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tiangen biotech co genomic dna
Numbers of different types of <t>DNA-RNA</t> differences detected in heads, thoraxes, or abdomens of honeybees.
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1) Product Images from "Evolutionary forces on A-to-I RNA editing revealed by sequencing individual honeybee drones"

Article Title: Evolutionary forces on A-to-I RNA editing revealed by sequencing individual honeybee drones

Journal: bioRxiv

doi: 10.1101/2020.01.15.907287

Numbers of different types of DNA-RNA differences detected in heads, thoraxes, or abdomens of honeybees.
Figure Legend Snippet: Numbers of different types of DNA-RNA differences detected in heads, thoraxes, or abdomens of honeybees.

Techniques Used:

Identification and annotation of the A-to-I RNA editing sites in the honeybee. (A) Workflow of sample collection, dissection and library construction. (B) Schematic diagram illustrating the identification of A-to-I RNA editing sites. (C) Numbers of different types of DNA-RNA differences detected in honeybee. (D) A summary of A-to-I editing sites according to their functional annotations.
Figure Legend Snippet: Identification and annotation of the A-to-I RNA editing sites in the honeybee. (A) Workflow of sample collection, dissection and library construction. (B) Schematic diagram illustrating the identification of A-to-I RNA editing sites. (C) Numbers of different types of DNA-RNA differences detected in honeybee. (D) A summary of A-to-I editing sites according to their functional annotations.

Techniques Used: Dissection, Functional Assay

2) Product Images from "Co-expression of AaPMT and AaTRI effectively enhances the yields of tropane alkaloids in Anisodus acutangulus hairy roots"

Article Title: Co-expression of AaPMT and AaTRI effectively enhances the yields of tropane alkaloids in Anisodus acutangulus hairy roots

Journal: BMC Biotechnology

doi: 10.1186/1472-6750-11-43

DNA identification and gene transcripts analysis . A) PCR analyses of transgenic hairy root lines. Analyses for the presence of AaPMT (1269 bp) and AaTRI genes (1074 bp) in PT lines (1), AaPMT gene in P lines (2) and AaTRI gene in T lines (3), respectively. M, DL-2000 Marker. PC, positive control (vector contains corresponding gene). BC, blank control (hairy root generate from blank-vector transformation). B) Effects of transformation on the expression of related genes. AaPMT T and AaTRI T represent total gene of AaPMT and AaTRI respectively, AaPMT E and AaTRI E represent endogenous AaPMT and AaTRI respectively. C) Real-time fluorescence quantitative PCR analysis of the expressions of AaPMT and AaTRI in transgenic hairy roots.
Figure Legend Snippet: DNA identification and gene transcripts analysis . A) PCR analyses of transgenic hairy root lines. Analyses for the presence of AaPMT (1269 bp) and AaTRI genes (1074 bp) in PT lines (1), AaPMT gene in P lines (2) and AaTRI gene in T lines (3), respectively. M, DL-2000 Marker. PC, positive control (vector contains corresponding gene). BC, blank control (hairy root generate from blank-vector transformation). B) Effects of transformation on the expression of related genes. AaPMT T and AaTRI T represent total gene of AaPMT and AaTRI respectively, AaPMT E and AaTRI E represent endogenous AaPMT and AaTRI respectively. C) Real-time fluorescence quantitative PCR analysis of the expressions of AaPMT and AaTRI in transgenic hairy roots.

Techniques Used: Polymerase Chain Reaction, Transgenic Assay, Marker, Positive Control, Plasmid Preparation, Transformation Assay, Expressing, Fluorescence, Real-time Polymerase Chain Reaction

3) Product Images from "Epigenetic down regulation of G protein-coupled estrogen receptor (GPER) functions as a tumor suppressor in colorectal cancer"

Article Title: Epigenetic down regulation of G protein-coupled estrogen receptor (GPER) functions as a tumor suppressor in colorectal cancer

Journal: Molecular Cancer

doi: 10.1186/s12943-017-0654-3

Promoter methylation and histone H3 deacetylation were involved in GPER down regulation in CRC cell and tissues. The mRNA ( a ) and protein ( b ) levels of GPER in CRC cell lines, human colon mucosal epithelial NCM460 cells were measured by qRT-PCR and western blot analysis, respectively; ( c ) Methylation status of GPER promoter in CRC cell lines was determined by bisulfite genomic DNA sequencing. Each dot represents a CpG site. White dots represent unmethylated CpG dinucleotides whereas each black dots represents a methylated cytosine residue within the CpG islands; ( d ) Methylation statuses of GPER promoter in five pairs of CRCs tissues and patient-matched normal tissues (Cohort 1) were determined by bisulfite genomic DNA sequencing; ( e ) HCT-116 or SW480 cells were treated with 5 μM 5-Aza for the different times, then mRNA of GPER was measured by use of qRT-PCR; ( f ) ChIP analysis of NCM460 and CRC cell lines were conducted on the GPER promoter regions by use of antiacetyl histone H3 antibody; ( g ) The correlation between relative acet-H3 enrichment and GPER mRNA expression in LS147T, HCT-116, SW620, HCT8, and SW480 cells; ( h ) ChIP analysis of five pairs of CRCs tissues and patient-matched normal tissues (Cohort 1) were conducted on the GPER promoter regions by use of antiacetyl histone H3 antibody. Data were presented as means ± SD of three independent experiments. * p
Figure Legend Snippet: Promoter methylation and histone H3 deacetylation were involved in GPER down regulation in CRC cell and tissues. The mRNA ( a ) and protein ( b ) levels of GPER in CRC cell lines, human colon mucosal epithelial NCM460 cells were measured by qRT-PCR and western blot analysis, respectively; ( c ) Methylation status of GPER promoter in CRC cell lines was determined by bisulfite genomic DNA sequencing. Each dot represents a CpG site. White dots represent unmethylated CpG dinucleotides whereas each black dots represents a methylated cytosine residue within the CpG islands; ( d ) Methylation statuses of GPER promoter in five pairs of CRCs tissues and patient-matched normal tissues (Cohort 1) were determined by bisulfite genomic DNA sequencing; ( e ) HCT-116 or SW480 cells were treated with 5 μM 5-Aza for the different times, then mRNA of GPER was measured by use of qRT-PCR; ( f ) ChIP analysis of NCM460 and CRC cell lines were conducted on the GPER promoter regions by use of antiacetyl histone H3 antibody; ( g ) The correlation between relative acet-H3 enrichment and GPER mRNA expression in LS147T, HCT-116, SW620, HCT8, and SW480 cells; ( h ) ChIP analysis of five pairs of CRCs tissues and patient-matched normal tissues (Cohort 1) were conducted on the GPER promoter regions by use of antiacetyl histone H3 antibody. Data were presented as means ± SD of three independent experiments. * p

Techniques Used: Methylation, Quantitative RT-PCR, Western Blot, DNA Sequencing, Chromatin Immunoprecipitation, Expressing

4) Product Images from "Deletion of CGLD1 Impairs PSII and Increases Singlet Oxygen Tolerance of Green Alga Chlamydomonas reinhardtii"

Article Title: Deletion of CGLD1 Impairs PSII and Increases Singlet Oxygen Tolerance of Green Alga Chlamydomonas reinhardtii

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2017.02154

Genetic analysis of the x32 mutant. (A) DNA blot analysis of wild-type and x32 . Genomic DNA was digested with Hind III and Kpn I, fractionated by agarose gel electrophoresis and hybridized with a DNA probe of 407bp of APHVIII gene. (B) Mapping of the deletion in x32 caused by insertion of the paromomycin resistance cassette ( APHVIII ) by comparative analysis of wild-type and x32 DNA using PCR specific primer pairs. (C) Heterologous expression of recombinant CGLD1 protein and specificity of CGLD1 antibody. Coomassie-stained SDS-PAGE gel separating proteins extracted from Escherichia coli cells (left panel). M, protein molecular mass marker. 1 and 2, total proteins without or with isopropyl β- D -1-thiogalactopyranoside (IPTG) induction, respectively. 3, recombinant CGLD1 purified using Ni-NTA column. Specificity detection of the CGLD1 antibody by immunoblotting (right panel). Lane 1, purified recombinant CGLD1 protein (0.02 μg). Lanes 2 and 3, proteins (20 μg) extracted from wild-type and x32 , respectively. (D) Immunoblot detection of CGLD1 in wild-type, x32 and the complemented strains C15 and C19. Membrane proteins (20 μg per lane) were separated by SDS-PAGE (16%) followed by immunoblotting with the CGLD1 antibody.
Figure Legend Snippet: Genetic analysis of the x32 mutant. (A) DNA blot analysis of wild-type and x32 . Genomic DNA was digested with Hind III and Kpn I, fractionated by agarose gel electrophoresis and hybridized with a DNA probe of 407bp of APHVIII gene. (B) Mapping of the deletion in x32 caused by insertion of the paromomycin resistance cassette ( APHVIII ) by comparative analysis of wild-type and x32 DNA using PCR specific primer pairs. (C) Heterologous expression of recombinant CGLD1 protein and specificity of CGLD1 antibody. Coomassie-stained SDS-PAGE gel separating proteins extracted from Escherichia coli cells (left panel). M, protein molecular mass marker. 1 and 2, total proteins without or with isopropyl β- D -1-thiogalactopyranoside (IPTG) induction, respectively. 3, recombinant CGLD1 purified using Ni-NTA column. Specificity detection of the CGLD1 antibody by immunoblotting (right panel). Lane 1, purified recombinant CGLD1 protein (0.02 μg). Lanes 2 and 3, proteins (20 μg) extracted from wild-type and x32 , respectively. (D) Immunoblot detection of CGLD1 in wild-type, x32 and the complemented strains C15 and C19. Membrane proteins (20 μg per lane) were separated by SDS-PAGE (16%) followed by immunoblotting with the CGLD1 antibody.

Techniques Used: Mutagenesis, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Expressing, Recombinant, Staining, SDS Page, Marker, Purification

5) Product Images from "CRISPR-offinder: a CRISPR guide RNA design and off-target searching tool for user-defined protospacer adjacent motif"

Article Title: CRISPR-offinder: a CRISPR guide RNA design and off-target searching tool for user-defined protospacer adjacent motif

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.21312

Targeting androgen receptor (AR) gene by CRISPR/Cas9-based genome editing. A. Scheme of sgRNAs designed to target exon 1 of the AR gene using CRISPR-offinder. B C. Activity of 17-, 18-, 19-, or 20-nt sgRNAs targeted to the same genomic loci by cell sorting or unsorting after co-transfection with CMV-EGFP-hspCas9 plasmid. D. Indels mutation created by CRISPR/Cas9 and detected by DNA sequencing. AR, androgen receptor; M, the number of nucleotide mismatches (1M, 2M, 3M, 4M or 5M); 0M, perfect match to the on-target site, and if the number of 0M sites > 1, maybe off-target sites are contained; bp, base pair; WT, wild-type; OT, off-target; GFP, green fluorescent protein; CMV (Cytomegalovirus) promoter, a constitutive mammalian promoter; Control, cells for the negative control.
Figure Legend Snippet: Targeting androgen receptor (AR) gene by CRISPR/Cas9-based genome editing. A. Scheme of sgRNAs designed to target exon 1 of the AR gene using CRISPR-offinder. B C. Activity of 17-, 18-, 19-, or 20-nt sgRNAs targeted to the same genomic loci by cell sorting or unsorting after co-transfection with CMV-EGFP-hspCas9 plasmid. D. Indels mutation created by CRISPR/Cas9 and detected by DNA sequencing. AR, androgen receptor; M, the number of nucleotide mismatches (1M, 2M, 3M, 4M or 5M); 0M, perfect match to the on-target site, and if the number of 0M sites > 1, maybe off-target sites are contained; bp, base pair; WT, wild-type; OT, off-target; GFP, green fluorescent protein; CMV (Cytomegalovirus) promoter, a constitutive mammalian promoter; Control, cells for the negative control.

Techniques Used: CRISPR, Activity Assay, FACS, Cotransfection, Plasmid Preparation, Mutagenesis, DNA Sequencing, Negative Control

Targeting ADP ribosylation factor like GTPase 2 binding protein ( ARL2BP ) gene by CRISPR/Cpf1-based genome editing. A. Scheme of sgRNAs designed to target exon 3 of the ARL2BP gene using CRISPR-offinder. B. Activity of 18-, 19-, 20-, 21-, 22- or 23-nt sgRNA targeted to the different genomic loci after co-transfection with two members of the Cpf1 family, the AsCpf1 from Acidaminococcus sp. and the LbCpf1 from Lachnospiraceae bacterium. C. Indels mutation created by CRISPR/Cas9 and detected by DNA sequencing. D. Detection of the two Cpf1 off-target binding sites by the T7EN I cleavage assay. ARL2BP, ADP ribosylation factor like GTPase 2 binding protein; M, the number of nucleotide mismatches (1M, 2M, 3M, 4M or 5M); 0M, perfect match to the on-target site, and if the number of 0M sites > 1, maybe off-target sites are contained; bp, base pair; OT, off-target; POT, potential off-target sites; Control, cells for the negative control.
Figure Legend Snippet: Targeting ADP ribosylation factor like GTPase 2 binding protein ( ARL2BP ) gene by CRISPR/Cpf1-based genome editing. A. Scheme of sgRNAs designed to target exon 3 of the ARL2BP gene using CRISPR-offinder. B. Activity of 18-, 19-, 20-, 21-, 22- or 23-nt sgRNA targeted to the different genomic loci after co-transfection with two members of the Cpf1 family, the AsCpf1 from Acidaminococcus sp. and the LbCpf1 from Lachnospiraceae bacterium. C. Indels mutation created by CRISPR/Cas9 and detected by DNA sequencing. D. Detection of the two Cpf1 off-target binding sites by the T7EN I cleavage assay. ARL2BP, ADP ribosylation factor like GTPase 2 binding protein; M, the number of nucleotide mismatches (1M, 2M, 3M, 4M or 5M); 0M, perfect match to the on-target site, and if the number of 0M sites > 1, maybe off-target sites are contained; bp, base pair; OT, off-target; POT, potential off-target sites; Control, cells for the negative control.

