Journal: Cells

Article Title: Multiplexed Digital PCR Reference Gene Measurement for Genomic and Cell-Free DNA Analysis

doi: 10.3390/cells14191544

Figure Lengend Snippet: Reference gene measurement and HER2 ratio analysis of breast carcinoma cell line-derived hgDNA. ( A ) Reference gene panel was tested using HER2+ gDNA and its matched lymphoblast gDNA (HER2− gDNA). Error bar for the individual genes show range. The error bar for the average shows the expanded measurement uncertainty.; ( B ) HER2 multiplex panel was tested using HER2+ gDNA and its matched lymphoblast gDNA (HER2−). Symbols in black represent HER2+ gDNA, and symbols in grey represent HER2− gDNA. Circle, square, triangle, diamond, hexagon, and down-pointing triangle represent HBB, RPPH1, RPS27A, DCK, PMM1 and the average respectively. Each symbol shows individual measurements.

Article Snippet: Commercially available human genomic DNA (gDNA) isolated from the HCC1954 breast carcinoma cell line with HER2 copy number gain (ATCC CRL-2338D) and paired healthy control gDNA (ATCC CRL-2339D) isolated from the HCC1954 BL EBV-transformed lymphoblastoid cell line (both ATCC, Manassas, VA, USA) were also digested prior to dPCR application as described above.

Techniques: Derivative Assay, Multiplex Assay

Journal: Cell reports

Article Title: Hypoxia-induced CTCF mediates alternative splicing via coupling chromatin looping and RNA Pol II pause to promote EMT in breast cancer.

doi: 10.1016/j.celrep.2025.115267

Figure Lengend Snippet: Figure 3. Hypoxia-induced COL5A1 exon64A inclusion is regulated by DNA demethylation-dependent enrichment of CTCF on exon64A and is associated with RNA Pol II pausing (A) COL5A1 gene exon64 has two mutually exclusive isoforms, exon64A and exon64B. (B) Significant inclusion of exon64A of COL5A1 in GEO: GSE76250 breast cancer dataset stratified as hypoxic_high (H, n = 41) or hypoxic_low (L, n = 41). (C) Significant inclusion of exon64A of COL5A1 in microarray data in HCC1806 (GEO: GSE147516) post-hypoxic treatment (n = 2) and represented by splicing index values. (D) RT-qPCR analysis of COL5A1 exon64A and exon64B isoforms normalized to RPS16 and constitutive exon expression levels in normoxic and hypoxic MCF7 cells. (E) Schematic showing putative CTCF binding site on exon64A, MeDIP-qPCR, CTCF, and RNA Pol II ChIP-qPCR on exon64A and exon64B in normoxic and hypoxic MCF7 cells. (F and G) CTCF and RNA Pol II ChIP-qPCR on exon64A and exon64B (F) and RT-qPCR analysis of COL5A1 exon64A and exon64B isoforms normalized to RPS16 and constitutive exon expression levels (G) in MCF7 cells transduced with either shCTCF or shControl under hypoxia. (H and I) CTCF-ChIP-qPCR on COL5A1 promoter and CTCF and RNA Pol II ChIP-qPCR on COL5A1 exon64A (H) and RT-qPCR analysis of COL5A1 exon64A and exon64B isoforms normalized to RPS16 and constitutive exon expression levels (I) in MCF7 cells transduced with either shHIF1A or shControl under hypoxia. Error bars, mean ± SEM; two-tailed t test; *p < 0.05, **p < 0.01, and ***p < 0.001; n = 3 biological replicates.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Expand Long Template Polymerase mix Roche Cat #11681834001 Corning Collagen I, Rat Tail Corning Cat #354236 DpnII New England Biolabs Cat #R0543T NlaIII New England Biolabs Cat #R0125S Spermidine Sigma Cat #S2626-1G Digitonin Sigma Cat #D141-100MG Concavalin-A Cell Signaling Technology Cat #93569S pAG-MNase Cell Signaling Technology Cat #40366S GlycoBlueTM Coprecipitant (15 mg/mL) Thermo Fisher Scientific AM9515 Critical commercial assays QIAprep Spin Miniprep Kit Qiagen Cat# 27106 QIAquick PCR purification Kit Qiagen Cat# 28106 GenElute Mammalian Genomic DNA Miniprep Kits Sigma Cat# G1N70 QIAquick Gel extraction Kit Qiagen Cat# 28706 DNA Ligation kit ver2.1 TaKaRa Cat# 6022 NEBNext Ultra II DNA library Prep Kit for Illumina New England Biolabs Cat #E7645S CELL COUNTING KIT - 8 Sigma Cat #96992-500TESTS-F NEBNext Multiplex Oligos for Illumina (Index Primers Set 1) – 24 reactions New England Biolabs Cat #E7335S NEBNext Multiplex Oligos for Illumina (Index Primers Set 2) New England Biolabs Cat #E7500S Deposited data CTCF-CUT&RUN assay in normoxic and hypoxic MCF7 cell line This paper GSE216843 CTCF-ChIP-seq in normoxic and hypoxic MCF7 cell line This paper GSE216843 RNA-seq in normoxic and hypoxic MCF7 cell line Zhang et al.37 GSE166203 4C-seq This paper GSE216843 HTA in normoxic and hypoxic HCC1806 cell line Ahuja et al.38 GSE147516 HTA in triple negative breast cancer patient sample Liu et al.39 GSE76250 Unprocessed blots This paper Mendeley Data: https://doi.org/10.17632/ d2kvsgym55.2 Experimental models: Cell lines MCF7 ATCC Cat# HTB -22 HCC1806 ATCC Cat# CRL-2335 HEK293T ATCC Cat# CRL-3216 Oligonucleotides Molecular Cloning Primers, See Table S4 This paper N/A ChIP-qPCR and MeDIP-qPCR Primers, See Table S5 This paper N/A 3C and 4C Primers, See Table S5 This paper N/A qPCR Primers, See Table S6 This paper N/A shRNA, See Table S7 Sigma, Mission Human Genome shRNA Library N/A sgRNA, See Table S7 This paper N/A Off target primers, See Table S7 This paper N/A Recombinant DNA pGL3-Basic Promega E1751 pGL3-Control Promega E1741 pRL-TK Renilla luciferase plasmid Promega E2231 (Continued on next page) Cell Reports 44, 115267, February 25, 2025 19

