Drr037a

Total RNA was isolated from plants using the TRIzol reagent (Invitrogen) based on the manufacturer’s instructions. The concentration and purity of RNA was determined by using an Epoch UV-Vis microplate spectrophotometer (BioTek). The first-strand cDNA was synthesized using reverse transcriptase system (DRR037A; Takara, Dalian, China). All primers used for qRT-PCR are listed in Table 1. For real-time PCR, each reaction contained 2.0 μL of cDNA, 10 μL of SYBR Premix Ex Taq TM II, gene-specific primers 1.6 μL, and distilled deionized water up to a final volume of 20 μL. The PCR parameters were 95°C for 30 s; followed by 40 cycles of 95°C for 5 s, 60°C for 30 s. The melting curves were performed for the amplification of 95°C for 10 s, 65°C for 5 s, 95°C for 5 s. The wheat tubulin gene was used as a reference gene [64 (link)], and the reactions were performed using the Thermal Cycler Dice Real Time System III (Takarabio Inc., Otsu, Shiga, Japan). The baseline data were gathered between 18 and 32 cycles. All reactions were run in triplicate. The quantification of gene expression levels was caculated as 2^−ΔΔCT relative to the control.

Gene expression levels (mRNA) in cartilage were determined using RT-qPCR. Total RNA was extracted (Takara, China) and purified with 75% ethanol, and its concentration was determined by spectrophotometry. The purified total RNA (200 ng per sample) was added to a transcription kit (cat. no. DRR037A; Takara Bio, Inc.) and mixed to generate the first strand template (reverse transcription reaction). The sequences of the primers used for qPCR were the following: NOX1 forward, 5′-ATA TTT TGG AAT TGC AGA TGA ACA-3′ and reverse, 5′-ATA TTG AGG AAG AGA CGG TAG-3′; NOX2 forward, 5′-GGA GAA TTA ACC CCT GCC A-3′ and reverse, 5′-GGC TAG CTG GAG AAG ACC AC-3′; NOX4 forward, 5′-TGT TGG ATG ACT GGA AAC CA-3′ and reverse, 5′-TGG GTC CAC AAC AGA AAA CA-3′; and GADPH forward, 5′-CAA CAG CCT CAA GAT CAT CAG CA-3′ and reverse, 5′-TGG CAT GGT CTG TCA TGA GT-3′. qPCR was performed using a real-time PCR system (ABI 7300). Expression levels were determined quantitatively using SYBR Premix ExTaq (Takara Bio, Inc.).

Total RNAs of the shoots and roots were extracted from the samples of the three independent biological replicates for each of genotypes using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The first strand of cDNA was synthesized using 1.0 μg total RNA, 0.5 μg oligo d (T) 18 primer, and reverse transcriptase system (DRR037A; Takara, Dalian, China) at a total volume of 20 μL. The primers used for qPCR were designed according to the corresponding sequence on Primer 6, and the primers are listed in Table S1. Amplification through quantitative real-time polymerase chain reaction was performed with a Thermal Cycler Dice Real Time System III (Takarabio Inc., Otsu, Shiga, Japan) using an RT-PCR master mix (DRR820A; Takara) according to the user manual. The expression levels of the target genes were normalized using actin as an endogenous control. All reactions were run in triplicate. The quantification of gene expression levels relative to the control was determined by 2^−ΔΔCt method.

The gene expression level in brain tissues was determined by real-time PCR. Extracted total RNA was purified with 75% ethanol, and its concentration was determined by spectrophotometry. Then, the purified total RNA (200 ng) was retrotranscribed using a retrotranscription kit (DRR037A; Takara Bio, Inc.) and mixed to obtain the first-strand cDNA template. Subsequently, the expression levels of CD68, interleukin- (IL-)1β, and tumor necrosis factor- (TNF-) α were determined quantitatively by real-time PCR (ABI 7300) using the SYBR Premix Ex Taq kit (Takara Bio, Inc.).

Gene expression levels (mRNA) in lung tissue were determined by real-time PCR (Applied Biosystems). First, extracted total RNA were purified with 75% ethanol, and its concentration was determined by spectrophotometry. Then, the purified total RNA (200 ng each sample) was added into a transcription kit (DRR037A; TaKaRa, Dalian, China) and mixed to get the first strand template (reverse transcription reaction). GAPDH was used as the loading control. The primers were provided by GeneCopoeia Inc., as follows: CHOP forward, 5′-CAT ACA CCA CCA CAC CTG AAA G-3′; reverse, 5′-CAT ACA CCA CCA CAC CTG AAA G-3′; GRP78 forward, 5′-TCT CCA CGG CTT CCG ATA AT-3′; and reverse, 5′-GTA CCT TTG TCT TCA GCT GTC ACT C-3′. All oligonucleotide primers were designed by Sangon Biotech Co., Ltd (Shanghai, China). The Ct value of the target is normalized by subtracting the GAPDH Ct value to provide a ΔCt value. The relative expression level between treatments was then calculated using the following equation: relative gene expression = 2 − (ΔCt sample −ΔCt control).