Techniques Used: Binding Assay, CRISPR, Activity Assay, Cotransfection, Mutagenesis, DNA Sequencing, Cleavage Assay, Negative Control

6) Product Images from "Enhancement of grain number per spike by RNA interference of cytokinin oxidase 2 gene in bread wheat"

Article Title: Enhancement of grain number per spike by RNA interference of cytokinin oxidase 2 gene in bread wheat

Journal: Hereditas

doi: 10.1186/s41065-018-0071-7

PCR detection of 22 putative transgenic plants. 1~ 22 are putative transgenic plants, positive control (plasmid DNA as template, CK+) and negative control (non-transgenic plants, CK-)
Figure Legend Snippet: PCR detection of 22 putative transgenic plants. 1~ 22 are putative transgenic plants, positive control (plasmid DNA as template, CK+) and negative control (non-transgenic plants, CK-)

Techniques Used: Polymerase Chain Reaction, Transgenic Assay, Positive Control, Plasmid Preparation, Negative Control

Southern blotting analysis in selected T 0 primary wheat transformants. Genomic DNA digested with Eco R V and hybridized with FAD2 probe. The number of reactive bands in each lane represents the transgene copies in each transgenic line. Lane 1 is WT (NB1), lanes 2~ 6 are JW1-1A, JW5-1A, JW41-1B, JW39-3A and JW1-2B, respectively, M is the marker: λ-EcoT14 I digest (TaKaRa, Dalian, China)
Figure Legend Snippet: Southern blotting analysis in selected T 0 primary wheat transformants. Genomic DNA digested with Eco R V and hybridized with FAD2 probe. The number of reactive bands in each lane represents the transgene copies in each transgenic line. Lane 1 is WT (NB1), lanes 2~ 6 are JW1-1A, JW5-1A, JW41-1B, JW39-3A and JW1-2B, respectively, M is the marker: λ-EcoT14 I digest (TaKaRa, Dalian, China)

Techniques Used: Southern Blot, Transgenic Assay, Marker

7) Product Images from "Genome wide analyses uncover allele-specific RNA editing in human and mouse"

Article Title: Genome wide analyses uncover allele-specific RNA editing in human and mouse

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky613

Identification and characterization of allele-specific RNA editing sites in human tissues. ( A ) Overview of the approach for identifying allele-specific RNA editing sites. The pipeline uses raw DNA-seq and RNA-seq reads as source data and was compared to RNA editing sites from the RADAR database to assess allele-specific RNA editing. ( B ) Reads that contain both heterozygous SNPs and RNA-editing sites. These reads were used to identify allele-specific RNA editing sties. ( C ) Fisher's exact test and chi-square test evaluation of allele-specific RNA editing. ( D ) Heatmap showing the RNA editing efficiency for each allele associated with an allele-specific RNA editing site. RNA editing efficiency was defined as the ratio of G reads number to the sum of A and G reads number. Upper row represent low efficiency and the lower row high efficiency. ( E ) Heatmap of overlapping numbers of allele-specific RNA editing sites for tissues from one individual. Individual 1 is used as an example to indicate a high proportion of overlap between tissues. Tissues sampled are: bladder (BL), fat (FT), gastric (GA), lung (LG), ventricle (LV), psoas (PO), right ventricle (RV), small bowel (SB), Sigmoid colon (SG), spleen (SX) and thymus (TH).
Figure Legend Snippet: Identification and characterization of allele-specific RNA editing sites in human tissues. ( A ) Overview of the approach for identifying allele-specific RNA editing sites. The pipeline uses raw DNA-seq and RNA-seq reads as source data and was compared to RNA editing sites from the RADAR database to assess allele-specific RNA editing. ( B ) Reads that contain both heterozygous SNPs and RNA-editing sites. These reads were used to identify allele-specific RNA editing sties. ( C ) Fisher's exact test and chi-square test evaluation of allele-specific RNA editing. ( D ) Heatmap showing the RNA editing efficiency for each allele associated with an allele-specific RNA editing site. RNA editing efficiency was defined as the ratio of G reads number to the sum of A and G reads number. Upper row represent low efficiency and the lower row high efficiency. ( E ) Heatmap of overlapping numbers of allele-specific RNA editing sites for tissues from one individual. Individual 1 is used as an example to indicate a high proportion of overlap between tissues. Tissues sampled are: bladder (BL), fat (FT), gastric (GA), lung (LG), ventricle (LV), psoas (PO), right ventricle (RV), small bowel (SB), Sigmoid colon (SG), spleen (SX) and thymus (TH).

Techniques Used: DNA Sequencing, RNA Sequencing Assay

8) Product Images from "Phytophthora methylomes are modulated by 6mA methyltransferases and associated with adaptive genome regions"

Article Title: Phytophthora methylomes are modulated by 6mA methyltransferases and associated with adaptive genome regions

Journal: Genome Biology

doi: 10.1186/s13059-018-1564-4

PsDAMT3 contribute to P . sojae virulence. a Virulence reduced in psdamt3 mutants. psdamt3 -T3, psdamt3 -T9, and psdamt3 -T16 are psdamt3 individual mutants, T34 is the transformed strain but DNA sequence did not change, and WT is P . sojae wild-type strain P6497. The relative Phytophthora biomass of the infection tissues was compared with WT. *Significant differences ( P
Figure Legend Snippet: PsDAMT3 contribute to P . sojae virulence. a Virulence reduced in psdamt3 mutants. psdamt3 -T3, psdamt3 -T9, and psdamt3 -T16 are psdamt3 individual mutants, T34 is the transformed strain but DNA sequence did not change, and WT is P . sojae wild-type strain P6497. The relative Phytophthora biomass of the infection tissues was compared with WT. *Significant differences ( P

Techniques Used: Transformation Assay, Sequencing, Infection

9) Product Images from "A novel inducible mutagenesis screen enables to isolate and clone both embryonic and adult zebrafish mutants"

Article Title: A novel inducible mutagenesis screen enables to isolate and clone both embryonic and adult zebrafish mutants

Journal: Scientific Reports

doi: 10.1038/s41598-017-10968-w

Dox-dependent down-regulation of genes in three example mutants causes abnormal embryonic development. ( A ) Line pIDM-A11. pIDM-A, a β-act promoter was used to drive rtTA and Egfp genes. Diagram showing the position and orientation of pIDM in the faf1 genomic DNA. Black line, intron or intergenic DNA; Blue vertical bar, exon; Purple arrow, direction of Tet-on promoter; Red lines, position of the insertion. Total RNA was extracted at 48 hpf. The relative expression of the faf1 transcript was analyzed with qRT-PCR. β-actin was used to normalize the total RNA. The embryos treated and untreated with Dox (Ctr) were sampled at 5 dpf and subsequently subjected to alcian-blue-staining for fin (Top right panel) and pharyngeal cartilage (Bottom panel). Black arrow, position of caudal fin; Red star, ‘open-mouse’ phenotype. ( B ) Line pIDM-E10. pIDM-E, an elf1a promoter was used to drive rtTA and Egfp genes. Diagram of the position and orientation of pIDM in the grb14 genomic DNA (including two isoforms grb14X1 and X 3). The relative expression level of grb 1 4X 1 and X3 transcripts was analyzed by qRT-PCR with specific primers at 48 hpf. Pictures of WT and mutant embryos with different treatments at 36 hpf as indicated, noting that grb14 -MO morphants had similar defects as that in pIDM-E10 transgenic embryos upon Dox induction. ( C ) Line pIDM-A1. Diagram of the position and orientation of pIDM in the wu:fb77a09 ( nid2a ) genomic DNA. The relative expression level of the nid2a transcript was analyzed at 4 dpf. Pictures of WT and mutant embryos with differing treatments at 60 hpf as indicated. In A, B and C, representative embryos are shown, the number of embryos showing the displayed phenotype versus total embryos examined are provided in the corresponding panels. All statistically significant differences between samples were assessed with the independent-samples T -test (* P
Figure Legend Snippet: Dox-dependent down-regulation of genes in three example mutants causes abnormal embryonic development. ( A ) Line pIDM-A11. pIDM-A, a β-act promoter was used to drive rtTA and Egfp genes. Diagram showing the position and orientation of pIDM in the faf1 genomic DNA. Black line, intron or intergenic DNA; Blue vertical bar, exon; Purple arrow, direction of Tet-on promoter; Red lines, position of the insertion. Total RNA was extracted at 48 hpf. The relative expression of the faf1 transcript was analyzed with qRT-PCR. β-actin was used to normalize the total RNA. The embryos treated and untreated with Dox (Ctr) were sampled at 5 dpf and subsequently subjected to alcian-blue-staining for fin (Top right panel) and pharyngeal cartilage (Bottom panel). Black arrow, position of caudal fin; Red star, ‘open-mouse’ phenotype. ( B ) Line pIDM-E10. pIDM-E, an elf1a promoter was used to drive rtTA and Egfp genes. Diagram of the position and orientation of pIDM in the grb14 genomic DNA (including two isoforms grb14X1 and X 3). The relative expression level of grb 1 4X 1 and X3 transcripts was analyzed by qRT-PCR with specific primers at 48 hpf. Pictures of WT and mutant embryos with different treatments at 36 hpf as indicated, noting that grb14 -MO morphants had similar defects as that in pIDM-E10 transgenic embryos upon Dox induction. ( C ) Line pIDM-A1. Diagram of the position and orientation of pIDM in the wu:fb77a09 ( nid2a ) genomic DNA. The relative expression level of the nid2a transcript was analyzed at 4 dpf. Pictures of WT and mutant embryos with differing treatments at 60 hpf as indicated. In A, B and C, representative embryos are shown, the number of embryos showing the displayed phenotype versus total embryos examined are provided in the corresponding panels. All statistically significant differences between samples were assessed with the independent-samples T -test (* P

Techniques Used: Activated Clotting Time Assay, Expressing, Quantitative RT-PCR, Staining, Mutagenesis, Transgenic Assay

Schematics of the design and strategy of the inducible mutagenesis system for mutant screening. ( A ) Diagram showing the pIDM vector. Upper panel: ITR, inverted terminal repeats of Tol2 transposon; TRE-P, the third generation of tetracycline response element and CMV minimal promoter; I, the chicken β-globin insulator; β-act , beta-actin promoter;/elf1a, or elongation factor 1a promoter; rtTA, reverse tetracycline transcriptional activator; IRES, internal ribosome entry site; EGFP, enhanced green fluorescence gene; SV40, the SV40 transcriptional terminator. The purple arrow indicates the orientation of the promoter. Lower panel: upon Dox-treatment, TRE-P transcribes the flanked genomic DNA either antisense RNA (left panel), or sense RNA (middle panel), or non-coding RNA (ncRNA) (right panel), depending upon the inserted position. ( B ) Transgenic line with pIDM carrying a HA-DsRed gene and SV40 terminator downstream of the right ITR. The photos were taken at 36 hpf. Tg+/−, heterozygous transgenic fish; Ctr, untreated control sibling. Total protein was extracted at either 24 or 36 hpf. In the transgenic adult fish treated with Dox, total protein was extracted at 15 dpt. An HA monoclonal antibody was used to detect HA-DsRed. β-actin was used as the protein loading control. ( C ) Schematics of the screen strategy. F 0 transgenic founder fish were crossed with wild type zebrafish (WT) to generate F 1 . F 1 transgenic embryos in each line were divided into two groups. One group was treated with Dox at 12 hours post fertilization (hpf) to screen mutants with obvious abnormal developmental phenotypes until 5 days post fertilization (dpf). Another group was permitted to grow into adulthood in normal conditions. The 3 month-old F 1 transgenic fish were treated with Dox 14 days before caudal fin resection. The insertion sites were determined with linker-mediated PCR (LM-PCR) from pooled F 1 embryos. F 1 transgenic fish were crossed with WT zebrafish to generate F 2 . All mutants were further confirmed in a heterozygous F 2 progeny Transgenic fish are shown in green. Red arrows: Confirmation in F 2 generation.
Figure Legend Snippet: Schematics of the design and strategy of the inducible mutagenesis system for mutant screening. ( A ) Diagram showing the pIDM vector. Upper panel: ITR, inverted terminal repeats of Tol2 transposon; TRE-P, the third generation of tetracycline response element and CMV minimal promoter; I, the chicken β-globin insulator; β-act , beta-actin promoter;/elf1a, or elongation factor 1a promoter; rtTA, reverse tetracycline transcriptional activator; IRES, internal ribosome entry site; EGFP, enhanced green fluorescence gene; SV40, the SV40 transcriptional terminator. The purple arrow indicates the orientation of the promoter. Lower panel: upon Dox-treatment, TRE-P transcribes the flanked genomic DNA either antisense RNA (left panel), or sense RNA (middle panel), or non-coding RNA (ncRNA) (right panel), depending upon the inserted position. ( B ) Transgenic line with pIDM carrying a HA-DsRed gene and SV40 terminator downstream of the right ITR. The photos were taken at 36 hpf. Tg+/−, heterozygous transgenic fish; Ctr, untreated control sibling. Total protein was extracted at either 24 or 36 hpf. In the transgenic adult fish treated with Dox, total protein was extracted at 15 dpt. An HA monoclonal antibody was used to detect HA-DsRed. β-actin was used as the protein loading control. ( C ) Schematics of the screen strategy. F 0 transgenic founder fish were crossed with wild type zebrafish (WT) to generate F 1 . F 1 transgenic embryos in each line were divided into two groups. One group was treated with Dox at 12 hours post fertilization (hpf) to screen mutants with obvious abnormal developmental phenotypes until 5 days post fertilization (dpf). Another group was permitted to grow into adulthood in normal conditions. The 3 month-old F 1 transgenic fish were treated with Dox 14 days before caudal fin resection. The insertion sites were determined with linker-mediated PCR (LM-PCR) from pooled F 1 embryos. F 1 transgenic fish were crossed with WT zebrafish to generate F 2 . All mutants were further confirmed in a heterozygous F 2 progeny Transgenic fish are shown in green. Red arrows: Confirmation in F 2 generation.