Techniques: Microarray, Quantitative RT-PCR, Expressing, Binding Assay, Methylated DNA Immunoprecipitation, ChIP-qPCR, Transduction, Two Tailed Test

Journal:

Article Title: Genotypes Associated with Virulence in Environmental Isolates of Vibrio cholerae

doi: 10.1128/AEM.67.6.2421-2429.2001

Figure Lengend Snippet: Agarose gel electrophoresis of amplicons obtained using multiplex PCR. Lane M, molecular weight marker (lambda DNA HindIII); lanes 1, 3, 5, 11, and 14, V. cholerae O1 El Tor nontoxigenic ATCC 14033; lanes 2, 6, 9, 12, and 15, V. cholerae O1 Classical ATCC 14035; lane 4, RC224, V. cholerae O1 El Tor (Brazilian isolate from sewage, 1993); lane 7, TMA52, V. cholerae non-O1/non-O139 (Brazilian isolate from seawater, 1982); lane 8, negative control; lane 10, RC46, V. cholerae O139 (tcpI negative); lane 13, RC60, V. cholerae non-O1/non-O139 (Brazilian isolate from seawater, 1982); lane 16, RC66 (Brazilian isolate from sediment, 1982).

Article Snippet: Lane M, molecular weight marker (lambda DNA Hin dIII); lanes 1, 3, 5, 11, and 14, V. cholerae O1 El Tor nontoxigenic ATCC 14033; lanes 2, 6, 9, 12, and 15, V. cholerae O1 Classical ATCC 14035; lane 4, RC224, V. cholerae O1 El Tor (Brazilian isolate from sewage, 1993); lane 7, TMA52, V. cholerae non-O1/non-O139 (Brazilian isolate from seawater, 1982); lane 8, negative control; lane 10, RC46, V. cholerae O139 ( tcpI negative); lane 13, RC60, V. cholerae non-O1/non-O139 (Brazilian isolate from seawater, 1982); lane 16, RC66 (Brazilian isolate from sediment, 1982).

Techniques: Agarose Gel Electrophoresis, Multiplex Assay, Molecular Weight, Marker, Lambda DNA Preparation, Negative Control

Journal: Gut Pathogens

Article Title: Development of multiplex PCR and multi-color fluorescent in situ hybridization ( m -FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease

doi: 10.1186/s13099-018-0278-1

Figure Lengend Snippet: Specificity and sensitivity of the IS900 nPCR in DNA extracts from the modified DNAzol ® and the standard phenol/chloroform/isoamyl-alcohol DNA extraction protocols. nPCR based on the IS900 specific to MAP was performed on DNA template extracted by the standard phenol/chloroform/isoamyl-alcohol DNA extraction (I) and the modified DNAzol ® DNA extraction technique (II). A 298 bp fragment on 2% agarose gel is positive for MAP. a (1) Non-pathogenic E. coli strain K-12; (2) S. aureus ; (3) L. monocytogenes ; (4) K. pneumoniae ; (5) M. smegmatis ; (6) M. avium subspecies avium ; (7) M. xenopi ; (8) M. fortuitum subspecies fortuitum ; (9) MAP Clinical Strain JF7. b (1) MAP Strain 1; (2) MAP Strain 3; (3) MAP Strain 8B; (4) MAP Para 18; (5) MAP UCF3; (6) MAP UCF5; (7) MAP UCF7; (8) MAP Linda; (9) MAP MS137. c Serial dilution of MAP UCF4 DNA concentrations were analyzed by nPCR. (1) 31.7 ng/μL; (2) 3.17 ng/μL; (3) 317 pg/μL; (4) 31.7 pg/μL; (5) 3.17 pg/μL; (6) 317 fg/μL; (7) 31.7 fg/μL; (8) 3.17 fg/μL; (9) 317 ag/μL; (10) 31.7 ag/μL. +: MAP UCF4; N: No DNA; M: molecular weight marker

Article Snippet: Non - pathogenic Escherichia coli Strain K-12 , ATCC 8739.

