Journal: Stem Cells International

Article Title: Comparison of the MicroRNA Expression Profiles of Male and Female Avian Primordial Germ Cell Lines

doi: 10.1155/2018/1780679

Figure Lengend Snippet: An overview of the results of LC chicken miRNA microarray analysis. (a) The microarray analysis was performed on 4 PGC lines (2 female and 2 male) (Supplementary Tables – ). On the microarray, 991 chicken-specific miRNAs, 42 plate controls, and 6 gga-5Sb rRNA probes were placed (Supplementary ). From the 991 miRNAs, 27 miRNAs were expressed in all samples and altogether 153 miRNAs were expressed in PGC lines. (b) The Venn diagram introduces the similarities and differences between the cell lines. (c) Paired t -test analysis was conducted between the male (4ZP, FS101) and female (5ZP, FS111) PGC lines by LC Sciences Company. Paired t -test was conducted between the male and female PGC samples at the 0.05 significance level, to analyse the differences in the expression level of all expressing miRNAs in the samples. The heat map represents the result of the analysis. Only 6 differentially expressing miRNAs were found: gga-miR-1354, gga-miR-1767, gga-mir-30c-5p, gga-miR-1584, gga-miR-1599, and gga-miR-2127 (Supplementary , Supplementary Fig. ).

Article Snippet: As microRNAs (miRNAs) have been proved to play a key role in the maintenance of pluripotency and the cell cycle regulation of stem cells, a complex miRNA analysis (microarray-based, μ Paraflo® Microfluidic Biochip Technology, LC Sciences, Houston, Texas, USA) was performed to determine the difference in miRNA expression profile between 2 female and 2 male PGC lines (Supplementary Tables – ).

Techniques: Microarray, Expressing

Journal: Stem Cells International

Article Title: Comparison of the MicroRNA Expression Profiles of Male and Female Avian Primordial Germ Cell Lines

doi: 10.1155/2018/1780679

Figure Lengend Snippet: Expression pattern of miRNAs, identified in all examined samples by LC chicken miRNA microarray analysis. The microarray analysis was performed on 4 PGC lines (2 female (FS111 and 5ZP) and 2 male (FS101 and 4ZP)). (a) The expression values of miRNAs, expressing in all PGC samples, were visualized in a heat map (using GenEx software, complete linkage and Spearman correlation analysis were performed). Simultaneously, cluster analysis was performed. According to the analysis, the male cells were more related to each other. The 5ZP PGC line was the most different from the others. (b), (c) Using GenEx software, we performed a scatterplot analysis to identify the upregulated miRNAs. (b) represents the group of upregulated miRNAs in the 5ZP PGC line. These miRNAs compose cluster 1, while (c) shows the upregulated miRNAs in the highly proliferating PGC lines. These miRNAs formed cluster 2. The results of the clustering analysis highlighted a portion of miRNAs in cluster 2 which belong to the miRNA-302 cluster.

Article Snippet: As microRNAs (miRNAs) have been proved to play a key role in the maintenance of pluripotency and the cell cycle regulation of stem cells, a complex miRNA analysis (microarray-based, μ Paraflo® Microfluidic Biochip Technology, LC Sciences, Houston, Texas, USA) was performed to determine the difference in miRNA expression profile between 2 female and 2 male PGC lines (Supplementary Tables – ).

Techniques: Expressing, Microarray, Software

Journal: Stem Cells International

Article Title: Comparison of the MicroRNA Expression Profiles of Male and Female Avian Primordial Germ Cell Lines

doi: 10.1155/2018/1780679

Figure Lengend Snippet: Expression pattern of stem cell- and germ cell-specific markers in RNA samples examined at LC miRNA microarray analysis. We performed qPCR analysis to check the RNA expression profile in samples sent to LC microarray analysis. We compared one female (5ZP) and one male (4ZP) GFP PGC sample, and one female (FS111) and one male (FS101) PC PGC sample using 3 parallel samples at qPCR analysis. (a), (b) Expression of CVH, cDAZL, cPOUV, and cNANOG (relative to cGAPDH as the reference gene) and gga-miR-302a, gga-miR-302b-3p, and gga-miR-302b-5p (relative to U6 as the reference gene) was analysed. Relative gene expression values were calculated relative to the FS101 sample in each case. (c) The proliferation rate of PGCs was measured on 3 different days using CCK-8 proliferation assay. The doubling time was calculated according to the measured optical densities (OD). (d) The miR-302b-5p/miR-302b-3p ratio was calculated from the average delta Ct values of samples.