Techniques Used: Mutagenesis, Plasmid Preparation, Activated Clotting Time Assay, Fluorescence, Transgenic Assay, Fluorescence In Situ Hybridization, Polymerase Chain Reaction

10) Product Images from "Curing of Plasmid pXO1 from Bacillus anthracis Using Plasmid Incompatibility"

Article Title: Curing of Plasmid pXO1 from Bacillus anthracis Using Plasmid Incompatibility

Journal: PLoS ONE

doi: 10.1371/journal.pone.0029875

PCR analysis of pXO1 in B. anthracis vaccine strain A16R. PCR analysis of the vaccine strain A16R (1) and the putative pXO1-cured strain A16R (2) with 14 primer pairs specific for plasmid pXO1. M, DNA marker IV (Real-Times Biotechnology).
Figure Legend Snippet: PCR analysis of pXO1 in B. anthracis vaccine strain A16R. PCR analysis of the vaccine strain A16R (1) and the putative pXO1-cured strain A16R (2) with 14 primer pairs specific for plasmid pXO1. M, DNA marker IV (Real-Times Biotechnology).

Techniques Used: Polymerase Chain Reaction, Plasmid Preparation, Marker

Colony PCR screening for pXO1 elimination with three plasmid-specific primer pairs. Results of using recombinant plasmids pKS11K (A), pKS4K (B), and pKS5K (C) to eliminate the large plasmid pXO1 from B. anthracis vaccine strain A16R by plasmid incompatibility. The presence of anthrax toxin genes pagA , lef , and cya was determined by PCR analysis of the vaccine strain A16R (1) and the putative pXO1-cured strain A16R (2). M, DNA marker IV (Real-Times Biotechnology, Beijing, China).
Figure Legend Snippet: Colony PCR screening for pXO1 elimination with three plasmid-specific primer pairs. Results of using recombinant plasmids pKS11K (A), pKS4K (B), and pKS5K (C) to eliminate the large plasmid pXO1 from B. anthracis vaccine strain A16R by plasmid incompatibility. The presence of anthrax toxin genes pagA , lef , and cya was determined by PCR analysis of the vaccine strain A16R (1) and the putative pXO1-cured strain A16R (2). M, DNA marker IV (Real-Times Biotechnology, Beijing, China).

Techniques Used: Polymerase Chain Reaction, Plasmid Preparation, Recombinant, Marker

11) Product Images from "All-Trans Retinoic Acid Induces CD4+CD25+FOXP3+ Regulatory T Cells by Increasing FOXP3 Demethylation in Systemic Sclerosis CD4+ T Cells"

Article Title: All-Trans Retinoic Acid Induces CD4+CD25+FOXP3+ Regulatory T Cells by Increasing FOXP3 Demethylation in Systemic Sclerosis CD4+ T Cells

Journal: Journal of Immunology Research

doi: 10.1155/2018/8658156

DNA methylation levels at the FOXP3 promoter in SSc CD4+ T cells. The average methylation level of the FOXP3 gene promoter sequence was cloned and sequenced as described in Materials and Methods. (a) The bar graph shows the mean DNA methylation status of the FOXP3 promoter in different groups. (b) The broken line shows the mean methylation status of 8 CG pairs in the promoter region of FOXP3 within 10 sequenced clones in different groups. The DNA methylation status of the FOXP3 promoter region was significantly decreased in SSc CD4+ T cells with the stimulation of the ATRA alone group or the ATRA and TGF- β combined group compared with negative controls ( ∗ p
Figure Legend Snippet: DNA methylation levels at the FOXP3 promoter in SSc CD4+ T cells. The average methylation level of the FOXP3 gene promoter sequence was cloned and sequenced as described in Materials and Methods. (a) The bar graph shows the mean DNA methylation status of the FOXP3 promoter in different groups. (b) The broken line shows the mean methylation status of 8 CG pairs in the promoter region of FOXP3 within 10 sequenced clones in different groups. The DNA methylation status of the FOXP3 promoter region was significantly decreased in SSc CD4+ T cells with the stimulation of the ATRA alone group or the ATRA and TGF- β combined group compared with negative controls ( ∗ p

Techniques Used: DNA Methylation Assay, Methylation, Sequencing, Clone Assay

12) Product Images from "Apolipoprotein E deficiency accelerates atherosclerosis development in miniature pigs"

Article Title: Apolipoprotein E deficiency accelerates atherosclerosis development in miniature pigs

Journal: Disease Models & Mechanisms

doi: 10.1242/dmm.036632

CRISPR/Cas9 mediates ApoE gene targeting in PFFs. (A) Schematic diagram of Cas9 -sgRNA targeting sites of the pig ApoE locus. The sgRNA targeting sequences are shown in red, and the protospacer-adjacent motif (PAM) sequences are shown in green and underlined. (B) T7E1 assay for Cas9-mediated cleavage at ApoE targeting sites in PFFs. M: DNA marker; Controls: PCR products of untransfected PFFs treated with T7E1; sgRNA1 and sgRNA2: PCR products of PFFs transfected with Cas9-sgRNA1 and Cas9-sgRNA2 treated with T7E1, respectively. (C) Genotypes of homozygous ApoE biallelic-modified colonies. The WT sequence is shown at the top. Deletion (Δ); insertion (+); italic letter denotes the inserted base pair.
Figure Legend Snippet: CRISPR/Cas9 mediates ApoE gene targeting in PFFs. (A) Schematic diagram of Cas9 -sgRNA targeting sites of the pig ApoE locus. The sgRNA targeting sequences are shown in red, and the protospacer-adjacent motif (PAM) sequences are shown in green and underlined. (B) T7E1 assay for Cas9-mediated cleavage at ApoE targeting sites in PFFs. M: DNA marker; Controls: PCR products of untransfected PFFs treated with T7E1; sgRNA1 and sgRNA2: PCR products of PFFs transfected with Cas9-sgRNA1 and Cas9-sgRNA2 treated with T7E1, respectively. (C) Genotypes of homozygous ApoE biallelic-modified colonies. The WT sequence is shown at the top. Deletion (Δ); insertion (+); italic letter denotes the inserted base pair.

Techniques Used: CRISPR, Marker, Polymerase Chain Reaction, Transfection, Modification, Sequencing

13) Product Images from "Characteristics of dihydroflavonol 4-reductase gene promoters from different leaf colored Malus crabapple cultivars"

Article Title: Characteristics of dihydroflavonol 4-reductase gene promoters from different leaf colored Malus crabapple cultivars

Journal: Horticulture Research

doi: 10.1038/hortres.2017.70

Cis -element binding ability and yeast one-hybrid assay of the McDFR1 promoters with McMYB10. ( a ) Interaction of the McMYB10 protein with the McDFR1-F1 , McDFR1-F2 , McDFR1-R , McDFR1-Ra1 , McDFR1- Ra2 and McDFR1-Ra2 (without F4) promoter regions, as revealed using yeast one-hybrid assays. The panel shows yeast cells containing distinct effector and reporter constructs grown on an SD/-Trp/-Ura medium plate. The interaction of McMYB10, fused to the GAL4 activation domain (pJG4-5-McMYB10), with LacZ driven by McDFR promoters (pLacZi-promoters of McDFR1 ) is shown in the bottom panel. Yeast transformed with pJG4-5-McMYB10/pLacZi, pJG4-5/pLacZi- McDFR1 promoters and pJG4-5/pLacZi were used as controls. ( b ) Electrophoretic mobility shift assay (EMSA) of four the cis -elements, MYB1AT, MYBGAHV, MYB1LEPR, MYBST1, with McMYB10. 100×Unlabeled probe refers to the control of adding 100 times the concentration of a competing non-labeled specific probe. The black arrow indicates protein-DNA complexes, and the white arrow shows the positions of free probes. In lanes with competitor DNA, there was an excess of unlabeled probe.
Figure Legend Snippet: Cis -element binding ability and yeast one-hybrid assay of the McDFR1 promoters with McMYB10. ( a ) Interaction of the McMYB10 protein with the McDFR1-F1 , McDFR1-F2 , McDFR1-R , McDFR1-Ra1 , McDFR1- Ra2 and McDFR1-Ra2 (without F4) promoter regions, as revealed using yeast one-hybrid assays. The panel shows yeast cells containing distinct effector and reporter constructs grown on an SD/-Trp/-Ura medium plate. The interaction of McMYB10, fused to the GAL4 activation domain (pJG4-5-McMYB10), with LacZ driven by McDFR promoters (pLacZi-promoters of McDFR1 ) is shown in the bottom panel. Yeast transformed with pJG4-5-McMYB10/pLacZi, pJG4-5/pLacZi- McDFR1 promoters and pJG4-5/pLacZi were used as controls. ( b ) Electrophoretic mobility shift assay (EMSA) of four the cis -elements, MYB1AT, MYBGAHV, MYB1LEPR, MYBST1, with McMYB10. 100×Unlabeled probe refers to the control of adding 100 times the concentration of a competing non-labeled specific probe. The black arrow indicates protein-DNA complexes, and the white arrow shows the positions of free probes. In lanes with competitor DNA, there was an excess of unlabeled probe.

Techniques Used: Binding Assay, Y1H Assay, Construct, Activation Assay, Transformation Assay, Electrophoretic Mobility Shift Assay, Concentration Assay, Labeling

14) Product Images from "The heat shock protein 90 of Toxoplasma gondii is essential for invasion of host cells and tachyzoite growth"

Article Title: The heat shock protein 90 of Toxoplasma gondii is essential for invasion of host cells and tachyzoite growth

Journal: Parasite

doi: 10.1051/parasite/2017023

Growth of Hsp90 knockout parasite ( ΔHsp90 ) in vitro. The parasites were cultured in African green monkey kidney (Vero) cells, 10 5 T. gondii were added to the 6-well plates, and the infection ratio was 1:1. Observation of RH Δku80 (A), ΔHsp90 (B), and complemented (C) parasites by inverted microscope. The plaque produced by ΔPKAR strains (Fig. 6B) was significantly smaller than that of RH Δku80 and complemented parasites (Figs. 6A, 6C), scale bar = 20 μm. T. gondii tachyzoites and Vero cells were indicated by arrows, T. gondii (black arrow), Vero cells (white arrow). (D) The parasites were collected at the same time, and genomic DNA was extracted by TIANGEN kit. T. gondii DNA was detected by SYBR-green real-time PCR using B1 primer pairs, the standard curve was obtained by the known concentration of the RH Δku80 parasites with the primers (B1), and the parasite number was calculated by interpolation from this standard curve. ** p
Figure Legend Snippet: Growth of Hsp90 knockout parasite ( ΔHsp90 ) in vitro. The parasites were cultured in African green monkey kidney (Vero) cells, 10 5 T. gondii were added to the 6-well plates, and the infection ratio was 1:1. Observation of RH Δku80 (A), ΔHsp90 (B), and complemented (C) parasites by inverted microscope. The plaque produced by ΔPKAR strains (Fig. 6B) was significantly smaller than that of RH Δku80 and complemented parasites (Figs. 6A, 6C), scale bar = 20 μm. T. gondii tachyzoites and Vero cells were indicated by arrows, T. gondii (black arrow), Vero cells (white arrow). (D) The parasites were collected at the same time, and genomic DNA was extracted by TIANGEN kit. T. gondii DNA was detected by SYBR-green real-time PCR using B1 primer pairs, the standard curve was obtained by the known concentration of the RH Δku80 parasites with the primers (B1), and the parasite number was calculated by interpolation from this standard curve. ** p

Techniques Used: Knock-Out, In Vitro, Cell Culture, Infection, Inverted Microscopy, Produced, SYBR Green Assay, Real-time Polymerase Chain Reaction, Concentration Assay

15) Product Images from "MYCT1-TV, A Novel MYCT1 Transcript, Is Regulated by c-Myc and May Participate in Laryngeal Carcinogenesis"

Article Title: MYCT1-TV, A Novel MYCT1 Transcript, Is Regulated by c-Myc and May Participate in Laryngeal Carcinogenesis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0025648