Techniques: Modification, DNA Extraction, Agarose Gel Electrophoresis, Serial Dilution, Molecular Weight, Marker

Journal: Gut Pathogens

Article Title: Development of multiplex PCR and multi-color fluorescent in situ hybridization ( m -FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease

doi: 10.1186/s13099-018-0278-1

Figure Lengend Snippet: Validation of multiplex PCR using individual and combined oligonucleotide primer sets and DNA extracts from mixed bacterial culture. Multiplex PCR was performed on DNA extracts from mixed bacterial cultures. DNA template was extracted using the modified DNAzol ® . (1) Non-pathogenic E. coli strain K-12 18s primers were used (171 bp); (2) MAP UCF4 IS900 AV1/AV2 primers were used (298 bp); (3) K. pneumoniae 23s primers were used (493 bp); (4) Mycobacterium avium complex (MAC) IS1311 primers were used (534 bp); (5) a cocktail of the 4 primer sets mentioned above were used. M: DNA molecular weight marker

Article Snippet: Non - pathogenic Escherichia coli Strain K-12 , ATCC 8739.

Techniques: Biomarker Discovery, Multiplex Assay, Modification, Molecular Weight, Marker

Journal: Gut Pathogens

Article Title: Development of multiplex PCR and multi-color fluorescent in situ hybridization ( m -FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease

doi: 10.1186/s13099-018-0278-1

Figure Lengend Snippet: Multiplex PCR and m -FISH results for intestinal tissue from IBD

Article Snippet: Non - pathogenic Escherichia coli Strain K-12 , ATCC 8739.

Techniques: Multiplex Assay

Journal: Gut Pathogens

Article Title: Development of multiplex PCR and multi-color fluorescent in situ hybridization ( m -FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease

doi: 10.1186/s13099-018-0278-1

Figure Lengend Snippet: Validation of multiplex PCR using individual and combined oligonucleotide primer sets and DNA extracts from IBD tissue. Multiplex PCR was performed on DNA extracts from intestinal tissue samples. DNA template was extracted using the modified DNAzol ® . RS1: ulcerative colitis (UC) patient; RS2: Crohn’s disease (CD) patient; (1) Non-pathogenic E. coli strain K-12 18s primers were used (171 bp); (2) MAP UCF4 IS900 AV1/AV2 primers were used (298 bp); (3) AIEC strain LF82 g ipA primers were used (357 bp); (4) K. pneumoniae 23s primers were used (493 bp); (5) Mycobacterium avium complex (MAC) IS1311 primers were used (534 bp); (6) a cocktail of the 5 primer sets mentioned above were used. M: DNA molecular weight marker

Article Snippet: Non - pathogenic Escherichia coli Strain K-12 , ATCC 8739.

Techniques: Biomarker Discovery, Multiplex Assay, Modification, Molecular Weight, Marker

Journal: Gut Pathogens

Article Title: Development of multiplex PCR and multi-color fluorescent in situ hybridization ( m -FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease

doi: 10.1186/s13099-018-0278-1

Figure Lengend Snippet: Gram stain, acid-fast stain and m -FISH detection of bacterial cultures. Gram stain (1), acid-fast stain (2), and oligonucleotide m -FISH (3) images of bacterial cultures: a , b Non-pathogenic E. coli strain K-12; c MAP UCF4; and d K. pneumoniae . For - m -FISH detection: a 3 m -FISH using non-pathogenic E. coli K-12 18s probe labeled with AF647 fluorophore; b 3 m -FISH using AIEC strain LF82 g ipA probe labeled with AF568 fluorophore; c 3 m -FISH using MAP UCF4 IS900 AV1 probe labeled with AF488 fluorophore; and d 3 m-FISH using K. pneumoniae 23s probe labeled with AF546 fluorophore. All microscopic images were obtained at 1000 × magnification. White measurement bar found in D3 represents 100 μm. All m -FISH images were obtained using CSLM

Article Snippet: Non - pathogenic Escherichia coli Strain K-12 , ATCC 8739.

Techniques: Staining, Ziehl-Neelsen Stain, Labeling

Journal: Gut Pathogens

Article Title: Development of multiplex PCR and multi-color fluorescent in situ hybridization ( m -FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease

doi: 10.1186/s13099-018-0278-1

Figure Lengend Snippet: Use of m -FISH using individual probes to detect multiple pathogens in intestinal tissue. Oligonucleotide m -FISH analysis in tissue sections from patients with UC (RS1) and CD (RS2) was done targeting for: a Non-pathogenic E. coli strain K-12; b AIEC strain LF82; c MAP; and d K. pneumoniae . Images illustrating DAPI are in red. The individual m -FISH probes that were used were: a non-pathogenic E. coli strain K-12 18s probe labeled with AF647 fluorophore; b AIEC strain LF82 g ipA probe labeled with AF568 fluorophore; c MAP UCF4 IS900 AV1 probe labeled with AF488 fluorophore; and d K. pneumoniae 23s probe labeled with AF546 fluorophore. White measurement bars found in D1 and D2 represents 20 μm. All m -FISH images were obtained using CSLM

Article Snippet: Non - pathogenic Escherichia coli Strain K-12 , ATCC 8739.

