DNase I (New England BioLabs) and the complementary DNA (cDNA) was synthesized using the SuperScript^TM III First-Strand Synthesis System (Thermo Fisher Scientific). Applied Biosystems™ SYBR™ Green PCR Master Mix (Thermo Fisher Scientific) was used for qRT-PCR. Three technical replicates were carried out for each sample using a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad Laboratories). AtEf1α was used as the reference gene for Arabidopsis and OsTubulin was used as the reference gene for rice. Protoplasts transformed with the T-DNA vector containing LbCas12a without crRNAs were used as controls for rice. Relative expression level of each gene was calculated using the 2^−ΔΔCt method.

Quantitative real‐time reverse transcription polymerase chain reaction (RT‐qPCR) was performed on kidneys for kidney injury molecule 1, neutrophilic gelatinase‐associated lipocalin (NGAL), and interleukin‐6 (IL‐6), and on lung tissue for zona occludens‐1 (ZO‐1) and metalloproteinase‐9 (MMP‐9). The primer sequences are shown in Table S1. Slices from the right superior lung lobe and right kidney were sampled in cryotubes and stored at −80°C after immersion in liquid nitrogen. Extraction of total RNA was achieved with an RNA total SV system (Promega). RNA concentration was measured using Nanodrop ND‐1000 spectrophotometry. cDNA was synthesized and amplified from total RNA with a GoTaq 2‐STEP RT‐qPCR system (Promega). The RT‐PCR reaction was performed with Applied Biosystems SYBR green PCR Master Mix (Thermo Fisher Scientific), and relative mRNA was measured using an SYBR green detection system (ABI 7500 Real‐Time PCR; Applied Biosystems). For each sample, the expression of each gene was normalized to that of the housekeeping gene 36B4 (Akamine et al., 2007 (link)) and expressed as fold change relative to the nonventilated group, using the 2^−∆∆Ct method, where ΔCt = Ct (reference gene) − Ct (target gene). All analyses were performed by M.A.A. who was blinded to the group assignment.