A role for E-box sites in basal human MTCT1-TV promoter activity. A. Binding of MYCT1-TV E-box sites to c-Myc in vitro detected by EMSAs. The symbol “ * ” means the oligonucleotides labled by biotin. Lanes 1 to 10 stand for the results from Hep2 cells and Lanes 11 to 20 the results from HEK293 cells. Lanes 1, 6, 11 and 16 represent biotin-labled oligonucleotides. Lanes 2, 7, 13 and 18 represent each probe incubated with nuclear extracts. Lanes 3, 8, 14 and 19 represent each probe incubated with a 100-fold excess of the unlabeled competitor oligonucleotides. Lanes 4, 9, 15 and 20 represent each probe incubated with a 100-fold excess of the unlabeled mutant competitor oligonucleotides. Lanes 5, 10, 12 and 17 represent the EMSA results in the presence of anti-c-Myc antibody. The experiments were repeated three times. B. Binding of E-box sites to c-Myc in vivo detected by ChIP. The Input lanes correspond to PCR products derived from chromatin prior to immunoprecipitation. The IgG lanes correspond to PCR products containing chromatin immunoprecipitated with antibodies against control IgG. The c-Myc lanes correspond to PCR products containing chromatin immunoprecipitated with antibodies against c-Myc. M indicates DNA 2000 marker. The 242 bp PCR product of c-Myc A or the 215 bp PCR product of c-Myc B is obtained corresponding to the sequence either E-box A or B binding site of the MYCT1-TV promoter. Results of Hep2 cells shown in the left figure are in line with those of HEK293 cells in the right one.
Figure Legend Snippet: A role for E-box sites in basal human MTCT1-TV promoter activity. A. Binding of MYCT1-TV E-box sites to c-Myc in vitro detected by EMSAs. The symbol “ * ” means the oligonucleotides labled by biotin. Lanes 1 to 10 stand for the results from Hep2 cells and Lanes 11 to 20 the results from HEK293 cells. Lanes 1, 6, 11 and 16 represent biotin-labled oligonucleotides. Lanes 2, 7, 13 and 18 represent each probe incubated with nuclear extracts. Lanes 3, 8, 14 and 19 represent each probe incubated with a 100-fold excess of the unlabeled competitor oligonucleotides. Lanes 4, 9, 15 and 20 represent each probe incubated with a 100-fold excess of the unlabeled mutant competitor oligonucleotides. Lanes 5, 10, 12 and 17 represent the EMSA results in the presence of anti-c-Myc antibody. The experiments were repeated three times. B. Binding of E-box sites to c-Myc in vivo detected by ChIP. The Input lanes correspond to PCR products derived from chromatin prior to immunoprecipitation. The IgG lanes correspond to PCR products containing chromatin immunoprecipitated with antibodies against control IgG. The c-Myc lanes correspond to PCR products containing chromatin immunoprecipitated with antibodies against c-Myc. M indicates DNA 2000 marker. The 242 bp PCR product of c-Myc A or the 215 bp PCR product of c-Myc B is obtained corresponding to the sequence either E-box A or B binding site of the MYCT1-TV promoter. Results of Hep2 cells shown in the left figure are in line with those of HEK293 cells in the right one.

Techniques Used: Activity Assay, Binding Assay, In Vitro, Incubation, Mutagenesis, In Vivo, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Derivative Assay, Immunoprecipitation, Marker, Sequencing

Transfection of MYCT1-TV and MYCT1 in Hep2 and HEK293 cells. A. mRNA levels of MYCT1-TV/MYCT1 in Hep2 and HEK293 cells transfected with MYCT1-TV-GFP/MYCT1-GFP. PCR produce a 929 bp DNA fragment for MYCT1-TV , a 726 bp DNA fragment for MYCT1 and a 511 bp DNA fragment for β-actin. In Hep2 cells, the gray-scale ratios of MYCT1-TV to β-actin mRNA levels (black bars) in blank, GFP and MYCT1-TV-GFP groups are 0.4735±0.0335, 0.4315±0.0303 and 23.5188±2.0896, and the gray-scale ratios of MYCT1 to β-actin mRNA levels (gray bars) in blank, GFP and MYCT1-GFP groups are 0.4157±0.1080, 0.4242±0.0658 and 25.7520±1.3244, respectively. In HEK293 cells, the counterpart gray-scale ratios of MYCT1-TV to β-actin mRNA levels are 1.3071±0.2223, 1.2523±0.1002 and 32.0339±2.2903, and the counterpart gray-scale ratios of MYCT1 to β-actin mRNA levels are 0.9950±0.0725, 1.1448±0.1346 and 31.5161±1.9808, respectively. B. Transfection efficiency and expression of MYCT1-TV/MYCT1 protein in Hep2 and HEK293 cells. Transfection efficiency and expression of MYCT1-TV/MYCT1 protein in GFP, MYCT1-TV-GFP and MYCT1-GFP groups are revealed by contrast and fluorescence microscopy under the same phase. M, DNA marker; blank, control cells before transfection; GFP, control cells after transfection with GFP only; MYCT1-TV-GFP, cells transfected with MYCT1-TV-GFP; MYCT1-GFP, cells transfected with MYCT1-GFP (**, p
Figure Legend Snippet: Transfection of MYCT1-TV and MYCT1 in Hep2 and HEK293 cells. A. mRNA levels of MYCT1-TV/MYCT1 in Hep2 and HEK293 cells transfected with MYCT1-TV-GFP/MYCT1-GFP. PCR produce a 929 bp DNA fragment for MYCT1-TV , a 726 bp DNA fragment for MYCT1 and a 511 bp DNA fragment for β-actin. In Hep2 cells, the gray-scale ratios of MYCT1-TV to β-actin mRNA levels (black bars) in blank, GFP and MYCT1-TV-GFP groups are 0.4735±0.0335, 0.4315±0.0303 and 23.5188±2.0896, and the gray-scale ratios of MYCT1 to β-actin mRNA levels (gray bars) in blank, GFP and MYCT1-GFP groups are 0.4157±0.1080, 0.4242±0.0658 and 25.7520±1.3244, respectively. In HEK293 cells, the counterpart gray-scale ratios of MYCT1-TV to β-actin mRNA levels are 1.3071±0.2223, 1.2523±0.1002 and 32.0339±2.2903, and the counterpart gray-scale ratios of MYCT1 to β-actin mRNA levels are 0.9950±0.0725, 1.1448±0.1346 and 31.5161±1.9808, respectively. B. Transfection efficiency and expression of MYCT1-TV/MYCT1 protein in Hep2 and HEK293 cells. Transfection efficiency and expression of MYCT1-TV/MYCT1 protein in GFP, MYCT1-TV-GFP and MYCT1-GFP groups are revealed by contrast and fluorescence microscopy under the same phase. M, DNA marker; blank, control cells before transfection; GFP, control cells after transfection with GFP only; MYCT1-TV-GFP, cells transfected with MYCT1-TV-GFP; MYCT1-GFP, cells transfected with MYCT1-GFP (**, p

Techniques Used: Transfection, Polymerase Chain Reaction, Expressing, Fluorescence, Microscopy, Marker

Expression of MYCT1-TV and MYCT1 in human cells and tissues. A. MYCT1-TV mRNA levels in human cells. The gray-scale ratios of MYCT1-TV to β-actin mRNA levels in Hep2, HeLa, BGC823, SGC7901, Bel7402, GES1, HEK293, human blood and MKN1 cells are 0.2890±0.0521, 0.3113±0.0138, 0.2985±0.0130, 0.2964±0.0427, 0.3512±0.0407, 1.0522±0.0808, 1.1159±0.1467, 1.1641±0.0665 and 0.2348±0.0147, respectively. B. MYCT1-TV and MYCT1 mRNA levels in LSCC and paired adjacent normal laryngeal tissues. PCR produce a 929 bp DNA fragment for MYCT1-TV , a 726 bp DNA fragment for MYCT1 and a 511 bp DNA fragment for β-actin. The gray-scale ratios of MYCT1-TV to β-actin mRNA levels (black bars) in LSCC and paired adjacent normal laryngeal tissues are 0.4172±0.0324 and 0.8073±0.0478, and the counterpart gray-scale ratios of MYCT1 to β-actin mRNA levels (gray bars) are 0.4304±0.0304 and 0.8416±0.0499. M, T and R indicate DNA marker, LSCC tumor tissue and paired adjacent normal tissue, respectively.
Figure Legend Snippet: Expression of MYCT1-TV and MYCT1 in human cells and tissues. A. MYCT1-TV mRNA levels in human cells. The gray-scale ratios of MYCT1-TV to β-actin mRNA levels in Hep2, HeLa, BGC823, SGC7901, Bel7402, GES1, HEK293, human blood and MKN1 cells are 0.2890±0.0521, 0.3113±0.0138, 0.2985±0.0130, 0.2964±0.0427, 0.3512±0.0407, 1.0522±0.0808, 1.1159±0.1467, 1.1641±0.0665 and 0.2348±0.0147, respectively. B. MYCT1-TV and MYCT1 mRNA levels in LSCC and paired adjacent normal laryngeal tissues. PCR produce a 929 bp DNA fragment for MYCT1-TV , a 726 bp DNA fragment for MYCT1 and a 511 bp DNA fragment for β-actin. The gray-scale ratios of MYCT1-TV to β-actin mRNA levels (black bars) in LSCC and paired adjacent normal laryngeal tissues are 0.4172±0.0324 and 0.8073±0.0478, and the counterpart gray-scale ratios of MYCT1 to β-actin mRNA levels (gray bars) are 0.4304±0.0304 and 0.8416±0.0499. M, T and R indicate DNA marker, LSCC tumor tissue and paired adjacent normal tissue, respectively.

Techniques Used: Expressing, Polymerase Chain Reaction, Marker

16) Product Images from "The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis"

Article Title: The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis

Journal: Veterinary Research

doi: 10.1186/s13567-018-0589-8

Identification of the mutant strain RA1062. A PCR amplification. M: Takara DL2000 marker; lanes 1–2: R. anatipestifer 16S rRNA was amplified from the WT strain CH3 (lane 1), the mutant strain RA1062 (lane 2), showing a 744-bp fragment of 16S rRNA; lanes 4–5: a 678-bp fragment of M949_RS01035 was amplified from the WT strain CH3 (lane 4), but not the mutant strain RA1062 (lane 5); lanes 7–8: the 644-bp fragment of erm gene was not amplified from the WT strain CH3 (lane 7), but amplified from the mutant strain RA1062 (lane 8); lanes 3, 6 and 9: the avian pathogenic E. coli strain (APEC, CVCC1547), as negative controls. B Southern blot analysis of the transposon Tn4351 insertion. Lane 1, 10 μg of pEP 4351 digested with Xba I (positive control); Lane 2, 10 μg of chromosomal DNA from mutant strain RA1062 digested with Xba I; Lane 3, 10 μg of chromosomal DNA from the WT strain CH3 digested with Xba I (negative control). The digested sample was resolved on a 0.7% agarose gel and Southern blot analysis was performed using a TnDIG-labeled probe. C Schematic chart of Tn4351 insertion in RA1062 chromosome at 318 bp of the gene, which is 678 nucleotides in length. D qPCR analysis. The expression of the mRNAs were expressed as fold change and calculated using the comparative C T (2 −∆∆CT ) method. Data were normalized to the housekeeping gene ldh and expressed as fold changes. The expression of M949_RS01035 in the mutant strain RA1062 was disrupted. However, no change was shown for its upstream M949_RS10475 gene and downstream M949_RS01030 gene. Error bars represent standard deviations from three replicates (*** p
Figure Legend Snippet: Identification of the mutant strain RA1062. A PCR amplification. M: Takara DL2000 marker; lanes 1–2: R. anatipestifer 16S rRNA was amplified from the WT strain CH3 (lane 1), the mutant strain RA1062 (lane 2), showing a 744-bp fragment of 16S rRNA; lanes 4–5: a 678-bp fragment of M949_RS01035 was amplified from the WT strain CH3 (lane 4), but not the mutant strain RA1062 (lane 5); lanes 7–8: the 644-bp fragment of erm gene was not amplified from the WT strain CH3 (lane 7), but amplified from the mutant strain RA1062 (lane 8); lanes 3, 6 and 9: the avian pathogenic E. coli strain (APEC, CVCC1547), as negative controls. B Southern blot analysis of the transposon Tn4351 insertion. Lane 1, 10 μg of pEP 4351 digested with Xba I (positive control); Lane 2, 10 μg of chromosomal DNA from mutant strain RA1062 digested with Xba I; Lane 3, 10 μg of chromosomal DNA from the WT strain CH3 digested with Xba I (negative control). The digested sample was resolved on a 0.7% agarose gel and Southern blot analysis was performed using a TnDIG-labeled probe. C Schematic chart of Tn4351 insertion in RA1062 chromosome at 318 bp of the gene, which is 678 nucleotides in length. D qPCR analysis. The expression of the mRNAs were expressed as fold change and calculated using the comparative C T (2 −∆∆CT ) method. Data were normalized to the housekeeping gene ldh and expressed as fold changes. The expression of M949_RS01035 in the mutant strain RA1062 was disrupted. However, no change was shown for its upstream M949_RS10475 gene and downstream M949_RS01030 gene. Error bars represent standard deviations from three replicates (*** p

Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, Marker, Southern Blot, Positive Control, Negative Control, Agarose Gel Electrophoresis, Labeling, Real-time Polymerase Chain Reaction, Expressing

17) Product Images from "Klebsiella pneumoniae SnebYK Mediates Resistance Against Heterodera glycines and Promotes Soybean Growth"

Article Title: Klebsiella pneumoniae SnebYK Mediates Resistance Against Heterodera glycines and Promotes Soybean Growth

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.01134

Amplification and analysis of the nifH gene of SnebYK. (A) Amplicon of SnebYK nifH (363 bp) in the left lane and DNA ladder in the right lane. (B) Transcriptional analysis of the SnebYK nifH gene in nitrogen-free medium determined with RT-PCR. The lane labeled “-N” shows the expression of the 16S rRNA and nifH gene of SnebYK grown in ACCC55 nitrogen-free medium; the lane labeled “+N” shows the expression of the 16S rRNA and nifH gene of SnebYK grown in nutrient agar medium. The transcript level of the 16S rRNA was used as a loading control; the transcript level of nifH of SnebYK grown in nutrient agar medium was used as a negative control. (C) Dendrogram based on the nifH sequences of SnebYK and other strains in the genus Klebsiella with similar nifH sequences. This analysis was performed using the neighbor-joining method in Mega 7.0.26 with a bootstrap value of n = 1000.
Figure Legend Snippet: Amplification and analysis of the nifH gene of SnebYK. (A) Amplicon of SnebYK nifH (363 bp) in the left lane and DNA ladder in the right lane. (B) Transcriptional analysis of the SnebYK nifH gene in nitrogen-free medium determined with RT-PCR. The lane labeled “-N” shows the expression of the 16S rRNA and nifH gene of SnebYK grown in ACCC55 nitrogen-free medium; the lane labeled “+N” shows the expression of the 16S rRNA and nifH gene of SnebYK grown in nutrient agar medium. The transcript level of the 16S rRNA was used as a loading control; the transcript level of nifH of SnebYK grown in nutrient agar medium was used as a negative control. (C) Dendrogram based on the nifH sequences of SnebYK and other strains in the genus Klebsiella with similar nifH sequences. This analysis was performed using the neighbor-joining method in Mega 7.0.26 with a bootstrap value of n = 1000.