Techniques: Labeling

Journal: Gut Pathogens

Article Title: Development of multiplex PCR and multi-color fluorescent in situ hybridization ( m -FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease

doi: 10.1186/s13099-018-0278-1

Figure Lengend Snippet: Use of m -FISH using combined probes to detect multiple pathogens in intestinal tissue from CD. Oligonucleotide m -FISH images of intestinal tissue sections from four CD patients ( a RS3, b RS4, c RS5, and d RS6). A combined probes mixture including (2) EC647 (non-pathogenic E. coli strain K-12 18s probe labeled with AF647 fluorophore) and (3) MAP488 (MAP UCF4 IS900 AV1 probe labeled with AF488 fluorophore) were used in all tissue sections. (1) DAPI staining and (4) merged images between DAPI and the corresponding probe. White measurement bar located in B3 represents 20 μm. All m -FISH images were obtained using CSLM

Article Snippet: Non - pathogenic Escherichia coli Strain K-12 , ATCC 8739.

Techniques: Labeling, Staining

Journal: Gut Pathogens

Article Title: Development of multiplex PCR and multi-color fluorescent in situ hybridization ( m -FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease

doi: 10.1186/s13099-018-0278-1

Figure Lengend Snippet: Nucleotide primers used in multiplex PCR and m -FISH probes

Article Snippet: Non - pathogenic Escherichia coli Strain K-12 , ATCC 8739.

Techniques: Multiplex Assay

Journal: Gut Pathogens

Article Title: Development of multiplex PCR and multi-color fluorescent in situ hybridization ( m -FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease

doi: 10.1186/s13099-018-0278-1

Figure Lengend Snippet: Microorganisms used in this study

Article Snippet: Non - pathogenic Escherichia coli Strain K-12 , ATCC 8739.

Techniques:

Journal: Cell Reports

Article Title: Chromatin accessibility governs the differential response of cancer and T cells to arginine starvation

doi: 10.1016/j.celrep.2021.109101

Figure Lengend Snippet:

Article Snippet: Human tumor cell lines NB4 (female; Cancer Research UK; RRID CVCL_0005), MOLM13 (male; DSMZ, ACC-554; RRID CVCL_2119), RT112 (female; CRUK; RRID CVCL_1670), LNCaP (male; CRUK; RRID CVCL_0395), OCI-AML3 (male; DSMZ, ACC-582; RRID CVCL_1844), THP1 (male; ATCC, TIB-202; RRID CVCL_0006), HL60 (female; ATCC, CCL-240; RRID CVCL_0002) and RS4;11 (female; ATCC, CRL-1873; RRID CVCL_0093) cells were cultured in RPMI-1640 supplemented with 10% fetal calf serum and GlutaMAX (ThermoFisher Scientific).

Techniques: Recombinant, Multiplex sample analysis, Cell Isolation, Activation Assay, Staining, Flow Cytometry, Expressing, Reverse Transcription, Transfection, TA Cloning, Plasmid Preparation, Methylation, Immunoprecipitation, Purification, DNA Library Preparation, Library Quantification, Control, Sequencing, Methylation Sequencing, Amplification, Software

Journal: Frontiers in Medicine

Article Title: Syndrome Evaluation System for Simultaneous Detection Pathogens Causing Acute Encephalitic Syndrome in India, Part-1: Development and Standardization of the Assay

doi: 10.3389/fmed.2018.00208

Figure Lengend Snippet: Source of pathogens used in the development of SES.

Article Snippet: 6 , S. pneumoniae , 6301 , ATCC.

Techniques: Virus, Plasmid Preparation

Journal: Frontiers in Medicine

Article Title: Syndrome Evaluation System for Simultaneous Detection Pathogens Causing Acute Encephalitic Syndrome in India, Part-1: Development and Standardization of the Assay

doi: 10.3389/fmed.2018.00208

Figure Lengend Snippet: Name of the pathogen, Gene targets chosen for primer design and the gene bank accession numbers.

Article Snippet: 6 , S. pneumoniae , 6301 , ATCC.

Techniques: Transformation Assay

Journal: Frontiers in Medicine

Article Title: Syndrome Evaluation System for Simultaneous Detection Pathogens Causing Acute Encephalitic Syndrome in India, Part-1: Development and Standardization of the Assay

doi: 10.3389/fmed.2018.00208

Figure Lengend Snippet: (A) Template depicting the position of various probes used for detection of DNA pathogens on the Syndrome Evaluation System platform. Row 1:HgD = Glycoprotein D of HSV 1 and 2; HUL = UL 44 of HSV 1; HDP = DNA polymerase of HSV 1 and 2; 1st Comp = complementary strand of HgD gene of HSV 1 and 2; Row 2: CGO = Glycoprotein O of CMV; CUL = UL 63 of CMV; CMT = Morphological transformation Region of CMV; 5th Comp = complementary strand of CMT gene of CMV; Row 3: VO = ORF 29 gene of VZV; VDP = DNA polymerase gene of VZV; MBT = M. tuberculosis; TG = T.gondii; Row4: HI = H. influenzae; Nm = N. meningitidis; SP = S. Pneumoniae and JC = JC virus; Row 5: CN = C. neoformans; Row 6: HHV-6 = Human Herpes Vvirus-6; IC = Internal control. (B) Template depicting the position of various probes used for detection of RNA pathogens on the Syndrome Evaluation System platform. Row 1: FLA1 = Flavivirus; MES = Measles; FLA2 = Complimentary strand of FLA1 gene of Flavivirus; MUM = Mumps; Row 2: CHIK = Chikungunya; JEV = Japanese encephalitis virus; NIP = Nipah; CHA = Chandipura; Row 3: RAB = Rabies; ENT1 = Enteroviruses; RUB = Rubella; ENT 2 = Complimentary strand of ENT1 of Enteroviruses; Row 4: Den1 = Dengue type1and 3 virus; Den2 = Dengue Type 2 and 4 virus and IC = Internal control.

