polymerase chain reaction  (Vazyme Biotech Co)

Journal: Clinical Epigenetics

Article Title: An epigenomic investigation of atrial fibrillation in a matched left and right atrial human cohort

doi: 10.1186/s13148-026-02076-2

Figure Lengend Snippet: Targeted validation of gene expression levels at candidate loci in an independent replication cohort. Locus-specific comparison of gene expression levels in AF and SR samples for selected candidate loci in the discovery cohort (top boxplots in each category, RNA-seq), and their validation in an independent replication set (bottom barplots in each category, RT-qPCR). RT-qPCR results are represented as relative expression levels over those of TBP as a reference gene. Gene expression differences across disease status were considered as validated in independent samples when the direction of change was maintained with a p-value < 0.2 (one-sided t-test). a Gene expression levels in the discovery cohort (top boxplots) and validation cohort (bottom barplots) across 8 AF-enriched candidate loci. b As in a. for five SR-enriched candidate loci. See also Figure S5 and Table S7

Article Snippet: 1000 ng of RNA were reversed transcribed into complementary DNA (cDNA) using the High-Capacity cDNA Reverse Transcription Kit (Fisher Scientific), and RT-qPCR reactions were performed using 5 ng of template cDNA.

Techniques: Biomarker Discovery, Gene Expression, Comparison, RNA Sequencing, Quantitative RT-PCR, Expressing

Journal: Cell Death Discovery

Article Title: CSF1R T567M mutation induces microglial dysfunction and synaptic impairment in patient iPSC-derived cerebral organoids of CSF1R-related disorder

doi: 10.1038/s41420-026-02995-2

Figure Lengend Snippet: CSF1R-T567M mutant (MT) and corrected (Ctrl) iPSCs derived from patients were used to differentiate into microglia (iMGL). A Representative images of P2RY12 + and TMEM119 + iMGL. Scale bar = 10 µm. B Comparison of area, branch number, and maximum branch length between Ctrl and MT iMGL with IBA1 + TMEM119 + staining. Cells from 50 fields of view were counted for each group. Scale bar = 5 µm. Statistical analysis of IBA1 + iMGL cell area ( C ), branches per cell ( D ), and maximum branch length ( E ) in ( B ). F Cytokine profiling assay after 24 h of stimulation with 100 ng/mL LPS, showing significant upregulation of candidate cytokines. RT-qPCR of CCL2 ( G ) and IL-18 ( H ) transcription in Ctrl and MT iMGL. N = 3. I Migration assay of Ctrl and MT iMGL after stimulation with ATP. White arrowheads indicate some of the migrated iMGL. Cells from 15 fields of view were counted for each group. Scale bar = 5 µm. J Effect of the CSF1R-MT on the phagocytic ability of green fluorescent latex beads (white arrowheads). The iMGL were stained with IBA1 (red signals). N = 8. Scale bar = 10 µm. K Effect of the CSF1R-MT on the phagocytic ability of the pHrodo-labeled myelin (red signals) in iMGL. Number of cells quantified: Ctrl = 76, MT = 61. Scale bar = 10 µm. Two-tailed Student’s t -test was used to compare the differences between the two groups. Data are presented as mean ± SD. The statistical significance levels were set at * p < 0.05 and *** p < 0.001.

Article Snippet: For RT-PCR, 1 μg of total RNA was converted to complementary DNA (cDNA) using the iScript cDNA Synthesis kit (Bio-Rad, #170-8891). qPCR was conducted in a LightCycler system (Roche) with the 2x All-in-One qPCR reaction mix (GeneCopoeia, #QP001-01). β-actin was used as the internal control.

Techniques: Mutagenesis, Derivative Assay, Comparison, Staining, Quantitative RT-PCR, Migration, Labeling, Two Tailed Test

Journal: Cell Death Discovery

Article Title: CSF1R T567M mutation induces microglial dysfunction and synaptic impairment in patient iPSC-derived cerebral organoids of CSF1R-related disorder

doi: 10.1038/s41420-026-02995-2

Figure Lengend Snippet: Three batches of D30 iMGL were used to conduct bulk RNA-sequencing. A Venn diagram showing co-expressed and independently expressed genes in Ctrl and MT iMGL. B Volcano plot of differentially expressed genes between Ctrl and MT iMGL. C Gene Ontology analysis of top-downregulated genes. Some biological processes, cellular components, and molecular functions are downregulated in the MT iMGL. D KEGG pathway analysis of downregulated genes. ECM-receptor interaction, axon guidance, and PI3K-Akt signaling pathway are downregulated in CSF1R-MT iMGL. E Gene Ontology analysis of upregulated genes. Immune receptor activity is upregulated in the MT iMGL. F qPCR analysis of NLRP1 and TUBB4A mRNA expression. Two-tailed Student’s t -test was used to compare the differences between the two groups, N = 3. Data are presented as mean ± SD. The statistical significance levels were set at * p < 0.05 and ** p < 0.01.

Article Snippet: For RT-PCR, 1 μg of total RNA was converted to complementary DNA (cDNA) using the iScript cDNA Synthesis kit (Bio-Rad, #170-8891). qPCR was conducted in a LightCycler system (Roche) with the 2x All-in-One qPCR reaction mix (GeneCopoeia, #QP001-01). β-actin was used as the internal control.

Techniques: RNA Sequencing, Activity Assay, Expressing, Two Tailed Test