Techniques Used: Amplification, Reverse Transcription Polymerase Chain Reaction, Labeling, Expressing, Negative Control

18) Product Images from "CRISPR/Cas9-Mediated Generation of Guangxi Bama Minipigs Harboring Three Mutations in α-Synuclein Causing Parkinson’s Disease"

Article Title: CRISPR/Cas9-Mediated Generation of Guangxi Bama Minipigs Harboring Three Mutations in α-Synuclein Causing Parkinson’s Disease

Journal: Scientific Reports

doi: 10.1038/s41598-018-30436-3

Production and genotyping of gene-edited Guangxi Bama minipigs. ( A ) A total of nine cloned piglets from three litters appeared healthy at birth. ( B ) Bmg BI digestion analysis showed that eight of nine piglets were monoallelic mutant at the SCNA locus (labeled by red asterisks), and the remaining one (1–4#) was a wild-type (WT). WT minipig were used as a negative control and the repair vector as a positive control. ( C ) Genotype-confirmed mutant minipigs (labeled by red asterisks) and the one wild-type minipig remained clinically healthy and showed normal growth and development at 3 months of age. ( D ) One mutant minipig (1–2#) and the age-matched wild type (1–4#) were used to confirm the SCNA mutations at the transcriptional level. The SCNA mRNA-coding sequence was obtained by RT-PCR. ( E ) The presence of the desired mutations at the transcriptional level (sequence coding for SCNA mRNA) was confirmed by DNA sequencing.
Figure Legend Snippet: Production and genotyping of gene-edited Guangxi Bama minipigs. ( A ) A total of nine cloned piglets from three litters appeared healthy at birth. ( B ) Bmg BI digestion analysis showed that eight of nine piglets were monoallelic mutant at the SCNA locus (labeled by red asterisks), and the remaining one (1–4#) was a wild-type (WT). WT minipig were used as a negative control and the repair vector as a positive control. ( C ) Genotype-confirmed mutant minipigs (labeled by red asterisks) and the one wild-type minipig remained clinically healthy and showed normal growth and development at 3 months of age. ( D ) One mutant minipig (1–2#) and the age-matched wild type (1–4#) were used to confirm the SCNA mutations at the transcriptional level. The SCNA mRNA-coding sequence was obtained by RT-PCR. ( E ) The presence of the desired mutations at the transcriptional level (sequence coding for SCNA mRNA) was confirmed by DNA sequencing.

Techniques Used: Clone Assay, Mutagenesis, Labeling, Negative Control, Plasmid Preparation, Positive Control, Sequencing, Reverse Transcription Polymerase Chain Reaction, DNA Sequencing

19) Product Images from "Analysis of promoter methylation and epigenetic regulation of miR-32 in colorectal cancer cells"

Article Title: Analysis of promoter methylation and epigenetic regulation of miR-32 in colorectal cancer cells

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2019.7328

Bisulfate sequencing PCR of the promoter region of microRNA-32. (A) CpG island regions identified in the promoter of the host gene transmembrane protein 245. (B) The methylation status of 94 CpG sites in the promoter was investigated using genomic DNA from HCT-116. A total of 10 individual clones were randomly picked for sequencing. The figures below each column of the circle represents the position of the CG site in the sequence. Solid and hollow circles represent methylated and unmethylated sites, respectively. ‘X’ denotes unmethylated sites, as continuous repeated bases appeared in some regions following bisulfite modifications, so success rate was reduced when detected by this instrument. These regions were thought as ‘base loss’ by the instrument. PCR, polymerase chain reaction.
Figure Legend Snippet: Bisulfate sequencing PCR of the promoter region of microRNA-32. (A) CpG island regions identified in the promoter of the host gene transmembrane protein 245. (B) The methylation status of 94 CpG sites in the promoter was investigated using genomic DNA from HCT-116. A total of 10 individual clones were randomly picked for sequencing. The figures below each column of the circle represents the position of the CG site in the sequence. Solid and hollow circles represent methylated and unmethylated sites, respectively. ‘X’ denotes unmethylated sites, as continuous repeated bases appeared in some regions following bisulfite modifications, so success rate was reduced when detected by this instrument. These regions were thought as ‘base loss’ by the instrument. PCR, polymerase chain reaction.

Techniques Used: Sequencing, Polymerase Chain Reaction, Methylation, Clone Assay

20) Product Images from "Construction of Transgenic Plasmodium berghei as a Model for Evaluation of Blood-Stage Vaccine Candidate of Plasmodium falciparum Chimeric Protein 2.9"

Article Title: Construction of Transgenic Plasmodium berghei as a Model for Evaluation of Blood-Stage Vaccine Candidate of Plasmodium falciparum Chimeric Protein 2.9

Journal: PLoS ONE

doi: 10.1371/journal.pone.0006894

PCR and Southern analysis of the stability of the integrated locus in PfMSP1-19Pb8.7 clones. Genomic DNA was extracted from transgenic parasites at various times with wild type parasites as control. The primer pair of Tb5/Tf (upper line) was used to detect the integrated fragment in the transgenic parasite, while the primer pair of Tb5/T-Pb (lower line) was used to amplify the native sequence of PbMSP1 in the wild-type parasites used as controls (A). Ten micrograms of genomic DNA of wild type and transgenic P. berghei parasites collected at different times was digested with HincII and hybridized to probe PbM while the HincII-digested transfection plasmid served as positive control (B). M: base pair ladder; lane 1: the genomic DNA of wild type parasites; lanes 2–7: the genomic DNA of PfMSP1-19Pb 8.7 clone that was extracted at various times (days 0,7,14,21,28 and 35); lane 8: the DNA of transfection plasmid digested by HincII.
Figure Legend Snippet: PCR and Southern analysis of the stability of the integrated locus in PfMSP1-19Pb8.7 clones. Genomic DNA was extracted from transgenic parasites at various times with wild type parasites as control. The primer pair of Tb5/Tf (upper line) was used to detect the integrated fragment in the transgenic parasite, while the primer pair of Tb5/T-Pb (lower line) was used to amplify the native sequence of PbMSP1 in the wild-type parasites used as controls (A). Ten micrograms of genomic DNA of wild type and transgenic P. berghei parasites collected at different times was digested with HincII and hybridized to probe PbM while the HincII-digested transfection plasmid served as positive control (B). M: base pair ladder; lane 1: the genomic DNA of wild type parasites; lanes 2–7: the genomic DNA of PfMSP1-19Pb 8.7 clone that was extracted at various times (days 0,7,14,21,28 and 35); lane 8: the DNA of transfection plasmid digested by HincII.

Techniques Used: Polymerase Chain Reaction, Clone Assay, Transgenic Assay, Sequencing, Transfection, Plasmid Preparation, Positive Control

Generation of mutant P. berghei parasites and PCR analysis. Organizational maps of plasmid PyrFlu (A), the construction strategy of recombinant vector PyrFlu/PbfMSP-1/ PbM3′ (B), the gene map following the homologous integration of plasmid PyrFlu/PbfMSP-1/ PbM3′ at the MSP1 locus (C). Genomic DNA of PfMSP1-19Pb 8.7 clone (lanes 1–4 and 7) and wild-type P. berghei ANKA (lanes 5–6 and 8) were used as template; Test primers are indicated by arrows. lane 1: amplification of gfp with the primers gfp F/ gfp R; lane 2: verification of transfection using primers Tb/Tf; lanes 3 and 5: verification of the wild-type PbMSP-1 locus with the primers Tb5/T-Pb; lanes 4 and 6: verification of the predicted 5′ integration into the PbMSP-1 locus with the primers Tb5/Tf; lanes 7 and 8: verification of the predicted 3′ integration into the PbMSP-1 locus with the primers 3′veF/3′veR; M: base pair ladder (D). Probe PbM for Southern analysis was shown as black bar. The expected sizes of fragments resulting from digestion with HincII are shown. S ( Sac I), B ( BamH I), A ( Apa I), K ( Kpn I) and H2( HincII ).
Figure Legend Snippet: Generation of mutant P. berghei parasites and PCR analysis. Organizational maps of plasmid PyrFlu (A), the construction strategy of recombinant vector PyrFlu/PbfMSP-1/ PbM3′ (B), the gene map following the homologous integration of plasmid PyrFlu/PbfMSP-1/ PbM3′ at the MSP1 locus (C). Genomic DNA of PfMSP1-19Pb 8.7 clone (lanes 1–4 and 7) and wild-type P. berghei ANKA (lanes 5–6 and 8) were used as template; Test primers are indicated by arrows. lane 1: amplification of gfp with the primers gfp F/ gfp R; lane 2: verification of transfection using primers Tb/Tf; lanes 3 and 5: verification of the wild-type PbMSP-1 locus with the primers Tb5/T-Pb; lanes 4 and 6: verification of the predicted 5′ integration into the PbMSP-1 locus with the primers Tb5/Tf; lanes 7 and 8: verification of the predicted 3′ integration into the PbMSP-1 locus with the primers 3′veF/3′veR; M: base pair ladder (D). Probe PbM for Southern analysis was shown as black bar. The expected sizes of fragments resulting from digestion with HincII are shown. S ( Sac I), B ( BamH I), A ( Apa I), K ( Kpn I) and H2( HincII ).

Techniques Used: Mutagenesis, Polymerase Chain Reaction, Plasmid Preparation, Recombinant, Amplification, Transfection

21) Product Images from "Melon13-lipoxygenase CmLOX18 may be involved in C6 volatiles biosynthesis in fruit"

Article Title: Melon13-lipoxygenase CmLOX18 may be involved in C6 volatiles biosynthesis in fruit

Journal: Scientific Reports

doi: 10.1038/s41598-017-02559-6

Molecular analysis of T0 transegenic tomato liners expressing CmLOX18 . ( a ) Southern blot of wild-type control and four T0 transgenic tomato lines expression CmLOX18 . Genomic DNA was prepared from young leaf material from wild-type control and transgenic plants: 1, 2, and 3 and V (35S-overexpression CmLOX18 construct). The genomic DNA (10 μg/lane) was digested with HindШ and separated in a 0.8% (w/v) agarose gel. Blotted DNA was hybridized to a probe prepared from the bar gene. ( b ) Detection of CmLOX18 cDNA by PCR analysis. Genomic DNA was extracted from transgenic lines. ( c ) Western blot. Proteins were extracted and Western blot was carried according to standard procedures using anti-e GFP Mouse Monoclonal antibody. ( d ) Images of wild-type control and twoT0 transgenic tomato leaves.
Figure Legend Snippet: Molecular analysis of T0 transegenic tomato liners expressing CmLOX18 . ( a ) Southern blot of wild-type control and four T0 transgenic tomato lines expression CmLOX18 . Genomic DNA was prepared from young leaf material from wild-type control and transgenic plants: 1, 2, and 3 and V (35S-overexpression CmLOX18 construct). The genomic DNA (10 μg/lane) was digested with HindШ and separated in a 0.8% (w/v) agarose gel. Blotted DNA was hybridized to a probe prepared from the bar gene. ( b ) Detection of CmLOX18 cDNA by PCR analysis. Genomic DNA was extracted from transgenic lines. ( c ) Western blot. Proteins were extracted and Western blot was carried according to standard procedures using anti-e GFP Mouse Monoclonal antibody. ( d ) Images of wild-type control and twoT0 transgenic tomato leaves.