Article Snippet: 6 , S. pneumoniae , 6301 , ATCC.

Techniques: Transformation Assay, Virus, Control

Journal: Frontiers in Medicine

Article Title: Syndrome Evaluation System for Simultaneous Detection Pathogens Causing Acute Encephalitic Syndrome in India, Part-1: Development and Standardization of the Assay

doi: 10.3389/fmed.2018.00208

Figure Lengend Snippet: Comparative LOD for all the pathogens obtained in uniplex and multiplex assay formats of the SES.

Article Snippet: 6 , S. pneumoniae , 6301 , ATCC.

Techniques: Multiplex Assay

Journal: Frontiers in Medicine

Article Title: Syndrome Evaluation System for Simultaneous Detection Pathogens Causing Acute Encephalitic Syndrome in India, Part-1: Development and Standardization of the Assay

doi: 10.3389/fmed.2018.00208

Figure Lengend Snippet: Determination of Limit of detection (LOD) for DNA pathogen on the SES platform. DNA extracted from defined concentrations of viruses/bacteria /fungi/parasites were subjected to multiplex PCR and hybridization on the SES platform (Vide Materials and Methods for details). As indicated in the template provided on the right, multiple genes were amplified and hybridized with respective probes for HSV (row 1 of template), CMV (row 2 of template),VZV (row 3 of the template), and single genes were amplified and hybridized in case of M. tuberculosis and T. gondii (row 3 of the template), H influenza, N. meningitidis, S. pneumoniae and JCV (row 4 of template), C. neoformans (row 5 of the template), and HHV-6 and Internal control (row6 of the template). Each panel depicts NC = negative control. PC = Positive Control and the numbers written below each SES indicate the number of particles/CFU/organisms/ml that has been used for DNA extraction, amplification and hybridization in a multiplex format. (refer to Table ).

Article Snippet: 6 , S. pneumoniae , 6301 , ATCC.

Techniques: Bacteria, Multiplex Assay, Hybridization, Amplification, Control, Negative Control, Positive Control, DNA Extraction

Journal: NPJ Genomic Medicine

Article Title: Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection

doi: 10.1038/s41525-017-0034-3

Figure Lengend Snippet: a Optimization of DNA extraction from rare and fixed cells using Qiagen QIAamp DNA Micro Kit. ~200–300 cells/experiment, N ≥ 2 per condition. ① Fresh cells using Cell Protocol yielded 6–7 pg of DNA/cell and were used as a control. ② Cell Protocol did not work on cells fixed with 4% PFA. ③ Tissue Protocol significantly increased the DNA yield when Proteinase K digestion was extended from 4 to 36 h, with significant advantage of overnight incubation. ④ Increasing the Proteinase K digestion temperature from 56 to 60 °C further increased the DNA yield from 60 to 80%. ⑤ The optimized protocol was then verified on fixed, fixed+permeabilized and fixed+permeabilized+stained HCT116 cells to mimic the output from CTC enrichment platforms. b Validation of an optimal DNA extraction protocol for low number of fixed cells. Different amounts of HCT116 cells fixed with 4% PFA were seeded inside a 96 well-plate, imaged and counted. DNA was extracted from these cells using the optimized protocol defined in A (Qiagen QIAamp DNA Micro Kit, Tissue Protocol, Proteinase K digestion overnight at 60 °C) and quantitated using qPCR

Article Snippet: When applied on 40 ng of DNA from fresh HCT116 cells (Fig. ), eight variants were detected, including KRAS (p.G13D), PIK3CA (p.H1047R), and CTNNB1 ( p.S45del), with all of them being reported in ATCC or in COSMIC (Catalog of Somatic Mutations In Cancer).

Techniques: DNA Extraction, Control, Incubation, Staining, Biomarker Discovery

Journal: NPJ Genomic Medicine

Article Title: Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection

doi: 10.1038/s41525-017-0034-3

Figure Lengend Snippet: Mutation detection using GeneRead DNAseq targeted CRC panel sequencing. a Mutation detection in HCT116 cancer cells. Forty nanogram of DNA extracted from fresh HCT116 cells were subjected to multiplex PCR, library preparation and MiSeq sequencing. Mutations and corresponding allele frequencies were called using Ingenuity variant analysis software and compared to results reported from Cosmic* (catalog of somatic mutations in cancer) and ATCC websites. b Mutation detection in CRC patient biopsy tissues. Forty nanogram of DNA extracted from primary tumor and liver metastasis were subjected to multiplex PCR, library preparation and MiSeq sequencing. Mutations and corresponding Mutation Allele Fraction (MAF) were analyzed and compared between primary tumor and liver metastasis. In both tables, the blue color highlights the detected mutation and the number inside each cell represents the MAF of the mutation

Article Snippet: When applied on 40 ng of DNA from fresh HCT116 cells (Fig. ), eight variants were detected, including KRAS (p.G13D), PIK3CA (p.H1047R), and CTNNB1 ( p.S45del), with all of them being reported in ATCC or in COSMIC (Catalog of Somatic Mutations In Cancer).

