Rnasey mini kit

The tibialis anterior muscle sample at the different time points was homogenized, and RNA was extracted with RNasey Mini Kit (Qiagen, San Francisco, CA, United States) according to the manufacturer’s instructions. Microarray analysis was performed using an Agilent SurePrint G3 Rat GE (8 × 60K, Design ID: 028279), as previously described (Shen et al., 2019 (link)). Microarray analysis was performed on an Agilent Gene Chip platform and scanned by Agilent Scanner G2505C (Agilent Technologies). Data were extracted from scanned images using Agilent Feature Extraction Software (version 10.7.1.1, Agilent Technologies). The raw data were normalized by Genespring Software (version 13.1, Agilent Technologies). This dataset is available at the NCBI Gene Expression Omnibus (GEO) repository with GEO accession: GSE201025.

Total RNA was isolated from 20 samples (collected from Henan Provincial People’s Hospital), including ten tumor tissues and ten adjacent normal tissues, using an mirVana™ RNA isolation kit (AM1561, Thermo Fisher, USA). RNA clean-up was performed using RNasey Mini Kit (Qiagen p/n 74,104). Quantification and quality control were achieved by NanoDrop ND-2100 (ThermoFisher) and Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, USA). cDNA synthesis and biotin labeling of cRNA were performed using One-Cycle Target Labeling and Control Reagents (Affymetrix, Santa Clara, USA) and the GeneChip™ Hybridization, Wash and Stain Kit (Affymetrix) following the manufacturer’s protocol. The purified and fragmented labeled cRNAs were hybridized onto the Affymetrix PrimeView™ Human Gene Expression microarray. After washing and staining, the arrays were scanned and analyzed using Affymetrix Scanner 3000 (Affymetrix). GeneChip Command Console Software (version 4.0, Affymetrix) and Genespring software (version 14.9, Agilent Technologies) were used to analyze the transcriptome data. Differentially expressed genes (DEGs) from TCGA were analyzed using DESeq2 package within R language (version 3.4.3).

Total RNAs were extracted from the hippocampal tissues using RNAiso Plus Reagent (TaKaRa, Japan), and purified through RNasey Mini Kit (QIAGEN). The total RNAs were identified and quantified by NanoDrop spectrophotometry (Thermo Scientific, Wilmington, USA), and 1–2 μg of total RNAs was used to construct the RNA-Seq libraries according to the manufacturer's protocol. Sequencing library was detected using an Illumina HiSeq platform, and paired-end reads were generated followed by cluster generation. The raw sequencing data were processed by in-house Perl scripts after quality control. Thereafter, the differentially expressed genes (FDR < 0.05, fold change >2) were analyzed using R studio software. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were performed using String online tools (https://string-db.org). Then, the RNA-seq data files were deposited in the NCBI Sequence Read Archive (SRA) database (SRA accession: PRJNA781082).

Total RNA was isolated from cultured cells at 70% confluency using QIAGEN RNasey mini kit according to manufacturer’s instruction. cDNA was then synthesized using iscript reverse transcriptase (BIORAD 1708840). Real-time quantitative PCR analysis was performed in 20 uL reaction using SYBR Green Master mix (BIORAD 1725270) and CFX Real Time PCR system (BIORAD). Primer efficiency was verified by linear regression to standard curve using 2, 1, ½, ¼, 1/8, 1/16, 1/32, 1/64 and1/128 dilution. Reactions were carried out in quadruplicate and values were standardized to housekeeping genes actin, 18S rRNA, and GAPDH. To normalize values obtained in the samples, control actin Ct values were subtracted from the target gene Ct values for each sample (ΔC_T) then, Ct of the unstimulated sample was subtracted from ΔC_T of the stimulated sample (ΔΔC_T). The relative levels of target gene were calculated as 2 ^–ΔΔCT.

Total RNA was extracted using the TRIzol reagent (Invitrogen, Foster City, CA, USA) according to the manufacturer's instructions. RNA was cleaned using the RNasey Mini Kit (p/n 74104, Qiagen, Germany), and labeled with Quick Amp Labeling Kit One-Color (p/n 5190-0442, Agilent, USA). lncRNA expression was detected using the lncRNA microarray (Arraystar, Rockville, MD, USA). Microarray data were extracted using Agilent Feature Extraction Software.
During functional enrichment analysis, the online software Database for Annotation, Visualization, and Integrated Discovery (DAVID; https://david.ncifcrf.gov/) was utilized to perform GO and KEGG pathway analyses to investigate potential pathways and networks associated with differentially expressed genes [36 (link), 37 (link)]. Terms with P<0.05 were considered significantly enriched.

Total RNA was extracted from tissue cells using Trizol Reagent (Invitrogen life technologies) according to the manufacturer's instructions; total RNA was extracted from whole blood using TRI reagent BD (MRCgene, TB-126). Total RNA was extracted from plasma and serum exosomes using TRIzol LS. RNAsey Mini Kit (QIAGEN) was used for RNA purification; NanoDrop ND-1000 was employed for measurement of RNA concentration after purification. RNA integrity was detected by electrophoresis.