Techniques Used: Expressing, Southern Blot, Transgenic Assay, Over Expression, Construct, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Western Blot

22) Product Images from "Riemerella anatipestifer AS87_RS09170 gene is responsible for biotin synthesis, bacterial morphology and virulence"

Article Title: Riemerella anatipestifer AS87_RS09170 gene is responsible for biotin synthesis, bacterial morphology and virulence

Journal: Scientific Reports

doi: 10.1038/s41598-018-32905-1

Analyses of Riemerella anatipestifer AS87_RS09170 gene and deduced amino acid sequence. ( A ) Polymerase chain reaction (PCR) analysis. The AS87_RS09170 gene was amplified from all R . anatipestifer strains tested. Lane M, DL2000 DNA Marker (Takara); lanes 1–5: R . anatipestifer serotype 1 strains GTB1, GTB2, CQ1, CQ3 and NJ4; lanes 6–10, R . anatipestifer serotype 2 strains GDO-3, JY1, JY2, NJ3 and SC2; lanes 11–15, R . anatipestifer serotype 10 strains GDO-1, HXb2, YXb1, YXb11 and YXL1; lanes 16–20, R . anatipestifer serotype 15 strains SQ007, SQ003, SQ004, YGT002 and MA001; lanes 21–25, R . anatipestifer undefined serotype strains GDO-6, GDO-7, G46, G77 and JY-6; lane 26, negative control of Yb2ΔbioF; lane 27, positive control of Yb2. A 1158-bp fragment was amplified from all 25 R . anatipestifer . ( B ) Identity and divergence analysis. The predicted R . anatipestifer 8-amino-7-oxononanoate synthase (AONS) and other known AONS were compared. The crystallized AONS sequences [ Paraburkholderia xenovorans (pdb5JAY), Francisella tularensis (pdb4IW7), Escherichia coli (pdb1DJE), Burkholderia multivorans (pdb5VNX) and Mycobacterium smegmatis (pdb3WY7)] were retrieved from the Uniprot database and later aligned with Clustal W algorithm in the MegAlign program from the DNASTAR Lasergene suite. ( C ) Multiple sequence alignment. Strictly conserved residues in all sequences are boxed in black. Residues forming the conserved pyridoxal 5′-phosphate binding pocket are indicated by a black inverted triangle (G97, Y98, N101, E168, D197, H200, T228 and K231).
Figure Legend Snippet: Analyses of Riemerella anatipestifer AS87_RS09170 gene and deduced amino acid sequence. ( A ) Polymerase chain reaction (PCR) analysis. The AS87_RS09170 gene was amplified from all R . anatipestifer strains tested. Lane M, DL2000 DNA Marker (Takara); lanes 1–5: R . anatipestifer serotype 1 strains GTB1, GTB2, CQ1, CQ3 and NJ4; lanes 6–10, R . anatipestifer serotype 2 strains GDO-3, JY1, JY2, NJ3 and SC2; lanes 11–15, R . anatipestifer serotype 10 strains GDO-1, HXb2, YXb1, YXb11 and YXL1; lanes 16–20, R . anatipestifer serotype 15 strains SQ007, SQ003, SQ004, YGT002 and MA001; lanes 21–25, R . anatipestifer undefined serotype strains GDO-6, GDO-7, G46, G77 and JY-6; lane 26, negative control of Yb2ΔbioF; lane 27, positive control of Yb2. A 1158-bp fragment was amplified from all 25 R . anatipestifer . ( B ) Identity and divergence analysis. The predicted R . anatipestifer 8-amino-7-oxononanoate synthase (AONS) and other known AONS were compared. The crystallized AONS sequences [ Paraburkholderia xenovorans (pdb5JAY), Francisella tularensis (pdb4IW7), Escherichia coli (pdb1DJE), Burkholderia multivorans (pdb5VNX) and Mycobacterium smegmatis (pdb3WY7)] were retrieved from the Uniprot database and later aligned with Clustal W algorithm in the MegAlign program from the DNASTAR Lasergene suite. ( C ) Multiple sequence alignment. Strictly conserved residues in all sequences are boxed in black. Residues forming the conserved pyridoxal 5′-phosphate binding pocket are indicated by a black inverted triangle (G97, Y98, N101, E168, D197, H200, T228 and K231).

Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification, Marker, Negative Control, Positive Control, Binding Assay

23) Product Images from "Detection of Infectious Laryngotracheitis Virus by Real-Time PCR in Naturally and Experimentally Infected Chickens"

Article Title: Detection of Infectious Laryngotracheitis Virus by Real-Time PCR in Naturally and Experimentally Infected Chickens

Journal: PLoS ONE

doi: 10.1371/journal.pone.0067598

Distribution and quantity of ILTV DNA in each tissue of the chickens at different days post infection. Ten-week-old SPF chickens were inoculated intratracheally with 10 4 EID 50 of ILTV strain LJS09. The viral loads were given as ILTV DNA copy number per g in each tissue. The tissues included the heart, liver, spleen, lung, kidney, larynx, tongue, thymus, glandular stomach, duodenum, pancreatic gland, small intestine, large intestine, cecum, cecal tonsil, bursa of Fabricius, and brain. A: The mean viral load of three chickens in the infection group and the standard deviation for each time point. B: The mean viral load of two chickens in the contact exposure group and the standard deviation for each time point.
Figure Legend Snippet: Distribution and quantity of ILTV DNA in each tissue of the chickens at different days post infection. Ten-week-old SPF chickens were inoculated intratracheally with 10 4 EID 50 of ILTV strain LJS09. The viral loads were given as ILTV DNA copy number per g in each tissue. The tissues included the heart, liver, spleen, lung, kidney, larynx, tongue, thymus, glandular stomach, duodenum, pancreatic gland, small intestine, large intestine, cecum, cecal tonsil, bursa of Fabricius, and brain. A: The mean viral load of three chickens in the infection group and the standard deviation for each time point. B: The mean viral load of two chickens in the contact exposure group and the standard deviation for each time point.

Techniques Used: Infection, Standard Deviation

Specificity of the real-time PCR. Six other avian pathogens were used for the specificity test. Dilutions of 10 5 , 10 4 , 10 3 , 10 2 were the standard DNA; 1: ILTV LJS09 strain DNA; 2–7: DNA and RNA samples of IBDV, CAV, REV, ARV, NDV, MDV; 8: H 2 O.
Figure Legend Snippet: Specificity of the real-time PCR. Six other avian pathogens were used for the specificity test. Dilutions of 10 5 , 10 4 , 10 3 , 10 2 were the standard DNA; 1: ILTV LJS09 strain DNA; 2–7: DNA and RNA samples of IBDV, CAV, REV, ARV, NDV, MDV; 8: H 2 O.

Techniques Used: Real-time Polymerase Chain Reaction

24) Product Images from "Genome wide analyses uncover allele-specific RNA editing in human and mouse"

Article Title: Genome wide analyses uncover allele-specific RNA editing in human and mouse

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky613

Identification and characterization of allele-specific RNA editing sites in human tissues. ( A ) Overview of the approach for identifying allele-specific RNA editing sites. The pipeline uses raw DNA-seq and RNA-seq reads as source data and was compared to RNA editing sites from the RADAR database to assess allele-specific RNA editing. ( B ) Reads that contain both heterozygous SNPs and RNA-editing sites. These reads were used to identify allele-specific RNA editing sties. ( C ) Fisher's exact test and chi-square test evaluation of allele-specific RNA editing. ( D ) Heatmap showing the RNA editing efficiency for each allele associated with an allele-specific RNA editing site. RNA editing efficiency was defined as the ratio of G reads number to the sum of A and G reads number. Upper row represent low efficiency and the lower row high efficiency. ( E ) Heatmap of overlapping numbers of allele-specific RNA editing sites for tissues from one individual. Individual 1 is used as an example to indicate a high proportion of overlap between tissues. Tissues sampled are: bladder (BL), fat (FT), gastric (GA), lung (LG), ventricle (LV), psoas (PO), right ventricle (RV), small bowel (SB), Sigmoid colon (SG), spleen (SX) and thymus (TH).
Figure Legend Snippet: Identification and characterization of allele-specific RNA editing sites in human tissues. ( A ) Overview of the approach for identifying allele-specific RNA editing sites. The pipeline uses raw DNA-seq and RNA-seq reads as source data and was compared to RNA editing sites from the RADAR database to assess allele-specific RNA editing. ( B ) Reads that contain both heterozygous SNPs and RNA-editing sites. These reads were used to identify allele-specific RNA editing sties. ( C ) Fisher's exact test and chi-square test evaluation of allele-specific RNA editing. ( D ) Heatmap showing the RNA editing efficiency for each allele associated with an allele-specific RNA editing site. RNA editing efficiency was defined as the ratio of G reads number to the sum of A and G reads number. Upper row represent low efficiency and the lower row high efficiency. ( E ) Heatmap of overlapping numbers of allele-specific RNA editing sites for tissues from one individual. Individual 1 is used as an example to indicate a high proportion of overlap between tissues. Tissues sampled are: bladder (BL), fat (FT), gastric (GA), lung (LG), ventricle (LV), psoas (PO), right ventricle (RV), small bowel (SB), Sigmoid colon (SG), spleen (SX) and thymus (TH).

Techniques Used: DNA Sequencing, RNA Sequencing Assay

25) Product Images from "Transgenic Cotton Plants Expressing Double-stranded RNAs Target HMG-CoA Reductase (HMGR) Gene Inhibits the Growth, Development and Survival of Cotton Bollworms"

Article Title: Transgenic Cotton Plants Expressing Double-stranded RNAs Target HMG-CoA Reductase (HMGR) Gene Inhibits the Growth, Development and Survival of Cotton Bollworms

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.12463

Molecular analysis for the putative transgenic cotton plants. (A) PCR analysis for HMGi1 and HMGi2 putative transgenic plants. M:Marker; N:Negative control; P: Positive control; Numbers marked above the gel indicating the corresponding T0 transgenic plants. (B) Southern blotting analysis of transgenic T0 plants. M: DNA molecular weight marker DIG-labeled (0.12-23.1 kb)(Roche, Germany); P: positive control; B: blank lane (no DNA loading); N: negative control plant DNA; Lane: 1-10 different HMGi transgenic lines.
Figure Legend Snippet: Molecular analysis for the putative transgenic cotton plants. (A) PCR analysis for HMGi1 and HMGi2 putative transgenic plants. M:Marker; N:Negative control; P: Positive control; Numbers marked above the gel indicating the corresponding T0 transgenic plants. (B) Southern blotting analysis of transgenic T0 plants. M: DNA molecular weight marker DIG-labeled (0.12-23.1 kb)(Roche, Germany); P: positive control; B: blank lane (no DNA loading); N: negative control plant DNA; Lane: 1-10 different HMGi transgenic lines.

Techniques Used: Transgenic Assay, Polymerase Chain Reaction, Marker, Negative Control, Positive Control, Southern Blot, Molecular Weight, Labeling

26) Product Images from "In vitro subminimum inhibitory concentrations of macrolide antibiotics induce macrolide resistance in Mycoplasma pneumoniae"

Article Title: In vitro subminimum inhibitory concentrations of macrolide antibiotics induce macrolide resistance in Mycoplasma pneumoniae

Journal: Hippokratia

doi:

A) Polymerase chain reaction (PCR) amplification of ribosomal protein L4 gene from M. pneumoniae clinical iso- lates. 1: DNA marker; 2: M. pneumoniae reference strain M129; 3-11: PCR products (464 bp) of ribosomal protein L4 gene; 12: Negative control.
Figure Legend Snippet: A) Polymerase chain reaction (PCR) amplification of ribosomal protein L4 gene from M. pneumoniae clinical iso- lates. 1: DNA marker; 2: M. pneumoniae reference strain M129; 3-11: PCR products (464 bp) of ribosomal protein L4 gene; 12: Negative control.

Techniques Used: Polymerase Chain Reaction, Amplification, Marker, Negative Control

A) Polymerase chain reaction (PCR) amplification of 23SrRNA gene including 2063 and 2064 from M. pneumoniae clinical isolates. 1: DNA marker; 2: M. pneumoniae reference strain M129; 3-11: PCR products (303 bp) of 23SrRNA gene (including 2063 and 2064).
Figure Legend Snippet: A) Polymerase chain reaction (PCR) amplification of 23SrRNA gene including 2063 and 2064 from M. pneumoniae clinical isolates. 1: DNA marker; 2: M. pneumoniae reference strain M129; 3-11: PCR products (303 bp) of 23SrRNA gene (including 2063 and 2064).

Techniques Used: Polymerase Chain Reaction, Amplification, Marker

27) Product Images from "Apolipoprotein E deficiency accelerates atherosclerosis development in miniature pigs"

Article Title: Apolipoprotein E deficiency accelerates atherosclerosis development in miniature pigs

Journal: Disease Models & Mechanisms

doi: 10.1242/dmm.036632

CRISPR/Cas9 mediates ApoE gene targeting in PFFs. (A) Schematic diagram of Cas9 -sgRNA targeting sites of the pig ApoE locus. The sgRNA targeting sequences are shown in red, and the protospacer-adjacent motif (PAM) sequences are shown in green and underlined. (B) T7E1 assay for Cas9-mediated cleavage at ApoE targeting sites in PFFs. M: DNA marker; Controls: PCR products of untransfected PFFs treated with T7E1; sgRNA1 and sgRNA2: PCR products of PFFs transfected with Cas9-sgRNA1 and Cas9-sgRNA2 treated with T7E1, respectively. (C) Genotypes of homozygous ApoE biallelic-modified colonies. The WT sequence is shown at the top. Deletion (Δ); insertion (+); italic letter denotes the inserted base pair.
Figure Legend Snippet: CRISPR/Cas9 mediates ApoE gene targeting in PFFs. (A) Schematic diagram of Cas9 -sgRNA targeting sites of the pig ApoE locus. The sgRNA targeting sequences are shown in red, and the protospacer-adjacent motif (PAM) sequences are shown in green and underlined. (B) T7E1 assay for Cas9-mediated cleavage at ApoE targeting sites in PFFs. M: DNA marker; Controls: PCR products of untransfected PFFs treated with T7E1; sgRNA1 and sgRNA2: PCR products of PFFs transfected with Cas9-sgRNA1 and Cas9-sgRNA2 treated with T7E1, respectively. (C) Genotypes of homozygous ApoE biallelic-modified colonies. The WT sequence is shown at the top. Deletion (Δ); insertion (+); italic letter denotes the inserted base pair.

