Total rna extraction kit

HK-2 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, United States). The ALR shRNA lentiviral and control shRNA lentiviral were purchased from Genechem Co. Ltd (Shanghai, China). The JC-1 fluorescence kit and Annexin V-FITC/PI apoptosis detection kit were obtained from Sigma-Aldrich (MO, United States). MitoSOX was obtained from Invitrogen (Thermo Fisher, MA, United States). 4,6-Diamidino-2-phenylindole was acquired from KeyGen Biotech. Co. Ltd (Nanjing, China). The ALR primer was designed by Sangon Biotech Co., Ltd (Shanghai, China). Total RNA extraction kit and its reverse transcription kit were obtained from Takara Biotechnology (Shiga, Japan). Anti-PINK1, anti-Parkin, anticytochrome c oxidase subunit IV (anti-COX IV), antitranslocase of outer mitochondrial membrane 20 (anti-TOMM20), antitranslocase of inner mitochondrial membrane 23 (anti-TIMM23), anti-P62, and anticleaved caspase-3 (CASP-3) were purchased from Abcam (Cambridge, United Kingdom). Anti-ALR and anti-LC3B were purchased from Thermo Scientific (Thermo Fisher) and Cell Signaling Technology (CST, United States), respectively. Antibeta actin (anti-β-actin) was from Tianjin Sungene Biotech Co., Ltd (Tianjin, China). Cell mitochondrial extraction kits and all secondary antibodies for fluorescent labeling or immunoblot analysis were from Beyotime Biotech Co., Ltd (Shanghai, China).

Total RNA extraction was conducted using the Total RNA Extraction Kit (TaKaRa) and first-strand cDNA was synthesized with the PrimeScript RT Master Mix (TaKaRa). Real-time RT-PCR was performed as described previously (Li et al., 2015 (link)) by using SYBR Premix Ex TaqII (TaKaRa) on an Applied Biosystems QuanStudio 3 Real-Time PCR System. The following standard thermal profile was used for all PCRs: 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and 60 °C for 34 s. All reactions were done at least in triplicates. The primers used for qRT-PCR analysis were listed in Table S4.

To analyze gene expression of stemness and differentiation, qRT-PCR was performed. 3D co-axial cells were extracted by dissociation solution as described above and 2D cell were also collected. Total RNA was isolated and extracted by total RNA extraction kit (Takara, Beijing, China). RNA was reversely transcribed into cDNA with reverse transcription kit (Beyotime Biotechnology, Shanghai, China) according to the protocol. For qPCR, pairing primer sequences [20 (link)] were used (Supplementary Table S1), including alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP2), osteopontin (OPN), Runt-related transcription factor 2 (Runx-2) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). PCR amplification was performed using SYBR Green qPCR SuperMix (C11733046, Invitrogen, NY, USA) with an ABI PRISM^® 7500 Sequence Detection System (ThermoFisher Scientific) with the procedure of 95°C 2 min; 95°C 15 s, 60°C 30 s, reading plate, 40cycles. Each sample was repeated three times.

We performed RNA-seq analysis using the Illumina RNA-seq (separate samples) and PacBio full-length transcript (mixed samples) sequencing methods to improve the predictive ability of gene models. Six different tissues from M. polymorpha, including seeds, roots, stems, leaves, flowers, and pods, were collected and frozen in liquid nitrogen. Total RNA was extracted using a Takara Total RNA Extraction Kit (Takara, Dalian, China). Transcriptome libraries were constructed using a NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer’s procedures. Then, 150-bp paired-end sequencing was performed on the Illumina HiSeq X Ten platform. For PacBio sequencing, cDNA was synthesized from the mixed RNA samples used for Illumina sequencing using a SMARTer PCR cDNA Synthesis Kit. After PCR amplification, the products were sequenced on the PacBio RSII platform.

Total RNA of OSCC cells was isolated using a Total RNA Extraction Kit (Suzhou, China) and then transcribed into cDNA using Prime Script RT Reagent Kit (Takara, Shiga, Japan). Subsequently, the SYBR Green Master Mix (TOYOBO, Japan) was applied to conduct quantitative real-time polymerase chain reaction (qRT-PCR) on the ABIPRISM 7900HT instrument (Applied Biosystems, United States). The above experiments were repeated 3 times. Primer sequences used were as follows:

HOTAIRM1

F: 5′-TTG​ACC​TGG​AGA​CTG​GTA​GC-3′

R: 5′-TTC​AGT​GCA​CAG​GTT​CAA​GC-3′;

β-actin

F:5′ ATG​AAC​TGG​CGA​GAG​GTC​TGT3′

R:5′ CCA​GGA​ATG​AGT​AAC​ACG​GAG​T3′.

Total RNA from BEND cells was extracted using a Total RNA Extraction Kit (TaKaRa Biotechnology Co., Ltd., Tokyo, Japan) according to the manufacturer’s instructions. The RNA concentration and purity were determined using a nucleic acid protein detector (Bio-Rad Instruments). Reverse transcription of the extracted RNA into single-stranded cDNA was carried out using a reverse transcription kit (TaKaRa Biotechnology Co., Ltd.) according to the manufacturer’s instructions. Quantitative real-time PCR (RT-PCR) was performed using SYBR Premix Ex Taq II (TaKaRa Biotechnology Co., Ltd.) to assess mRNA expression levels of TNF-α, IL-6, IL-8, and NF-κB p65. All amplifications were repeated three times. RT-PCR data were calculated using the gene expression formula 2^−ΔΔCt. The relative expression of each gene was normalized to β-actin levels. The primers used for RT-PCR are listed in Table 1. Primers for IL-8 and TNF-α were designed using Primer Express Version 5.0 software (New York, NY, USA), and primers for NF-κB p65, IL-6, and β-actin were identical to those used by Shi et al. [40 (link)]. All primers were commercially synthesized by Sangon Biotech Institute Co., Ltd. (Beijing, China).

Total RNA was extracted from both root and leaves tissue of control and treated plant samples using the total RNA extraction kit (Takara, Dalian, China) according to the recommended protocol of manufacturers. RNA abundance was quantified with Nanodrop, while purity was confirmed on 1% agarose gel. For real-time RT-PCR analysis, 500 μg of total RNA was reverse transcribed by using the primer Script RT reagent Kit (Takara, Dalian, China). The cDNA samples were assayed by quantitative real-time PCR (qRT-PCR) in the Lightcycler480 (Roche International Diagnostics Systems, Switzerland) as follows: initial denaturation at 95 °C for 30 s, 40 cycles for denaturation and annealing (95 °C for 5 s and 60 °C for 30 s, respectively), followed by steps for Melt-Curve analysis (60−95 °C). The assay was performed using SYBR Green PCR Mastermix (Applied Biosystem, Hangzhou, China). List of gene-specific primers’ sequence of both wheat and barley plants are given in supplementary Table S1. 2^-ΔΔCt method was used to compare the transcript levels between different samples [66 (link)], as follows: $2^{-\mathsf{\Delta }\mathsf{\Delta }\mathrm{Ct} }=\left(C{t}_{gene}-C{t}_{endo Ref}\right) \mathrm{treated} \mathrm{sample} -\left(C{t}_{gene}-C{t}_{endo Ref}\right)non treated sample$