mrna sequencing
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2022 ◽  
Vol 12 ◽  
Author(s):  
Zongwei Zhang ◽  
Wei Liang ◽  
Qiang Luo ◽  
Hongtu Hu ◽  
Keju Yang ◽  
...  

BackgroundGlycolysis dysfunction is an important pathogenesis of podocyte injury in diabetic kidney disease (DKD). Foot process fusion of podocytes and increased albuminuria are markers of early DKD. Moreover, cytoskeletal remodeling has been found to be involved in the foot process fusion of podocytes. However, the connections between cytoskeletal remodeling and alterations of glycolysis in podocytes in DKD have not been clarified.MethodsmRNA sequencing of glomeruli obtained from db/db and db/m mice with albuminuria was performed to analyze the expression profiling of genes in glucose metabolism. Expressions of phosphofructokinase platelet type (PFKP) in the glomeruli of DKD patients were detected. Clotrimazole (CTZ) was used to explore the renal effects of PFKP inhibition in diabetic mice. Using Pfkp siRNA or recombinant plasmid to manipulate PFKP expression, the effects of PFKP on high glucose (HG) induced podocyte damage were assessed in vitro. The levels of fructose-1,6-bisphosphate (FBP) were measured. Targeted metabolomics was performed to observe the alterations of the metabolites in glucose metabolism after HG stimulation. Furthermore, aldolase type b (Aldob) siRNA or recombinant plasmid were applied to evaluate the influence of FBP level alteration on podocytes. FBP was directly added to podocyte culture media. Db/db mice were treated with FBP to investigate its effects on their kidney.ResultsmRNA sequencing showed that glycolysis enzyme genes were altered, characterized by upregulation of upstream genes (Hk1, and Pfkp) and down-regulation of downstream genes of glycolysis (Pkm, and Ldha). Moreover, the expression of PFKP was increased in glomeruli of DKD patients. The CTZ group presented more severe renal damage. In vitro, the Pfkp siRNA group and ALDOB overexpression group showed much more induced cytoskeletal remodeling in podocytes, while overexpression of PFKP and suppression of ALDOB in vitro rescued podocytes from cytoskeletal remodeling through regulation of FBP levels and inhibition of the RhoA/ROCK1 pathway. Furthermore, targeted metabolomics showed FBP level was significantly increased in HG group compared with the control group. Exogenous FBP addition reduced podocyte cytoskeletal remodeling and renal damage of db/db mice.ConclusionsThese findings provide evidence that PFKP may be a potential target for podocyte injury in DN and provide a rationale for applying podocyte glycolysis enhancing agents in patients with DKD.


2021 ◽  
Author(s):  
Hiroki Ura ◽  
Sumihito Togi ◽  
Yo Niida

Abstract Background mRNA sequencing is a powerful technique, which is used to investigate the transcriptome status of a gene of interest, such as its transcription level and splicing variants. Presently, several RNA sequencing (RNA-Seq) methods have been developed; however, the relative advantage of each method has remained unknown. Here we used three commercially available RNA-Seq library preparation kits; the traditional method (TruSeq), in addition to full-length double-stranded cDNA methods (SMARTer and TeloPrime) to investigate the advantages and disadvantages of these three approaches in transcriptome analysis. Results We observed that the number of expressed genes detected from the TeloPrime sequencing method was fewer than that obtained using the TruSeq and SMARTer. We also observed that the expression patterns between TruSeq and SMARTer correlated strongly. Alternatively, SMARTer and TeloPrime methods underestimated the expression of relatively long transcripts. Moreover, genes having low expression levels were undetected stochastically regardless of any three methods used. Furthermore, although TeloPrime detected a significantly higher proportion at the transcription start site (TSS), its coverage of the gene body was not uniform. SMARTer is proposed to be yielded for nonspecific genomic DNA amplification. In contrast, the detected splicing event number was highest in the TruSeq. The percent spliced in index (PSI) of the three methods was highly correlated. Conclusions TruSeq detected transcripts and splicing events better than the other methods and measured expression levels of genes, in addition to splicing events accurately. However, although detected transcripts and splicing events in TeloPrime were fewer, the coverage at TSS was highest. Additionally, SMARTer was better than TeloPrime among the understudied full-length double-stranded cDNA methods. In conclusion, for short-read sequencing, TruSeq has relative advantages for use in transcriptome analysis.