Techniques Used: CRISPR, Marker, Polymerase Chain Reaction, Transfection, Modification, Sequencing

28) Product Images from "High‐efficiency genome editing using a dmc1 promoter‐controlled CRISPR/Cas9 system in maize"

Article Title: High‐efficiency genome editing using a dmc1 promoter‐controlled CRISPR/Cas9 system in maize

Journal: Plant Biotechnology Journal

doi: 10.1111/pbi.12920

Identification of mutations in the zb7 gene by RFLP assay and Sanger sequencing. (a) Schematic illustration of the target site in the zb7 gene. Black box indicates exons, while the lines between them represent introns. Underlined sequence was selected for targeting; nucleotides marked in blue represent PAM (protospacer adjacent motif). (b) RFLP assay of the genomic DNA of four transgene‐positive calli (#1–#4) in the first batch. For each callus, three independent samples were collected and used. Lane 1–3, calli #1; lane 4–6, calli #2; lane 7–9, calli #3; lane 10–12, calli #4; lane 13, control (wild‐type DNA amplicons digested with Pvu II ). M, DNA marker. Primer pair zb7‐F/zb7‐R was used for PCR amplification, Pvu II was used for digestion. (c) Mutation analysis of two transgene‐positive calli by cloning followed by Sanger sequencing. #3, #4 were the two calli sample with homozygous or bi‐allelic mutations by RFLP assay. (d) Sanger sequencing results of mutations in regenerated seedlings. #1–3, #1–6, #3–8, #4–1 were selected seedlings used for analysis. Left, sequencing chromatograph and right, the edited sequences at the target site. The yellow box indicates the Pvu II site.
Figure Legend Snippet: Identification of mutations in the zb7 gene by RFLP assay and Sanger sequencing. (a) Schematic illustration of the target site in the zb7 gene. Black box indicates exons, while the lines between them represent introns. Underlined sequence was selected for targeting; nucleotides marked in blue represent PAM (protospacer adjacent motif). (b) RFLP assay of the genomic DNA of four transgene‐positive calli (#1–#4) in the first batch. For each callus, three independent samples were collected and used. Lane 1–3, calli #1; lane 4–6, calli #2; lane 7–9, calli #3; lane 10–12, calli #4; lane 13, control (wild‐type DNA amplicons digested with Pvu II ). M, DNA marker. Primer pair zb7‐F/zb7‐R was used for PCR amplification, Pvu II was used for digestion. (c) Mutation analysis of two transgene‐positive calli by cloning followed by Sanger sequencing. #3, #4 were the two calli sample with homozygous or bi‐allelic mutations by RFLP assay. (d) Sanger sequencing results of mutations in regenerated seedlings. #1–3, #1–6, #3–8, #4–1 were selected seedlings used for analysis. Left, sequencing chromatograph and right, the edited sequences at the target site. The yellow box indicates the Pvu II site.

Techniques Used: RFLP Assay, Sequencing, Marker, Polymerase Chain Reaction, Amplification, Mutagenesis, Clone Assay

Identification of mutations in the zyp1 gene by RFLP assay and Sanger sequencing. (a) Schematic illustration of the target site in the zyp1 gene. Black box indicates exons, while the lines between them represent introns. Underlined sequence was selected for targeting; nucleotides marked in blue represent PAM . (b) RFLP assay of the genomic DNA of transgenic seedlings. Lane 1, control (wild‐type DNA amplicons digested with Xba I). Lane 2–27, 26 random selected transgenic seedlings. M, DNA marker. Primer pair zyp1‐F/zyp1‐R was used for PCR amplification, Xba I was used for digestion. (c) Sanger sequencing results of three seedlings. Left shows sequencing chromatograph, and right shows the edited sequences at the target site. Nucleotides marked in blue represent PAM . The yellow box indicates the Xba I site.
Figure Legend Snippet: Identification of mutations in the zyp1 gene by RFLP assay and Sanger sequencing. (a) Schematic illustration of the target site in the zyp1 gene. Black box indicates exons, while the lines between them represent introns. Underlined sequence was selected for targeting; nucleotides marked in blue represent PAM . (b) RFLP assay of the genomic DNA of transgenic seedlings. Lane 1, control (wild‐type DNA amplicons digested with Xba I). Lane 2–27, 26 random selected transgenic seedlings. M, DNA marker. Primer pair zyp1‐F/zyp1‐R was used for PCR amplification, Xba I was used for digestion. (c) Sanger sequencing results of three seedlings. Left shows sequencing chromatograph, and right shows the edited sequences at the target site. Nucleotides marked in blue represent PAM . The yellow box indicates the Xba I site.

Techniques Used: RFLP Assay, Sequencing, Transgenic Assay, Marker, Polymerase Chain Reaction, Amplification

29) Product Images from "MT1G is Silenced by DNA Methylation and Contributes to the Pathogenesis of Hepatocellular Carcinoma"

Article Title: MT1G is Silenced by DNA Methylation and Contributes to the Pathogenesis of Hepatocellular Carcinoma

Journal: Journal of Cancer

doi: 10.7150/jca.25680

MT1G silencing is closely linked to DNA methylation. A) MT1G mRNA expression and B) methylation levels in HCC samples compared with the expression in their adjacent non-tumor tissues were analyzed in two genomic screening datasets (mRNA expression dataset: TCGA_LIHC_exp_HiSeqV2-2015-02-24 obtained from Cancer Browser; methylation levels dataset: lihc_tcga.tar obtained from CBioPortal; student's unpaired t-test, n = 50). C) The correlation between MT1G mRNA expression and methylation status was analyzed in HCC samples of the above two datasets (r = -0.4442, n = 371, p-value
Figure Legend Snippet: MT1G silencing is closely linked to DNA methylation. A) MT1G mRNA expression and B) methylation levels in HCC samples compared with the expression in their adjacent non-tumor tissues were analyzed in two genomic screening datasets (mRNA expression dataset: TCGA_LIHC_exp_HiSeqV2-2015-02-24 obtained from Cancer Browser; methylation levels dataset: lihc_tcga.tar obtained from CBioPortal; student's unpaired t-test, n = 50). C) The correlation between MT1G mRNA expression and methylation status was analyzed in HCC samples of the above two datasets (r = -0.4442, n = 371, p-value

Techniques Used: DNA Methylation Assay, Expressing, Methylation

30) Product Images from "Investigation of Plasma cell‐free cancer genome chromosomal instability as a tool for targeted minimally invasive biomarkers for primary liver cancer diagnoses, et al. Investigation of Plasma cell‐free cancer genome chromosomal instability as a tool for targeted minimally invasive biomarkers for primary liver cancer diagnoses"

Article Title: Investigation of Plasma cell‐free cancer genome chromosomal instability as a tool for targeted minimally invasive biomarkers for primary liver cancer diagnoses, et al. Investigation of Plasma cell‐free cancer genome chromosomal instability as a tool for targeted minimally invasive biomarkers for primary liver cancer diagnoses

Journal: Cancer Medicine

doi: 10.1002/cam4.3142

Circulating cancer genome of plasma cell‐free DNA from primary liver cancer patients. Circulating cell‐free chromosomal instability of 23 primary liver cancers (up) and 41 health controls (bottom). Chromosomes 1,2, …, and Y, are plotted from left to right. Each dot indicates the normalized coverage value of a 200K bin. Genes of interested are marked with dash lines.
Figure Legend Snippet: Circulating cancer genome of plasma cell‐free DNA from primary liver cancer patients. Circulating cell‐free chromosomal instability of 23 primary liver cancers (up) and 41 health controls (bottom). Chromosomes 1,2, …, and Y, are plotted from left to right. Each dot indicates the normalized coverage value of a 200K bin. Genes of interested are marked with dash lines.

Techniques Used:

31) Product Images from "Klebsiella pneumoniae SnebYK Mediates Resistance Against Heterodera glycines and Promotes Soybean Growth"

Article Title: Klebsiella pneumoniae SnebYK Mediates Resistance Against Heterodera glycines and Promotes Soybean Growth

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.01134

Amplification and analysis of the nifH gene of SnebYK. (A) Amplicon of SnebYK nifH (363 bp) in the left lane and DNA ladder in the right lane. (B) Transcriptional analysis of the SnebYK nifH gene in nitrogen-free medium determined with RT-PCR. The lane labeled “-N” shows the expression of the 16S rRNA and nifH gene of SnebYK grown in ACCC55 nitrogen-free medium; the lane labeled “+N” shows the expression of the 16S rRNA and nifH gene of SnebYK grown in nutrient agar medium. The transcript level of the 16S rRNA was used as a loading control; the transcript level of nifH of SnebYK grown in nutrient agar medium was used as a negative control. (C) Dendrogram based on the nifH sequences of SnebYK and other strains in the genus Klebsiella with similar nifH sequences. This analysis was performed using the neighbor-joining method in Mega 7.0.26 with a bootstrap value of n = 1000.
Figure Legend Snippet: Amplification and analysis of the nifH gene of SnebYK. (A) Amplicon of SnebYK nifH (363 bp) in the left lane and DNA ladder in the right lane. (B) Transcriptional analysis of the SnebYK nifH gene in nitrogen-free medium determined with RT-PCR. The lane labeled “-N” shows the expression of the 16S rRNA and nifH gene of SnebYK grown in ACCC55 nitrogen-free medium; the lane labeled “+N” shows the expression of the 16S rRNA and nifH gene of SnebYK grown in nutrient agar medium. The transcript level of the 16S rRNA was used as a loading control; the transcript level of nifH of SnebYK grown in nutrient agar medium was used as a negative control. (C) Dendrogram based on the nifH sequences of SnebYK and other strains in the genus Klebsiella with similar nifH sequences. This analysis was performed using the neighbor-joining method in Mega 7.0.26 with a bootstrap value of n = 1000.

Techniques Used: Amplification, Reverse Transcription Polymerase Chain Reaction, Labeling, Expressing, Negative Control

32) Product Images from "CircIBTK inhibits DNA demethylation and activation of AKT signaling pathway via miR-29b in peripheral blood mononuclear cells in systemic lupus erythematosus"

Article Title: CircIBTK inhibits DNA demethylation and activation of AKT signaling pathway via miR-29b in peripheral blood mononuclear cells in systemic lupus erythematosus

Journal: Arthritis Research & Therapy

doi: 10.1186/s13075-018-1618-8

CircIBTK could induce DNA methylation via miR-29b in systemic lupus erythematosus (SLE). a Global DNA methylation in peripheral blood mononuclear cells (PBMCs) from 42 patients with SLE and 35 healthy controls (HC) compared using the unpaired Student’s t test. b , c Correlation between global DNA methylation and expression of circIBTK or miR-29b analyzed with Spearman’s analysis. d DNA methylation in PBMCs from patients with SLE, transfected with miR-29b mimics, circIBTK expression plasmids, NC oligonucleotides, or empty vector. e DNA methylation in PBMCs from HC, transfected with miR-29b inhibitor, circIBTK siRNA or NC oligonucleotides. Results are represented as mean ± SD ( n = 3). * P
Figure Legend Snippet: CircIBTK could induce DNA methylation via miR-29b in systemic lupus erythematosus (SLE). a Global DNA methylation in peripheral blood mononuclear cells (PBMCs) from 42 patients with SLE and 35 healthy controls (HC) compared using the unpaired Student’s t test. b , c Correlation between global DNA methylation and expression of circIBTK or miR-29b analyzed with Spearman’s analysis. d DNA methylation in PBMCs from patients with SLE, transfected with miR-29b mimics, circIBTK expression plasmids, NC oligonucleotides, or empty vector. e DNA methylation in PBMCs from HC, transfected with miR-29b inhibitor, circIBTK siRNA or NC oligonucleotides. Results are represented as mean ± SD ( n = 3). * P

Techniques Used: DNA Methylation Assay, Expressing, Transfection, Plasmid Preparation

33) Product Images from "miR-29c-3p regulates DNMT3B and LATS1 methylation to inhibit tumor progression in hepatocellular carcinoma"

Article Title: miR-29c-3p regulates DNMT3B and LATS1 methylation to inhibit tumor progression in hepatocellular carcinoma

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-1281-7

miR-29c-3p directly targets DNA methyltransferase 3B (DNMT3B) and miR-29c-3p levels were inversely correlated with DNMT3B protein levels. a Venn diagram displaying miR-29c-3p computationally predicted to target DNMT3B by four different prediction algorithms: TargetScan, MiRanda, Oncomir, and miRWalk. b miR-29c-3p expression was negatively correlated with DNMT3B expression in hepatocellular carcinoma (HCC) tissues. Spearman's rank test ( r = −0.751, p
Figure Legend Snippet: miR-29c-3p directly targets DNA methyltransferase 3B (DNMT3B) and miR-29c-3p levels were inversely correlated with DNMT3B protein levels. a Venn diagram displaying miR-29c-3p computationally predicted to target DNMT3B by four different prediction algorithms: TargetScan, MiRanda, Oncomir, and miRWalk. b miR-29c-3p expression was negatively correlated with DNMT3B expression in hepatocellular carcinoma (HCC) tissues. Spearman's rank test ( r = −0.751, p