Techniques: Mutagenesis, Sequencing, Multiplex Assay, Variant Assay, Software

Journal: NPJ Genomic Medicine

Article Title: Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection

doi: 10.1038/s41525-017-0034-3

Figure Lengend Snippet: Mutation detection in HCT116 cancer cells with different DNA input using GeneRead DNAseq targeted CRC Panel. Forty and 1 ng DNA extracted from fresh HCT116 cells were compared to 1.0, 0.5, and 0.2 ng of DNA extracted from HCT116 cells fixed with 4% PFA. In all conditions, DNA was subjected to multiplex PCR, library preparation and MiSeq sequencing. Mutations and corresponding allele frequencies were analyzed and compared

Article Snippet: When applied on 40 ng of DNA from fresh HCT116 cells (Fig. ), eight variants were detected, including KRAS (p.G13D), PIK3CA (p.H1047R), and CTNNB1 ( p.S45del), with all of them being reported in ATCC or in COSMIC (Catalog of Somatic Mutations In Cancer).

Techniques: Mutagenesis, Multiplex Assay, Sequencing

Journal: NPJ Genomic Medicine

Article Title: Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection

doi: 10.1038/s41525-017-0034-3

Figure Lengend Snippet: Whole genome amplification by using GenomePlex (WGA4) and REPLI-g. a Comparison of two whole genome amplification methods: GenomePlex (WGA4) and REPLI-g. b Workflow of GenomePlex (WGA4) and REPLI-g kits. c Whole Genome Amplified DNA products. GenomePlex (WGA4) or REPLI-g WGA was performed on DNA from both fresh and fixed HCT116 cells following the vendor’s manuals. The amplified DNA was checked using agarose gel electrophoresis. d DNA yield comparison. GenomePlex (WGA4) or REPLI-g WGA was performed on both fresh and fixed HCT116 cells following the vendor’s manuals. The amplified DNA amount was measured using the Qubit. (*: standard protocol. **: increased DNA volume protocol)

Article Snippet: When applied on 40 ng of DNA from fresh HCT116 cells (Fig. ), eight variants were detected, including KRAS (p.G13D), PIK3CA (p.H1047R), and CTNNB1 ( p.S45del), with all of them being reported in ATCC or in COSMIC (Catalog of Somatic Mutations In Cancer).

Techniques: Whole Genome Amplification, Comparison, Amplification, Agarose Gel Electrophoresis

Journal: NPJ Genomic Medicine

Article Title: Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection

doi: 10.1038/s41525-017-0034-3

Figure Lengend Snippet: Sequencing comparison in WGA amplified and non-amplified DNA samples. a Mutation detection comparison for REPLI-g WGA. REPLI-g amplified or non-amplified DNA from both fresh and fixed HCT116 cells were subjected to the CRC targeted NGS. The blue color highlights the true mutations detected, while the number inside each cell represents the MAF of the mutation. The light red color highlights the false mutations called. b Gene coverage comparison for WGA. The coverage was compared among the following samples: ① Fresh cells; ② Fixed cells; ③ Fresh cells + REPLI-g; ④ Fixed cells + REPLI-g. ⑤ Fixed cells+WGA4. The reads were aligned using BWA-MEM and coverage was computed using GATK’s DepthOfCoverage and in-house scripts. The percentage of bases covered by at least 10 reads with minimum base quality score Q30 (%_bases_above_10) was calculated using data obtained from DepthOfCoverage and in-house scripts and summarized in the table. (*) In the case of GenomePlex (WGA4), the plotted data was trimmed of 10 bp at the beginning and 40 bp at the end because of a primer/adapter contamination

Article Snippet: When applied on 40 ng of DNA from fresh HCT116 cells (Fig. ), eight variants were detected, including KRAS (p.G13D), PIK3CA (p.H1047R), and CTNNB1 ( p.S45del), with all of them being reported in ATCC or in COSMIC (Catalog of Somatic Mutations In Cancer).

Techniques: Sequencing, Comparison, Amplification, Mutagenesis

Journal: NPJ Genomic Medicine

Article Title: Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection

doi: 10.1038/s41525-017-0034-3

Figure Lengend Snippet: Comparison of different commercial kits for DNA extraction from rare cells

Article Snippet: When applied on 40 ng of DNA from fresh HCT116 cells (Fig. ), eight variants were detected, including KRAS (p.G13D), PIK3CA (p.H1047R), and CTNNB1 ( p.S45del), with all of them being reported in ATCC or in COSMIC (Catalog of Somatic Mutations In Cancer).

Techniques: Comparison, DNA Extraction, Evaporation, Incubation

Journal: Science advances

Article Title: Pancreatic tumor microenvironmental acidosis and hypoxia transform gold nanorods into cell-penetrant particles for potent radiosensitization.

doi: 10.1126/sciadv.abm9729

Figure Lengend Snippet: Fig. 3. Qualitative and quantitative uptake of nanoparticles in cells. In vitro dark-field and fluorescence overlay images of KPC (A to C), PANC 1 (D to F), and HPDE (G to I) cells incubated with media, P(Glu-co-Lys)1:5-GNR at pH 7.4 and 6.5. Gold uptake was significantly higher in KPC and PANC 1 pancreatic cancer cells at pH 6.5 compared to pH 7.4 for both P(Glu-co-Lys)1:2.5-GNR and P(Glu-co-Lys)1:5-GNR. However, no observable increase in gold uptake was observed in case of HPDE cells. In vitro quantitative uptake of gold measured by ICP-MS analysis of KPC (J), PANC 1 (K), and HPDE (L) cells incubated with media alone (control), P(Glu-co-Lys)1:2.5-GNR, and P(Glu-co-Lys)1:5-GNR at pH 7.4 and 6.5. Gold uptake was significantly higher in KPC and PANC 1 pancreatic cancer cells at pH 6.5 compared to pH 7.4 for both P(Glu-co-Lys)1:2.5-GNR and P(Glu-co-Lys)1:5-GNR. However, no significant increase in gold uptake was observed in case of HPDE cells. Error bars = SEM.

