rna controls  (Twist Bioscience)

Journal: mSphere

Article Title: Targeted metatranscriptomic detection of viruses from floors for simultaneous evaluation of respiratory disease burden and viral variant identification

doi: 10.1128/msphere.00086-26

Figure Lengend Snippet: ( A ) Bubble chart showing the normalized read abundances of samples collected between the final week of November (week 4) 2022 and the final week of February (week 4) 2023 for all detected viruses in each sample. Samples are colored and ordered according to date, and the size of the bubble corresponds to abundance of normalized reads. HWA, hallway A; HWB, hallway B; NTC, no template control; OWB, outside waiting room B; PC, positive control; TRI, triage; WRA, waiting room A; WRB, waiting room B (a mixture of SARS-CoV-2, RSV, and influenza virus RNA). ( B ) Histogram showing the number of samples each virus was detected in (out of 38 swab samples). Samples were normalized using a scaling approach; the median number of viral target reads across all samples (not including controls) was determined, and a scaling factor for each sample was calculated compared to this median. The scaling factor was applied to all viral read counts within that sample.

Article Snippet: Influenza A, influenza B, and SARS-CoV-2 synthetic RNA controls were obtained from Twist Bioscience (catalog numbers 103001, 103003, and 103907, respectively).

Techniques: Control, Positive Control, Virus

Journal: RSC Advances

Article Title: Microfluidic toolbox using padlock probes and rolling circle amplification for direct detection and genotyping of viral RNA

doi: 10.1039/d6ra00912c

Figure Lengend Snippet: (A) Schematic of the hybridization-based rolling circle amplification (hybRCA), performed entirely inside a microfluidic device by capturing the RNA target in streptavidin-coated agarose beads using biotinylated “capture” oligonucleotides – the biotinylated anchors. (B) Dilution series of SARS-CoV-2 control 2 RNA detected using HybRCA. The shaded region corresponds to the LoD of the method (average of the negative control plus three times the standard deviation; n = 3); representative images for 3 detected concentrations (300 copies per µL, 3000 copies per µL and 30 000 copies per µL, respectively); scale bars correspond to 100 µm for each image.

Article Snippet: Direct viral RNA detection on the microfluidic device using a single round of RCA was performed using synthetic control 2 SARS-CoV-2 RNA (GISAID ID: MN908947.3 ; Twist Bioscience), supplied at approximately 1 × 10 6 copies per μL.

Techniques: Hybridization, Amplification, Control, Negative Control, Standard Deviation

Journal: RSC Advances

Article Title: Microfluidic toolbox using padlock probes and rolling circle amplification for direct detection and genotyping of viral RNA

doi: 10.1039/d6ra00912c

Figure Lengend Snippet: (A) Schematic of the circle-to-circle amplification (C2CA) method used for RNA detection and variant profiling, using T4 RNA ligase 2 (T4Rnl2) as the RNA ligase of choice, combined with “chimeric” padlock probes (PLPs) in the first amplification round. Detection oligonucleotides (DO) with different fluorophores were used to detect the different SARS-CoV-2 variants: Cy3 for Wu (Wuhan strain); Cy5 for Alpha (variant B.1.1.7); and AF488 for beta (variant B.1.351). (B) Counts of amplification products (RCPs) labelled with each of the different used DOs when detecting various concentrations of each SARS-CoV-2 variant. Each value corresponds to the sum of four independent 300 × 300 µm 2 images of 10 µL of labelled solution in positively charged glass slides. (C) Average fluorescent intensity (au) measured in the microchannels of the µACE for the different used DOs when detecting the various SARS-CoV-2 variants. Each value corresponds to the average grey scale intensity of the bead-packed region, normalized for the fluorescence of the empty channel. The shaded region corresponds to the determined LoD.

Article Snippet: Direct viral RNA detection on the microfluidic device using a single round of RCA was performed using synthetic control 2 SARS-CoV-2 RNA (GISAID ID: MN908947.3 ; Twist Bioscience), supplied at approximately 1 × 10 6 copies per μL.