Article Snippet: As microRNAs (miRNAs) have been proved to play a key role in the maintenance of pluripotency and the cell cycle regulation of stem cells, a complex miRNA analysis (microarray-based, μ Paraflo® Microfluidic Biochip Technology, LC Sciences, Houston, Texas, USA) was performed to determine the difference in miRNA expression profile between 2 female and 2 male PGC lines (Supplementary Tables – ).

Techniques: Expressing, Microarray, RNA Expression, Gene Expression, CCK-8 Assay, Proliferation Assay

Journal: Stem Cells International

Article Title: Comparison of the MicroRNA Expression Profiles of Male and Female Avian Primordial Germ Cell Lines

doi: 10.1155/2018/1780679

Figure Lengend Snippet: Most important targets of gga-miR-302b-5p and gga-miR-302-3p miRNAs. The main molecular targets for the studied miRNAs have been presented. The molecular pathways and targets for gga-miR-302b-5p are on the left side and for gga-miR-302b-3p on the right side. MAKP signalling is responsible for maintaining pluripotency and proliferation in mammals. It can be activated by a series of intrinsic and extrinsic stimulatory signals. In chickens, gga-miR-302b-5p controls the proliferation rate via MAPK signalling. In the case of low-proliferating PGC cell lines, gga-miR-302b-3p expression was high. It can be assumed that the high gga-miR-302b-3p expression somehow causes downregulation of pathways promoting proliferation, thereby causing cell cycle arrest at the G1 stage. gga-miR-302b-5p was found to be highly expressed in high-proliferating PGC lines. Hence, it can be hypothesized that probably the high expression of gga-miR-302b-5p is contributed in controlling throughout its molecular targets in the MAPK signalling pathway. miR-302b-5p probably by inhibiting the MAPK pathway components can cause high proliferation rate in PGC lines, and miR-302a is responsible for the fast transition from the G1 to S phase in the cell cycle .

Article Snippet: As microRNAs (miRNAs) have been proved to play a key role in the maintenance of pluripotency and the cell cycle regulation of stem cells, a complex miRNA analysis (microarray-based, μ Paraflo® Microfluidic Biochip Technology, LC Sciences, Houston, Texas, USA) was performed to determine the difference in miRNA expression profile between 2 female and 2 male PGC lines (Supplementary Tables – ).

Techniques: Expressing

Journal: Current Molecular Medicine

Article Title: microRNAs in the Regulation of Adipogenesis and Obesity

doi: 10.2174/156652411795677990

Figure Lengend Snippet: miRNA Microarray Platforms Used in Adipogenesis and Obesity Studies

Article Snippet: 3T3-L1 , Microfluidic biochip (LC Sciences) , [ ] .

Techniques: Microarray, TaqMan microRNA Assay

Journal: Small (Weinheim an Der Bergstrasse, Germany)

Article Title: On‐Chip De Novo Production of mRNA Vaccine in Lipid Nanoparticles

doi: 10.1002/smll.202500114

Figure Lengend Snippet: Design and characterization of on‐chip RNA synthesis. a) Schematics of universal template preparation. The CDS is located downstream of the T7P sequence and upstream of a T7T sequence. The template is then amplified using biotin‐tagged primers complementary to the T7P and T7T sequences. Following the PCR purification step, the DNA template is tagged with biotin both at the 5′ end and at the 3′ end. b) Design of the microfluidic device with the T junction motif for droplets encapsulating RNA. c) Illustration of the on‐chip IVT reaction, RNA purification, and encapsulation. i) SA and Ni‐NTA beads are trapped in the reaction compartment. Biotinylated PCR product is introduced to the device alongside HIS‐tagged T7RP and IVT reaction components. Following incubation at 37 °C, the IVT reaction components are allowed to flow through the device. The DNA template and HIS‐tagged T7RP are then immobilized onto the beads. ii, iii) brightfield images of the beads trapped in the compartment. The purified mRNA is allowed to flow toward the T junction motif, whereas iv) the oil phase intersects the aqueous mRNA phase, resulting in the formation of water‐in‐oil droplets. vi) Fluorescence microscopy image of DNA template tagged with Cy3, immobilized onto SA beads in the main compartment. Scale bar 200 µ m . vii) Fluorescence microscopy image of the on‐chip produced droplets encapsulating Mango RNA aptamer. Scale bar 200 µ m . d) Gel electrophoresis of RBD‐hFc mRNA and SARS‐CoV‐2 full spike protein mRNA produced on‐chip. e) On‐chip or column‐based purification yields of both non‐modified and modified SARS‐CoV‐2 full spike protein mRNA. ∗ p < 0.05, ∗∗ p < 0.01 (two‐tailed Student's t test, n = 3). f) On‐chip generated mRNA stability at different pH and with RNAse for up to 24 h.