2021 ◽  
Vol 32 (4) ◽  
Author(s):  
Ilona Holcomb ◽  
Nidhanjali Bansal ◽  
Tommy Duong ◽  
Paul Babb ◽  
Julie Laliberte ◽  
...  

2021 ◽  
Author(s):  
Mi K Trinh ◽  
Clarissa N Pacyna ◽  
Gerda K Kildisiute ◽  
Nathaniel D Anderson ◽  
Eleonora Khabirova ◽  
...  

A fundamental step of tumour single cell mRNA analysis is separating cancer and non-cancer cells. We show that the common approach to separation, using shifts in average expression, can lead to erroneous biological conclusions. By contrast, allelic imbalances representing copy number changes directly detect the cancer genotype and accurately separate cancer from non-cancer cells. Our findings provide a definitive approach to identifying cancer cells from single cell mRNA sequencing data.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4815-4815
Author(s):  
Mengyi Du ◽  
Heng Mei ◽  
Chenggong Li ◽  
Yinqiang Zhang ◽  
Lu Tang ◽  
...  

Abstract Background The development of mRNA sequencing has contributed greatly to the mechanism exploration in hematologic malignancies disease. With the advent of revolutionized single-cell mRNA sequencing (scRNA-seq), it is now possible to characterize every subset of expression programs and functional states in a comprehensive and unbiased manner. Here, we present a systematic evaluation of engineered chimeric antigen receptor T (CAR-T) products and patient bone marrow profiles in terms of primary resistance and severe cytokine release syndrome (CRS) at the single-cell level. Methods Using single-cell mRNA sequencing in conjunction with flow cytometry (FCM), we performed characterization of CD19-targeted CAR-T and mononuclear bone marrow cells from 4 on-trial B acute lymphoblastic leukemia (B-ALL) patients (NCT02965092). Bioinformatics analysis was utilized to explore diversity between patients with different grades of response or CRS. Basing on marker genes, CAR-T products were divided into four groups, which were double-positive T (DPT), CD4 positive T (CD4), CD8 positive T (CD8), and double-negative T (DNT) cells. Meanwhile, both the mononuclear bone marrow cells before and after CAR-T infusion were grouped into six clusters, which were B-ALL, stem, progenitor, B, T, and myeloid cells. The expression and enrichment analyses results were calculated by R (version 3.6.3) and then verified in a 22-sample conventional transcription sequencing cohort of the same clinical trial. Patient efficacy was assessed by the national comprehension cancer network guidelines version 2.2020 for acute lymphoblastic leukemia, and CRS was graded by CTCAE 5.0. Results By FCM detection, the variances of CAR-T infusion products between patients with different clinical outcomes were limited, and nor did mononuclear bone marrow cells. The scRNA sequencing results showed that distinct CAR-T and bone marrow cell subsets indicated differentiated expression in proliferation, cytotoxicity, and intercellular signaling pathways. Expression differentiation variances in CAR-T infusion products were minor than in mononuclear bone marrow cells. CD8+ CAR-T products of complete response (CR) patients were still significantly enriched in pathways such as cell killing (p adjust=0.0012), antigen processing and presentation (p adjust=0.0027), and cell cycle (p adjust=0.0231), exhibiting greater immune function when compared with no response patients. Also, DPT CAR-T products of the non-CRS patients were meaningfully enriched in negative regulation of cytokine production pathway (p adjust=0.0127) when compared with CRS ones. In mononuclear bone marrow cells, B-ALL cells before CAR-T treatment of CR patients presented negatively in cell-cycle (p adjust=0.0019), leading to a low malignant cell proliferation level; and stem-progenitor cells after CAR-T treatment of CR patients showed a stronger ability of neutrophil activation (p adjust<0.0001). As with comparisons between CRS and non-CRS, B-ALL cells before infusion manifested a cell cycle arrest profile (p adjust<0.0006) in non-CRS patients, whereas the immune cells at the same time point were enriched in positive regulation of cell cycle process (p adjust=0.0002). Conclusions Through single-cell RNA-seq profiling and unbiased canonical pathway analyses, our results unveil heterogeneities in the cell cycle, immune phenotype, and metabolic profiles of subsets during CAR-T therapy, providing a mechanistic basis for ameliorating clinical outcomes and individualized management. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.