Article Snippet: To quantitatively measure cellular internalization of P(Glu-co-Lys)GNR at different pH values (7.4 and 6.5), PANC 1 human pancreatic cancer cells (obtained from American Type Culture Collection, authenticated by STR-based DNA profiling) and KPC murine pancreatic cancer cells derived from the transgenic KrasLSL.G12D/+; p53R172H/+; PdxCretg/+ C57BL/6 mouse (a gift from T. Arumugam at MD Anderson Cancer Center, authenticated by IDEXX STR-based DNA profiling and multiplex polymerase chain reaction to detect both contamination and misidentification of your cell lines) were grown in 60-mm petri dishes (Corning 353004) and incubated with 2 ml of complete media (pH 7.4 and pH 6.5) containing 0.5 OD P(Glu-co-Lys)-GNR for 24 hours.

Techniques: In Vitro, Fluorescence, Incubation, Control

Journal: Science advances

Article Title: Pancreatic tumor microenvironmental acidosis and hypoxia transform gold nanorods into cell-penetrant particles for potent radiosensitization.

doi: 10.1126/sciadv.abm9729

Figure Lengend Snippet: Fig. 4. Radiosensitization of pancreatic cancer cells in vitro. Clonogenic surviv- al assay of irradiated KPC (A) and PANC 1 (B) cells treated with P(Glu-co-Lys)1:5-GNR at pH 7.4 or 6.5. P(Glu-co-Lys)1:5-GNR causes greater radiosensitization at pH 6.5 than at pH 7.4 in both cell lines. Clonogenic survival assay of irradiated KPC (C) and PANC 1 (D) cells treated with P(Glu-co-Lys)1:5-GNR in the presence or absence of chemical hypoxia (induced by CoCl2). P(Glu-co-Lys)1:5-GNR causes greater radio- sensitization under hypoxic conditions than normoxic conditions in both cell lines.

Article Snippet: To quantitatively measure cellular internalization of P(Glu-co-Lys)GNR at different pH values (7.4 and 6.5), PANC 1 human pancreatic cancer cells (obtained from American Type Culture Collection, authenticated by STR-based DNA profiling) and KPC murine pancreatic cancer cells derived from the transgenic KrasLSL.G12D/+; p53R172H/+; PdxCretg/+ C57BL/6 mouse (a gift from T. Arumugam at MD Anderson Cancer Center, authenticated by IDEXX STR-based DNA profiling and multiplex polymerase chain reaction to detect both contamination and misidentification of your cell lines) were grown in 60-mm petri dishes (Corning 353004) and incubated with 2 ml of complete media (pH 7.4 and pH 6.5) containing 0.5 OD P(Glu-co-Lys)-GNR for 24 hours.

Techniques: In Vitro, Irradiation, Clonogenic Cell Survival Assay

Journal: Science advances

Article Title: Pancreatic tumor microenvironmental acidosis and hypoxia transform gold nanorods into cell-penetrant particles for potent radiosensitization.

doi: 10.1126/sciadv.abm9729

Figure Lengend Snippet: Fig. 6. Radiosensitization of pancreatic cancers in vivo. (A) In vivo tumor regrowth delay curves of C57BL/6 mice harboring subcutaneous KPC tumors and treated with P(Glu-co-Lys)-GNRs, PEG-GNRs, and/or radiation. The greatest regression in tumor regrowth was observed in the P(Glu-co-Lys)-GNRs + RT group. (B) In vivo tumor growth delay curves of C57BL/6 mice harboring orthotopic KPC tumors and treated with P(Glu-co-Lys)-GNRs, PEG-GNRs, and/or radiation. The greatest regression in tumor re- growth was observed in the P(Glu-co-Lys)-GNRs + RT group. (C) A representative bioluminescence image of mice from each of the treatment groups on day 11.

Article Snippet: To quantitatively measure cellular internalization of P(Glu-co-Lys)GNR at different pH values (7.4 and 6.5), PANC 1 human pancreatic cancer cells (obtained from American Type Culture Collection, authenticated by STR-based DNA profiling) and KPC murine pancreatic cancer cells derived from the transgenic KrasLSL.G12D/+; p53R172H/+; PdxCretg/+ C57BL/6 mouse (a gift from T. Arumugam at MD Anderson Cancer Center, authenticated by IDEXX STR-based DNA profiling and multiplex polymerase chain reaction to detect both contamination and misidentification of your cell lines) were grown in 60-mm petri dishes (Corning 353004) and incubated with 2 ml of complete media (pH 7.4 and pH 6.5) containing 0.5 OD P(Glu-co-Lys)-GNR for 24 hours.