Article Snippet: The microfluidic biochips were fabricated from polydimethylsiloxane (PDMS, Dow Corning) using SU8 on silicon masters and standard soft lithography techniques.

Techniques: Sequencing, Amplification, Purification, Encapsulation, Incubation, Fluorescence, Microscopy, Produced, Nucleic Acid Electrophoresis, Modification, Two Tailed Test, Generated

Journal: Small (Weinheim an Der Bergstrasse, Germany)

Article Title: On‐Chip De Novo Production of mRNA Vaccine in Lipid Nanoparticles

doi: 10.1002/smll.202500114

Figure Lengend Snippet: On‐chip de novo production of mRNA in lipid nanoparticles. a) Fluorescence microscopy image of the microfluidic device with the Tesla mixer for LNP generation using a 0.01% (V/V) fluoresceine solution. Scale bar 5 mm. b) Fluorescence microscopy image of the intersection between the main compartment and the lipid mix using 0.01% (V/V) fluoresceine solution and EtOH. Scale bar 100 µm. c) Illustration of the on‐chip RNA‐LNP generation. i) mRNA is synthesized and purified as described in Figure . ii) lipid mixture is introduced from a second inlet. iii) the mRNA aqueous solution encounters the lipid solution and is mixed in the Tesla mixer to form LNP (iv). d) DLS characterization of the on‐chip produces LNPs pre‐ and post‐cryopreservation treatment, including size distribution and polydispersity index (PDI) e) Particle size (back) and PDI (green) of on‐chip produced over time. f) A representative Cryo‐EM image of on‐chip produced LNPs. Scale bar 100 nm. g) Agarose electrophoresis gel of naked mRNA and on‐chip produced LNP with or without RNAse treatment for up to 24 h.

Article Snippet: The microfluidic biochips were fabricated from polydimethylsiloxane (PDMS, Dow Corning) using SU8 on silicon masters and standard soft lithography techniques.

Techniques: Fluorescence, Microscopy, Synthesized, Purification, Produced, Cryo-EM Sample Prep, Electrophoresis

Journal: Blood Advances

Article Title: Reticulocyte and red blood cell deformation triggers specific phosphorylation events

doi: 10.1182/bloodadvances.2019000545

Figure Lengend Snippet: GSK3 and Lyn inhibition impacts capillary traversal of reticulocytes and red blood cells. (A) Percentage recovery of reticulocytes treated with the respective reagent after microsphiltration. After treatment, the cells were added to an untreated RBC suspension in a 5:95 ratio and the mixture then subjected to microsphiltration. Anti-GPA antibody was used as a positive control for negative effects on cell deformability. Bafetinib (3 µM) was used as a Lyn inhibitor and CHIR-98014 (10 nM) was used as a GSK3 inhibitor. Error bars correspond to the standard deviation (n = 3). All comparisons were performed with a paired 2-tailed Student t test. The P values for each comparison are shown below the bar graph. *P < .05, **P < 0.01. (B) Image of the microfluidic PDMS biochip used for the capillary traversal experiment. The magnified image shows a section of the microcapillary channels, through which reticulocytes and red blood cells were subjected to successive deformations. Frame sequences were obtained through the use of a high-speed camera and then subjected to image analysis for automated cell tracking with the use of TrackMate.51 Scale bars, 50 μm. (C) Percentage microcapillary traversal velocity of reticulocytes and red blood cells treated with the respective reagents. The cells were treated for a minimum period of 1 hour and subjected to passage through the microfluidic biochip. The graph shows the average cell microcapillary traversal velocity of each treated sample as a percentage of the respective DMSO control sample traversal velocity. All comparisons were performed with a 1-sample Student t test. The error bars correspond to the standard deviation (n = 3). The P values for each comparison are shown below the dot plot. *P < .05, **P < .01. A minimum of 1000 cell tracks were analyzed per sample. n.s.s., not statistically significant.

Article Snippet: The microfluidic biochip was designed and fabricated as described by Lizarralde Iragorri et al. 19 An MFCS-EZ microfluidic flow control system (Fluigent) was used to regulate the flow pressure in the microfluidic biochip.

Techniques: Inhibition, Positive Control, Standard Deviation, Cell Tracking Assay