Techniques: Amplification, RNA Detection, Variant Assay, Fluorescence

Journal: RSC Advances

Article Title: Microfluidic toolbox using padlock probes and rolling circle amplification for direct detection and genotyping of viral RNA

doi: 10.1039/d6ra00912c

Figure Lengend Snippet: Registered signal obtained for each different DO when detecting various samples containing various concentrations and SARS-CoV-2 variants using (A) positively charged glass slides or (B) µACE as a detection method. The solutions contain either (−) none of the variant, (+) 10 2 copies per µL of the variant, or (++) 10 4 copies per µL of the variant ( n = 3).

Article Snippet: Direct viral RNA detection on the microfluidic device using a single round of RCA was performed using synthetic control 2 SARS-CoV-2 RNA (GISAID ID: MN908947.3 ; Twist Bioscience), supplied at approximately 1 × 10 6 copies per μL.

Techniques: Variant Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Clusterin elaborated by renal tubular epithelial cells under high oxalate stress serves as a matrix protein to facilitate kidney stone formation

doi: 10.1007/s00018-026-06105-4

Figure Lengend Snippet: Targeting CLU mitigate CaOx crystal formation in vivo and in vitro. A Schematic drawing of the newly designed crystal pulldown and crystal-forming pulldown methods. B Crystal diameter increased after the addition of different concentrations of the recombinant CLU protein. C WB analysis of crystal pull-down and crystal-forming pull-down. D Knockdown of CLU in HK2 cells via Sh-siRNA reversed the trend of increased crystal adhesion; the black bar indicates 150 μm. E Expression profile of Cdh16 via single-cell RNA sequencing of the mouse kidney. H CaOx crystal deposition in mouse kidneys following conditional knockout of the Clu gene via the Cre‒LoxP system, I along with their statistic measurement. WB: Western blotting. The data are presented as the means ± SEMs; ns: not significant; ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: This study involved the transfection of TWIST1-RNAi, TWIST1-overexpression, CLU-RNAi, and plasmids utilized in a dual-luciferase reporter gene assay (shRNA from Shanghai, Genechem), along with the corresponding control RNA (shRNA-NC), into cells during the logarithmic growth phase.

Techniques: In Vivo, In Vitro, Recombinant, Knockdown, Expressing, Single Cell, RNA Sequencing, Knock-Out, Western Blot

Journal: Cancer Communications

Article Title: CEBPB Expression in Tumor Cells Drives Immune Evasion in Colorectal Cancer via CTLA4 Up-regulation in T Cells

doi: 10.34133/cancomm.0013

Figure Lengend Snippet: TME profile of tumor tissues from 30 CRC patients using scRNA-seq. (A) A total of 99,268 cells derived from scRNA-seq of tumor tissues from 30 CRC patients were clustered and annotated to identify main cell types, as displayed in UMAP. (B) Subclustering and annotation of T/NK cell subtypes. (C) Heatmap showing proportions of each cell type per patient, with MSI status, and APC , TP53 , and RAS mutation status, and bar graph displaying the total cell counts of the main cell types. (D) Violin plots showing proportions of CD4 + and CD8 + T cells according to TP53 and RAS mutation status (left), and proportions of CD4 + T cell subtypes according to TP53 mutation and CD8 + T cell subtypes according to RAS mutation (right). P values were obtained using the Wilcoxon rank-sum test. Red dots indicate the median. (E) Heatmap showing representative examples from the pathway enrichment analysis of DEGs from T cells according to MSI and mutation statuses . Abbreviations: Adj. P , adjusted P value; APC , adenomatosis polyposis coli; CRC, colorectal cancer; DEG, differentially expressed gene; MAIT, mucosal-associated invariant T cell; MSI, microsatellite instability; MSI-H, microsatellite instability-high; MSS, microsatellite stable; MUT, mutation; NF-κB, nuclear factor kappa B; NK, natural killer cell; RAS, rat sarcoma virus; scRNA-seq, single-cell RNA sequencing; T/NK, T cell/natural killer cell; Tcm, central memory T cell; Tem, effector memory T cell; Temra, terminally differentiated effector memory T cell; Tex, exhausted T cell; Tfh, T follicular helper cell; Th1, T helper 1; Th2, T helper 2; Th17, T helper 17 cell; TME, tumor microenvironment; Tnaive, naive T cell; TP53 , tumor protein 53; Treg, regulatory T cell; Trm, tissue-resident memory T cell; TNF-alpha, tumor necrosis factor-alpha; UMAP, uniform manifold approximation and projection; WT, wild type.

