Sybr premix ex taptm 2

HemECs seeded in 60-mm plates were treated with curcumin at 25 μM or DMSO control for 24 hours. The total RNA was isolated using TRIZOL Reagent (Invitrogen, Carlsbad, CA) and reverse transcripted to complementary DNA (cDNA) according to the RevetAidTMFirst Strand cDNA Synthesis Kit (Fermentas, Thermo Fisher Scientific, Shanghai, China) instructions. The gene expression was detected using SYBR Premix Ex TapTM II (TaKaRa) and glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene was served as the internal reference. All primers were synthesized by TaKaRa (Dalian, China) as shown in Table 1. Real-time polymerase chain reaction (RT-PCR) was performed in triplicate utilizing the StepOnePlusTM Real-Time PCR System (Applied Biosystems, Beijing, China). The results were normalized to gapdh level and the triplicate results were averaged for each sample. Data were analyzed using the comparative C_t method (2−ΔΔC_t).

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was performed to quantify the expression levels of the transcripts obtained from high-throughput sequencing. First, TRIzol^® (Cowin Biosciences) was used to extract total RNA, followed by RNA quality assessment using NanoDrop 2000. cDNA reverse transcription was performed using a TAKARA PrimeScriptTM RT reagent kit (TaKaRa). RT-qPCR was performed using SYBR Premix Ex Tap^TM II (TaKaRa) using LightCycler^®480 II (Roche Applied Science). The PCR reaction mixture and thermal profile were set up as described by Zhu et al. [21 ]. Gene relative expression levels were calculated using the 2^−ΔΔCt method [46 (link)]. Primers of candidate genes and an internal reference gene (GAPDH, Glyceraldehyde-3-phosphate dehydrogenase) were designed using Primer 5.0 software and synthesized at Sangon Biotech (Shanghai) Co., Ltd. The primer sequences used are listed in S1 File.

To analyze the gene expression pattern of stress-related genes in sugarcane, qRT-PCR was performed. In brief, the total RNA from all treated sugarcane plant leaves was extracted by Trizol reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s instructions. Extracted RNA was spectrophotometrically quantified and reverse-transcribed into cDNA through PrimeScriptTM RT Reagent Kit (TaKaRa, Shiga, Japan, China), following the manufacturer’s instructions. Further RT-PCR was performed with a final 20 µL reaction mixture, containing 2 µL of 10-fold diluted first-strand cDNA, 10 pM of sugarcane stress-specific primers of ScDREB2A, SuCAT, and SuSOD (Supplementary Table S1), and 10 µL of SYBR Premix Ex TapTMII (Takara, Kyoto, Japan). All RT-qPCR reactions were performed in a real-time PCR system (Bio-Rad, Hercules, CA, USA) with the following programming: initial denaturation at 95 °C for 3 min, followed by 35 cycles of 95 °C for 15 s, 58 °C for 30 s, and 72 °C for 30 s, with a subsequent melting cycle from 60 to 95 °C. All RT-qPCR reactions were performed with reference gene GAPDH in three replicates. To compare relative expression, the delta–delta CT method was used.

For validation of the relative expression levels of Luffa miRNAs and their targets, and the quality of high-throughput sequencing, RT-qPCR was performed. For validating target genes, we seperated total RNAs from pulps of the near-isogenic lines JAAS-BR and JAAS-BS at different DAP (0, 3, 5, 7, 9 and 11) with Trizol reagent (Invitrogen) and treated them using Superscript III First-Strand Synthesis System (Invitrogen) to reverse transcribe the RNAs into first-strand cDNA. Small RNAs were extracted from the above six pulp samples with a small RNA isolation kit (TaKaRa, Dalian, China). Then, we used a synthesis kit, the One Step PrimeScript miRNA cDNA, to reverse-transcribe the small RNAs into cDNAs. Each PCR reaction was performed with 0.2 μM of primer pairs, 10 μl of 2 × SYBR green PCR reaction mix, and 20 μl containing 2 μl of diluted cDNA, in which 5.8 S rRNAs were used as reference to normalize the expression level. We used Beacon Designer 7.0 (Premier Bio-soft International, Palo Alto, CA USA) to design the specific primers. We listed all the primer sequences of miRNAs and their target genes in Table S1. The RT-qPCR amplification was performed under the conditions described by previous reports^{75 (link)}. All reactions were performed on MyiQ RT-PCR (BIO-RAD, Hercules, CA, USA) using SYBR Premix Ex TapTM II (TaKaRa) with three biological replicates and technological replicates.

Total RNA was extracted from the skin tissue of cashmere goats using RNAiso Plus (TaKaRa Bio, Shiga, Japan). The integrity and molecular weight of the RNA were checked using denaturing agarose gel electrophoresis. The RNA concentration was determined using a Nanodrop 2000 spectrophotometer (Thermo Fisher). The cDNA was prepared from 500 ng of RNA using the PrimeScript^TMRT Reagent Kit (TaKaRa Bio), following the supplier's protocol. Quantitative real-time PCR was performed with SYBR Premix Ex Tap^TMII (TaKaRa Bio), and the relative expression of the target gene was calculated following the 2^-ΔΔCt method. The names and sequences of the primers are provided in S2.