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Identification and characterization analysis of the different genes in primary mouse microglia response to circDYM using RNA-seq.

ABSTRACT: Whole RNA-sequencing analysis was performed and analyzed by Lc. Biotech Co. Ltd. (Hangzhou, China). Primary mouse microglia from the Con + circCon group, Con + circDYM group, LPS + circCon group, and LPS + circDYM group were collected in TRIzol. Total RNAs were extracted from Trizol. UMI technology was used to label each sequence fragment with sequence tags, which minimized the interference of duplication caused by PCR amplification on the quantitative accuracy of the transcriptome. RNA sequencing reads were aligned to the mouse genome (GRCh37/hg19) using the software Hisat2 (2.0.4). Transcript abundance was evaluated by calculating fragments per kilo base of exon per million fragments mapped (FPKM).

ORGANISM(S): Mus musculus

PROVIDER: GSE159904 | GEO | 2022/01/31

REPOSITORIES: GEO

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Project description:The objective of the study was to examine the gene expression changes in the medial frontal cortices of P28 offspring from the breeding of Dnmt3l conditional knockin and Emx1-cre mice, strain of C57BL/6J. There were 12 samples in total- A-1, A-2, A-3, B-1, B-2, B-3, C-1, C-2, C-3, D-1, D-2, and D-3. A-1 to A-3 and B-1 to B-3 were medial frontal cortices from the male and female Emx1-cre mice respectively, which were used as the control groups (CON). C-1 to C-3 and D-1 to D-3 were medial frontal cortices from the male and female Dnmt3l overexpression mice respectively, which were used as the experimental groups (OE). The total RNA of each sample was extracted from the medial frontal cortices by using TRIzol reagent. Then the RNA samples were processed for high throughput transcriptome sequencing on Illumina HiSeq 3000 platform. There were two types of libraries: the circRNA, mRNA, and lncRNA were all constructed by removing rRNA (one library, three types of RNA were analyzed together), and the insert fragment was about 300bp; the small RNA was constructed and analyzed separately, mainly microRNA of about 22bp. Results: among 119414 cleaned mRNAs, 3015 were differentially expressed in OE group compared with CON group (p value ≤ 0.05 and expression change ≥2 fold). The differential expressed genes distributed in all chromosomes. Functional annotation with GO and KEGG enrichment revealed the top functional groups including ATP binding, zinc ion binding, glutamatergic synapse, dopaminergic synapse, MAPK signaling pathway, and PI3K-Akt signaling pathway. Among 23698 lncRNAs, 658 were differentially expressed in OE group compared with CON group (p value ≤ 0.05 and expression change ≥2 fold). Among 10673 circRNAs, 49 were differentially expressed in OE group compared with CON group (p value ≤ 0.05 and expression change ≥2 fold). Among 1821 microRNAs, 101 were differentially expressed in OE group compared with CON group (p value ≤ 0.05 and expression change ≥2 fold).

Project description:Mice and diets. One-month-old male mice of the long-lived B6C3F1 strain were purchased from Harlan (Indianapolis, IN) and maintained as described after weaning (15). Mice were housed in groups of 4 per cage and fed a non-purified diet, PMI Nutrition International Product #5001 (Purina Mills, Richmond, IN). At 5 months of age, the mice were individually housed, and randomly assigned to one of two groups, control (CON) or long-term CR (LTCR). CON mice were fed 93 kcal per week of a defined control diet (AIN-93M, Diet No. F05312, BIO-SERV). LTCR mice were fed 52.2 kcal per week of a defined CR diet (AIN-93M 40% Restricted, Diet No. F05314, BIO-SERV). The LTCR mice consumed approximately 40% fewer calories than mice in the CON mice. The CR diet was enriched so that the CR mice consumed approximately the same amount of protein, vitamins, and minerals per gram body weight as the control mice. Experimental design. At 20 months of age, cohorts of mice in the CON group were randomly assigned to one of 7 experimental groups (Fig. 1). A CON group continued to be fed 93 kcal per week of control diet for 8 weeks. A CR8 group was fed 77 kcal per week of CR diet for 2 weeks, followed by 52.2 kcal per week of CR diet for 6 weeks. The remaining dietary groups were fed the control diets containing, respectively, metformin (MET; Sigma, St. Louis, MO), glipizide (GLIP; Sigma), GLIP plus MET (GM), rosiglitazone (ROS; Avandia, SmithKline Beecham) or soy isoflavone extract (SOY; NOVASOY 400, Life Extension Foundation) for 8 weeks at the dosages indicated in Table 1. The drugs were mixed with powdered control diet and cold-pressed into one gram pellets (BIO-SERV Inc.). At 0900, all mice were fed 2/7 of the weekly allotment of food on Monday and Wednesday and 3/7 on Friday. The mice had free access to acidified tap water. They were fasted for 48 hours and killed by cervical dislocation at 22 months of age. No signs of pathology were detected in the animals used for the studies reported (n=4 per group). Organs were removed rapidly, flash frozen, and stored in liquid nitrogen. The weights of the mice were monitored bi-weekly. Mouse weights are given in Table 1. Measurement of specific mRNA levels. Liver total RNA was isolated as described from livers of pathology-free mice in each group (n=4 per group) (13). mRNA levels were measured using Affymetrix mouse U74Av2 arrays according to standard Affymetrix protocols (Affymetrix). Probe set expression measurement and normalization. After hybridization and scanning, raw image files were converted to probe set data (*.CEL files) using Microarray Suite (MAS 5.0). Probe set data from all 32 arrays were simultaneously analyzed with the Robust multichip Average (RMA) method to generate normalized expression measures for each probe set (Fig. 2; Ref. 24) The data were further filtered to exclude probe data sets that were “Absent” across all 32 arrays according to the MAS 5.0 detection algorithm (1,47). Keywords = CR mimetics Keywords = microarray profiling Keywords = drug discovery Keywords = gene-expression biomarkers Keywords: other

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Identification and characterization analysis of the different genes in primary mouse microglia response to circDYM using RNA-seq.

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