2021 ◽  
Author(s):  
Bryan Yoo ◽  
Jessica Griffiths ◽  
Sarkis Mazmanian
Keyword(s):  

Protocol for RNAseq used in Yoo et al 2021


2021 ◽  
Author(s):  
Weijian Li ◽  
Gaohuang Chen ◽  
Zhenyu Feng ◽  
Baoyi Zhu ◽  
Lilin Zhou ◽  
...  

Abstract Background Prostate cancer (PCa) is one of the most common malignancies in men. YTHDF1 may play an important role in promoting PCa progression, but there is no reports to date on YTHDF1 function in PCa. Objective This study explored whether YTHDF1 could regulate TRIM44 in PCa cells. Methods By querying the TCGA database, we evaluated YTHDF1 expression in PCa, the OS and DFS of YTHDF1, and the correlation between YTHDF1 and TRIM44 in PCa. We constructed vectors to interfere with YTHDF1 expression and overexpress TRIM44 to examine the role of YTHDF1 and TRIM44 in PCa cells. Differentially expressed mRNAs were identified by mRNA sequencing. The levels of YTHDF1, TRIM44, LGR4, SGTA, DDX20, and FZD8 were measured by qRT-PCR and WB was used to determine YTHDF1 and TRIM44 expression. A CCK-8 assay was used to assess cell proliferation. A Transwell chamber assay was used measure cell migration and invasion ability. Results YTHDF1 was highly expressed in both Pca tissues and cells. PCa patient prognosis with high YTHDF1 expression was relatively poor. Cell function experiments showed that inhibiting YTHDF1 expression decreased cell proliferation, migration, and invasion. RNA sequencing analysis revealed that YTHDF1 may promote PCa cell proliferation, migration, and invasion by modulating TRIM44 expression. Cell function experiments further verified that YTHDF1 promoted PCa cell proliferation, migration, and invasion by regulating TRIM44. Conclusions YTHDF1 enhances PCa cell proliferation, migration, and invasion by regulating TRIM44.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Nicholas J. Silva ◽  
Leah C. Dorman ◽  
Ilia D. Vainchtein ◽  
Nadine C. Horneck ◽  
Anna V. Molofsky

AbstractMicroglia are brain resident macrophages that play vital roles in central nervous system (CNS) development, homeostasis, and pathology. Microglia both remodel synapses and engulf apoptotic cell corpses during development, but whether unique molecular programs regulate these distinct phagocytic functions is unknown. Here we identify a molecularly distinct microglial subset in the synapse rich regions of the zebrafish (Danio rerio) brain. We found that ramified microglia increased in synaptic regions of the midbrain and hindbrain between 7 and 28 days post fertilization. In contrast, microglia in the optic tectum were ameboid and clustered around neurogenic zones. Using single-cell mRNA sequencing combined with metadata from regional bulk sequencing, we identified synaptic-region associated microglia (SAMs) that were highly enriched in the hindbrain and expressed multiple candidate synapse modulating genes, including genes in the complement pathway. In contrast, neurogenic associated microglia (NAMs) were enriched in the optic tectum, had active cathepsin activity, and preferentially engulfed neuronal corpses. These data reveal that molecularly distinct phagocytic programs mediate synaptic remodeling and cell engulfment, and establish the zebrafish hindbrain as a model for investigating microglial-synapse interactions.


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