Techniques: In Vivo

Journal: Systems biology in reproductive medicine

Article Title: Strategy for microbiome analysis using 16S rRNA gene sequence analysis on the Illumina sequencing platform.

doi: 10.3109/19396368.2011.555598

Figure Lengend Snippet: Figure 1. Location of variable and conserved regions in a typical bacterial 16S rRNA. A) location of variable (Vi, green) and conserved (Ci, pink) regions in a typical bacterial 16S rRNA gene (complete gene). The black bases in C4 are completely invariant in all bacteria. B) Specific example of the C2 to C4 region in Escherichia coli, with a similar color code to A. The C2, C3, and C4 conserved regions are the targets of universal bacterial ribo- somal DNA primers.

Article Snippet: Purified genomic DNA was obtained commercially for Escherichia coli (ATCC® cat. 10798D) and Desulfovibrio vulgaris(ATCC® cat. 29579D).

Techniques: Bacteria

Journal: Systems biology in reproductive medicine

Article Title: Strategy for microbiome analysis using 16S rRNA gene sequence analysis on the Illumina sequencing platform.

doi: 10.3109/19396368.2011.555598

Figure Lengend Snippet: Figure 3. PCR products amplified by complex primers. 16S rRNA gene PCR products amplified by complex primers, having anchor, index, and primer sequence, from many species. Primers were either C2X with C4Y or C2Y with C4X, as indicated. The C2Y/C4X reaction for Sp.01 failed for unknown reasons. Abbreviations: W: water control; Sp: S. pyogenes; Ec: E. coli; Bac: B. fragilis; Dsv: D. vulgaris.

Article Snippet: Purified genomic DNA was obtained commercially for Escherichia coli (ATCC® cat. 10798D) and Desulfovibrio vulgaris(ATCC® cat. 29579D).

Techniques: Amplification, Sequencing, Control

Journal: Systems biology in reproductive medicine

Article Title: Strategy for microbiome analysis using 16S rRNA gene sequence analysis on the Illumina sequencing platform.

doi: 10.3109/19396368.2011.555598

Figure Lengend Snippet: Figure 4. Sequence Basecall graphic summaries. Sequence information from the Percent Basecall graphic summaries of the Illumina analysis pipe- line for cycles 1 – 10 for three lanes for which the basecalls (‘Illumina data’) matched the species-specific sequence expected in that lane (‘Expected’ for lane 3, S. pyogenes; lane 4, E. coli; and lane 8, D. vulgaris, based on sequences in Genbank). Basecall colors are: A, gray; C, red; G, blue; and T, green. For lane 3, the ‘Illumina data’ basecalls are listed after subtracting the high baseline error rate for ‘C’ and ‘A’ (see text).

Article Snippet: Purified genomic DNA was obtained commercially for Escherichia coli (ATCC® cat. 10798D) and Desulfovibrio vulgaris(ATCC® cat. 29579D).

Techniques: Sequencing

Journal: Systems biology in reproductive medicine

Article Title: Strategy for microbiome analysis using 16S rRNA gene sequence analysis on the Illumina sequencing platform.

doi: 10.3109/19396368.2011.555598

Figure Lengend Snippet: Figure 5. Accuracy of multiplex sequencing. Output and accuracy of Illumina 16S rRNA gene multiplex sequencing for a target of known sequence (Lane 4, E. coli). A) typical text output from the analysis pipeline, shown for one cluster of lane 4, tile 43. The sequence calls are followed by quality estimates using characters ‘_’, ‘B’, etc (see text). B) Expected sequence for E. coli, with the primers shown for their respective read cycles. Bases in black were called correctly > 95% of the time; in red, either not called or mostly incorrectly. C) Summary of the percent of correct calls analyzed for 150 clusters from lane 4, tile 43. Brackets are the same as in (B).

Article Snippet: Purified genomic DNA was obtained commercially for Escherichia coli (ATCC® cat. 10798D) and Desulfovibrio vulgaris(ATCC® cat. 29579D).

Techniques: Multiplex Assay, Sequencing

Journal: The Brazilian journal of infectious diseases : an official publication of the Brazilian Society of Infectious Diseases

Article Title: Comparison of PCR-based methods for the simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in clinical samples.

doi: 10.1016/j.bjid.2016.04.005

Figure Lengend Snippet: Fig. 1 – Agarose gel of a multiplex PCR showing the detection of the three pathogens from three different samples. Lane 1: 100 bp DNA ladder; lane 2: Neisseria meningitidis (nspA gene with 481 bp); lane 3: Haemophilus influenzae (P6 gene with 273 bp); lane 4: Streptococcus pneumoniae

Article Snippet: Other 9 reference strains were also used as negative controls as they can also cause bacterial meningitis in humans: Neisseria lactamica (ATCC 23970), genes (ATCC 19615), Klebsiella pneumoniae (ATCC 13883), Listeria 2 0 1 m E E G a D t s t D t w c fi i P S t w d g d p i ( d t s fi g b r a z j i n f e c t d i s . onocytogenes (ATCC 15313), Acinetobacter sp. (ATCC 14293), scherichia coli (ATCC 11775). xtraction and purification of bacterial DNA enomic DNA extraction and purification from isolated strains nd negative clinical samples was carried out using the Neasy Blood and Tissue extraction kit (Qiagen) according o manufacturer instructions.

Techniques: Agarose Gel Electrophoresis, Multiplex Assay