Article Snippet: To silence TP53 , gene-specific and control small interfering RNA were purchased from Bioneer and transfected into CRC cancer cell lines using Lipofectamine RNAiMax (13778075, Invitrogen) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Mutagenesis, Virus, Single Cell, RNA Sequencing

Journal: Cancer Communications

Article Title: CEBPB Expression in Tumor Cells Drives Immune Evasion in Colorectal Cancer via CTLA4 Up-regulation in T Cells

doi: 10.34133/cancomm.0013

Figure Lengend Snippet: WT p53 suppresses mRNA and protein levels of C/EBPβ in CRC cell lines. (A) Protein and mRNA expression of C/EBPβ in CT26 cells stably expressing shCtrl or sh Trp53 were analyzed by Western blotting and real-time PCR, respectively. (B) C/EBPβ protein expression was determined in CRC cell lines harboring TP53 WT, nonsense mutation, and missense mutation treated with nutlin-3a (10 μmol/l) for WT p53 induction or vehicle for 24 h (left), as well as CEBPB mRNA levels in TP53 WT cell lines (right). (C) C/EBPβ protein expression was compared in TP53 WT HCT116 and SNU1544 cells after transfection with siCtrl or si TP53 , followed by nutlin-3a (10 μmol/l) or vehicle treatment for 24 h. (D) C/EBPβ protein expression was determined in TP53 nonsense SNUC1 and SNU1411 cells after transient transfection with EV or TP53 WT, followed by 24 h of nutlin-3a (10 μmol/l) treatment. (E) C/EBPβ protein levels were determined in SNU1544 cells pretreated with nutlin-3a (10 μmol/l) for 16 h and stimulated with 8-Br-cAMP (100 μmol/l), an inducer of CEBPB transcription, for 8 h. (F) C/EBPβ protein levels were determined in SNU1411 cells transiently transfected with EV, TP53 WT, or TP53 R175H for 24 h. (G) C/EBPβ protein (left) and mRNA (right) levels were assessed in p53-null Caco-2 cells stably transduced with doxycycline-inducible TP53 WT or TP53 R175H following treatment with 1 μg/ml doxycycline for 48 h. (H) C/EBPβ protein stability was compared in shCtrl and sh Trp53 CT26 cells after CHX treatment (5 μg/ml) for the indicated times. (I) SNU1544 were treated with nutlin-3a (10 μmol/l) for 21 h, followed by MG132 (2 μmol/l) for 3 h to block proteasomal degradation. Protein expression was analyzed by Western blotting, representative of 3 independent experiments, and the values presented underneath or in the graph (H) were generated by densitometric analyses using ImageJ. mRNA levels were determined using real-time PCR. The bar graphs represent the mean ± SD; each dot represents an independent biological replicate. P values were calculated using 2-tailed Student’s t tests. Abbreviations: 8-Br-cAMP, 8-bromoadenosine 3′,5′-cyclic monophosphate; CEBPB , CCAAT enhancer binding protein beta; CHX, cycloheximide; CRC, colorectal cancer; doxy, doxycycline; EV, empty vector; SD, standard deviation; shCtrl, control short hairpin RNA; sh Trp53 , short hairpin RNA for Trp53 ; si TP53 , small interfering RNA for TP53 ; WT, wild type.

Article Snippet: To silence TP53 , gene-specific and control small interfering RNA were purchased from Bioneer and transfected into CRC cancer cell lines using Lipofectamine RNAiMax (13778075, Invitrogen) according to the manufacturer’s instructions.

Techniques: Expressing, Stable Transfection, Western Blot, Real-time Polymerase Chain Reaction, Mutagenesis, Transfection, Transduction, Blocking Assay, Generated, Binding Assay, Plasmid Preparation, Standard Deviation, Control, shRNA, Small Interfering RNA