Nanodrop microspectrophotometer

Expression analysis was performed on samples taken from the microcarrier/cell suspension; RNA was collected using the RNeasy micro-kit Qiagen with DNAse treatment (Qiagen GmbH). RNA concentration was determined with the NanoDrop microspectrophotometer (Thermo Fisher Scientific). A total of 100 ng RNA per sample was subjected to reverse transcription with IScript cDNA synthese kit (Biorad, Veenendaal, Netherlands). Quantitative polymerase chain reaction (QPCR) was performed by iQ SYBR Green supermix (Biorad) and a primer concentration of 10 mM. Quantitative PCR reactions were run on the CFX Real-timePCR detection system (Biorad). Primers were designed with Mfold (www.idtdna.com/scitools/Application/mfold/) and were synthesized by Eurogentec (Liege, Belgium). Primer sequences are provided in Table 1. Samples were normalized for input based on both β-actin and GAPDH values.Table 1

Primer sequences used for Q-PCR

Gene	Fwd sequence	Rev sequence
GAPDH	TCC-CAA-CGT-GTC-TGT–TGT-GGA-TCT	TGT-TGA-AGT-CGC-AGG-AGA-CAA-CCT
β Actin	GGC-ACC-CAG-CAC-AAT-GAA-GAT-CAA	ATC-GTA-CTC-CTG-CTT-GCT-GAT-CCA
MyoD	TAG-GAG-AGG-CGA-AGG-AAC-TGT-TGT	TCT-GGC-CCA-CGG-AGT-AAC-ATC-AAA
Myogenin	AGC-CTC-CAA-ATC-CAC-TCC-CTG-AAA	AGC-CAC-TGG-CAT-AGG-AAG-AGA-TGA

The 100 mg of duodenum, jejunum, and ileum tissues were weighed, homogenized by liquid nitrogen grinding method, and RNA was extracted by EASYspin Plus tissue/cell RNA rapid extraction kit (Beijing Adlai Biotechnology Co., Ltd. Beijing China).
The RNA concentration was determined by NanoDrop microspectrophotometer (nd-1000uv0vis, Thermo Fisher Scientific Inc.). Samples (cDNA>100 ng/μl) with OD260:OD280 values of 2.0–2.2 were stored at −20°C for reverse transcription. cDNA was synthesized by reverse transcription Kit (Prime Script TM RT reagent kit and gDNA Eraser kit, RR047a, Takara, Japan).
Gene quantitative PCR (qPCR) took cDNA as a template, and the primer sequence is shown in Table 3. The reaction system was: 10 μl f SYBR® Premix Ex Taq (TaKaRa Biotechnology, Dalian, China), 2 μl f DNA template, 0.3 μl each of upstream and downstream primers, and 8.4 μl of water. Quantification was performed using the CFX96 PCR System (Bio-Rad, USA). Quantitative results used glyceraldehyde-3-phosphate dehydrogenase (GADPH) as the internal reference gene. Each sample was repeated three times, and the gene expression was analyzed by the 2^−ΔΔCt method.

Genomic DNA was extracted from umbilical cord tissue according to the manufacturer’s instruction (blood or tissue genomic DNA extraction kit, TIANGEN, Beijing). The DNA purity was measured by the Nanodrop microspectrophotometer (Thermo Fisher Scientific. Inc), and the A260/A280 ratio was controlled within 1.8–2.0. The DNA sample was then stored in at -20 °C until genotyping.
The DNA template containing the SNPs locus region was amplified by PCR technology, and then the PCR product was subjected to a single base extension reaction using specific extension primers. High-throughput genotyping of single nucleotide polymorphisms (SNPs) was performed using Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF–MS) in a gene mass spectrometry system (Sequenom, San Diego, CA, USA).

48 hours after transfection, the total RNA from untreated and transfected cell lines was extracted by RNX-Plus solution (Sinaclon, Iran) according to the manufacturer’s procedure manual. The quantity and quality of the extracted RNA were controlled by NanoDrop microspectrophotometer at 260 nm (Thermo Fisher Scientific, US) and electrophoresis on agarose gel respectively. Following this, 2 µg of total RNA after treatment by DNaseΙ (Thermo Fisher Scientific, Inc) was reverse transcribed into cDNA by using an AddScript cDNA Synthesis kit (AddBio, Korea). For measuring of SOX2-OT and other mRNA expression levels, oligo dT and random hexamer primers were utilized for the synthesis of the first strand of cDNA. For analysis of miR-122-3P and miR-194-5p expression levels, exclusive cDNA for each miRNA was synthesized from the specific Stem-loop RT primer. Stem-loop RT primers were designed according to Chen et al.^{44 (link)}.

Fresh shoots of B. pervariabilis × D. grandis were ground in liquid nitrogen, and total RNA was extracted from tissue cells using the PlantTrol RNA extraction kit (TransGen, TransGen Biotech Co., Beijing, China). The concentration and integrity of RNA were determined by Nanodrop microspectrophotometer (NANODROP, ThermoFisher Scientific-CN, Shanghai, China) and agarose gel electrophoresis (DYY-6D, LIUYI, Beijing, China), respectively. EasyScript^® All-in-One First-Strand cDNA Synthesis SuperMix (TransGen) of TransGen Biotech Co. China, was used for PCR, reverse transcription into cDNA was carried out, and the results were kept on standby at −20 °C.

Total RNA was extracted from leaves treated with different abiotic stresses using an RNA Prep Pure Plant Kit (Tiangen Biochemical Technology (Beijing, China) Co., Ltd.: DP201101X). The concentration and purity of the RNA were determined using a Nanodrop micro spectrophotometer (Thermo Scientific, Waltham, MA, USA) and agarose gel electrophoresis. The first strand of cDNA derived from mRNA was synthesized using HiScript^®Q RT SuperMix (Vazyme, Nanjing, China). Quantitative real-time PCR (qRT-PCR) was carried out by SYBR-green fluorescence with a QuantStudio^TM Real-Time PCR System (Thermo Fisher Scientific). Five TH genes were randomly selected to validate the expression patterns under drought, cold, and heat stresses (Table S11). The data were normalized by the internal control gene BnACTIN (BnaA03g55890D) and the 2^−ΔΔCT analysis method was utilized to calculate relative expression levels [62 (link)].