Techniques Used: Expressing

DNA methyltransferase 3B (DNMT3B) is upregulated and large tumor suppressor gene 1 (LATS1) is downregulated in hepatocellular carcinoma (HCC). a Quantitative real-time PCR (qRT-PCR) analysis of DNMT3B expression in 150 pairs of HCC tissues and paired normal adjacent tissues. b qRT-PCR analysis of LATS1 expression in 150 pairs of HCC tissues and paired normal adjacent tissues. c Immunohistochemical staining analysis of DNMT3B protein expression levels in HCC tissues. d Immunohistochemical staining analysis of LATS1 protein expression levels in HCC tissues. e Kaplan–Meier analysis of overall survival between HCC patients with high and low DNMT3B expression. f Kaplan–Meier analysis of overall survival between high and low LATS1 expression in HCC patients. g Kaplan–Meier analysis of overall survival between the high miR-29c-3p/low DNMT3B/high LATS1 expression group and low miR-29c-3p/high DNMT3B/low LATS1 expression group; ** p
Figure Legend Snippet: DNA methyltransferase 3B (DNMT3B) is upregulated and large tumor suppressor gene 1 (LATS1) is downregulated in hepatocellular carcinoma (HCC). a Quantitative real-time PCR (qRT-PCR) analysis of DNMT3B expression in 150 pairs of HCC tissues and paired normal adjacent tissues. b qRT-PCR analysis of LATS1 expression in 150 pairs of HCC tissues and paired normal adjacent tissues. c Immunohistochemical staining analysis of DNMT3B protein expression levels in HCC tissues. d Immunohistochemical staining analysis of LATS1 protein expression levels in HCC tissues. e Kaplan–Meier analysis of overall survival between HCC patients with high and low DNMT3B expression. f Kaplan–Meier analysis of overall survival between high and low LATS1 expression in HCC patients. g Kaplan–Meier analysis of overall survival between the high miR-29c-3p/low DNMT3B/high LATS1 expression group and low miR-29c-3p/high DNMT3B/low LATS1 expression group; ** p

Techniques Used: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Immunohistochemistry, Staining

Rescue experiments are performed to confirm that DNA methyltransferase 3B (DNMT3B) is the functional target of miR-29c-3p in hepatocellular carcinoma (HCC) progression. a Western blot revealed DNMT3B protein expression in MHCC-97H-miR-29c-3p cells and HepG2-miR-29c-3p that were transfected with DNMT3B vector and NC. b Proliferation of MHCC-97H-miR-29c-3p cells and HepG2-miR-29c-3p cells that were transfected with DNMT3B vector and negative control (NC) was determined by CCK-8 assay. c Wound healing assay was performed to determine the effects of DNMT3B on HCC cell migration. d Colony formation assays assessed the effects of DNMT3B on HCC cell proliferation. e The methylation status of large tumor suppressor gene 1 (LATS1) was detected in MHCC-97H-miR-29c-3p cells and HepG2-miR-29c-3p cells that were transfected with DNMT3B vector and NC. f Western blot revealed the expression of Hippo signaling pathway components, including proliferation- and apoptosis-related indicators in MHCC-97H-miR-29c-3p cells and HepG2-miR-29c-3p cells that were transfected with DNMT3B vector and NC; * p
Figure Legend Snippet: Rescue experiments are performed to confirm that DNA methyltransferase 3B (DNMT3B) is the functional target of miR-29c-3p in hepatocellular carcinoma (HCC) progression. a Western blot revealed DNMT3B protein expression in MHCC-97H-miR-29c-3p cells and HepG2-miR-29c-3p that were transfected with DNMT3B vector and NC. b Proliferation of MHCC-97H-miR-29c-3p cells and HepG2-miR-29c-3p cells that were transfected with DNMT3B vector and negative control (NC) was determined by CCK-8 assay. c Wound healing assay was performed to determine the effects of DNMT3B on HCC cell migration. d Colony formation assays assessed the effects of DNMT3B on HCC cell proliferation. e The methylation status of large tumor suppressor gene 1 (LATS1) was detected in MHCC-97H-miR-29c-3p cells and HepG2-miR-29c-3p cells that were transfected with DNMT3B vector and NC. f Western blot revealed the expression of Hippo signaling pathway components, including proliferation- and apoptosis-related indicators in MHCC-97H-miR-29c-3p cells and HepG2-miR-29c-3p cells that were transfected with DNMT3B vector and NC; * p

Techniques Used: Functional Assay, Western Blot, Expressing, Transfection, Plasmid Preparation, Negative Control, CCK-8 Assay, Wound Healing Assay, Migration, Methylation

Aberrant DNA hypermethylation and expression of large tumor suppressor gene 1 (LATS1) in hepatocellular carcinoma (HCC) and HCC cell lines. a The methylation status of LATS1 was randomly detected in 7 HCC and paired normal adjacent tissues. b The methylation status of LATS1 was detected in LO2, MHCC-97H, HepG2, SMMC-7721, and Huh7cell lines. c Bisulfite sequencing analysis was performed on LATS1 promoter methylation in HCC tissues compared with paired normal adjacent tissues. d The relative mRNA expression of miR-29c-3p in 7 HCC and paired normal adjacent tissues. e The relative mRNA expression of DNA methyltransferase 3B (DNMT3B) in 7 HCC and paired normal adjacent tissues. f The relative mRNA expression of LATS1 in 7 HCC and paired normal adjacent tissues. g The CpG islands (shaded area) of LATS1 promoter region. h YAP and LATS1 protein expression in HCC and paired normal adjacent tissues. i YAP and LATS1 protein expression in HCC and LO2 cells; * p
Figure Legend Snippet: Aberrant DNA hypermethylation and expression of large tumor suppressor gene 1 (LATS1) in hepatocellular carcinoma (HCC) and HCC cell lines. a The methylation status of LATS1 was randomly detected in 7 HCC and paired normal adjacent tissues. b The methylation status of LATS1 was detected in LO2, MHCC-97H, HepG2, SMMC-7721, and Huh7cell lines. c Bisulfite sequencing analysis was performed on LATS1 promoter methylation in HCC tissues compared with paired normal adjacent tissues. d The relative mRNA expression of miR-29c-3p in 7 HCC and paired normal adjacent tissues. e The relative mRNA expression of DNA methyltransferase 3B (DNMT3B) in 7 HCC and paired normal adjacent tissues. f The relative mRNA expression of LATS1 in 7 HCC and paired normal adjacent tissues. g The CpG islands (shaded area) of LATS1 promoter region. h YAP and LATS1 protein expression in HCC and paired normal adjacent tissues. i YAP and LATS1 protein expression in HCC and LO2 cells; * p

Techniques Used: Expressing, Methylation, Methylation Sequencing

34) Product Images from "A Δ-9 Fatty Acid Desaturase Gene in the Microalga Myrmeciaincisa Reisigl: Cloning and Functional Analysis"

Article Title: A Δ-9 Fatty Acid Desaturase Gene in the Microalga Myrmeciaincisa Reisigl: Cloning and Functional Analysis

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms17071143

( A ) Electrophoresis profiles of Δ9 FAD 5′-RACE and 3′-RACE product from Myrmecia incisa . M: D2000 marker; Lane 1: 5′-ends of Δ9 fatty acid desaturase (FAD); Lane 2: 3′-ends of Δ9 FAD; Lanes 3, 4, 5 and 6: the amplification products of Δ9 FAD using DNA as the template; ( B ) Schematic illustration of the gene structure of MiΔ9FAD . Black boxes: extrons; black lines: introns; gray lines: untranslated region (UTR).
Figure Legend Snippet: ( A ) Electrophoresis profiles of Δ9 FAD 5′-RACE and 3′-RACE product from Myrmecia incisa . M: D2000 marker; Lane 1: 5′-ends of Δ9 fatty acid desaturase (FAD); Lane 2: 3′-ends of Δ9 FAD; Lanes 3, 4, 5 and 6: the amplification products of Δ9 FAD using DNA as the template; ( B ) Schematic illustration of the gene structure of MiΔ9FAD . Black boxes: extrons; black lines: introns; gray lines: untranslated region (UTR).

Techniques Used: Electrophoresis, Marker, Amplification

35) Product Images from "The Adhesion of Lactobacillus salivarius REN to a Human Intestinal Epithelial Cell Line Requires S-layer Proteins"

Article Title: The Adhesion of Lactobacillus salivarius REN to a Human Intestinal Epithelial Cell Line Requires S-layer Proteins

Journal: Scientific Reports

doi: 10.1038/srep44029

Identification of S-layer proteins of L. salivarius REN. ( A ) SDS-PAGE of cell surface proteins extracted from L. salivarius REN. ( B ) Gene organization of the region surrounding the cbpA and nam - amidase locus. Arrows indicate the locations of primers used for PCR analysis. The red shaded regions represented the deletion target. The green and blue shaded regions represented the upstream and downstream fragments in target genes. The chromosomal map is not drawn to scale. ( C ) Schematic overview of the construction of Δ cbpA and Δ nam - amidase mutants. ( D ) PCR identification of double crossover recombinant. Lane M: DNA marker DL 2000, lane 1: PCR amplification of cbpA in Δ cbpA mutants, lane 2: PCR amplification of cbpA in wild type strain, lane 3: PCR amplification of nam - amidase in wild type strain, lane 4: PCR amplification of nam - amidase in Δ nam - amidase mutants. ( E ) Adhesion of L. salivarius REN, Δ cbpA and Δ nam - amidase mutants to the HT-29 cells. Data were analyzed by the paired Student’s t test (* P
Figure Legend Snippet: Identification of S-layer proteins of L. salivarius REN. ( A ) SDS-PAGE of cell surface proteins extracted from L. salivarius REN. ( B ) Gene organization of the region surrounding the cbpA and nam - amidase locus. Arrows indicate the locations of primers used for PCR analysis. The red shaded regions represented the deletion target. The green and blue shaded regions represented the upstream and downstream fragments in target genes. The chromosomal map is not drawn to scale. ( C ) Schematic overview of the construction of Δ cbpA and Δ nam - amidase mutants. ( D ) PCR identification of double crossover recombinant. Lane M: DNA marker DL 2000, lane 1: PCR amplification of cbpA in Δ cbpA mutants, lane 2: PCR amplification of cbpA in wild type strain, lane 3: PCR amplification of nam - amidase in wild type strain, lane 4: PCR amplification of nam - amidase in Δ nam - amidase mutants. ( E ) Adhesion of L. salivarius REN, Δ cbpA and Δ nam - amidase mutants to the HT-29 cells. Data were analyzed by the paired Student’s t test (* P

Techniques Used: SDS Page, Polymerase Chain Reaction, Recombinant, Marker, Amplification

36) Product Images from "Multi‐omics analyses reveal epigenomics basis for cotton somatic embryogenesis through successive regeneration acclimation process"

Article Title: Multi‐omics analyses reveal epigenomics basis for cotton somatic embryogenesis through successive regeneration acclimation process

Journal: Plant Biotechnology Journal

doi: 10.1111/pbi.12988

Active RNA ‐directed DNA methylation during the tissue culture and somatic embryogenesis process. (a) The DNA methylation patterns of Rd DM loci and their flanking upstream and downstream 5‐kb regions in NEC , EC and SE . (b) Pie chart showing proportions of overlapped and unique 24‐nt si RNA s during the continuous tissue culture process. (c) The CHH methylation patterns in cell‐specific and constitutive 24‐nt si RNA s in two groups, related to Fig. 5 b. (d) The gene expression levels of Rd DM pathway, demethylation and chromatin modification (left panel, A t ‐subgenome; right panel, D t ‐subgenome). The expression levels are normalized as Log2( FPKM ) for each gene. (e) The expression patterns of Rd DM components, DNA methyltransferases and de‐methyltransferases in NEC , EC and SE . GhUbQ7 was used as the reference gene. Error bars indicate the standard error (S.E) of three biological replicates (Student's t ‐test, * P
Figure Legend Snippet: Active RNA ‐directed DNA methylation during the tissue culture and somatic embryogenesis process. (a) The DNA methylation patterns of Rd DM loci and their flanking upstream and downstream 5‐kb regions in NEC , EC and SE . (b) Pie chart showing proportions of overlapped and unique 24‐nt si RNA s during the continuous tissue culture process. (c) The CHH methylation patterns in cell‐specific and constitutive 24‐nt si RNA s in two groups, related to Fig. 5 b. (d) The gene expression levels of Rd DM pathway, demethylation and chromatin modification (left panel, A t ‐subgenome; right panel, D t ‐subgenome). The expression levels are normalized as Log2( FPKM ) for each gene. (e) The expression patterns of Rd DM components, DNA methyltransferases and de‐methyltransferases in NEC , EC and SE . GhUbQ7 was used as the reference gene. Error bars indicate the standard error (S.E) of three biological replicates (Student's t ‐test, * P

Techniques Used: DNA Methylation Assay, Methylation, Expressing, Modification

Genomic landscape of DNA methylation in cotton somatic embryogenesis process and regenerated plants. (a) Percentages of methylated cytosines ( mC s) in the somatic embryogenesis and regenerated plants. (b) The relative content of mC s in CG , CHG and CHH contexts from seven samples. (c) Circos plot showing gene density, TE density, ratios of CG , CHG and CHH methylation in callus and leaves of regenerated plants using 1‐Mb sliding 200‐kb windows among A t ‐subgenome chromosomes (right) and D t ‐subgenome chromosomes (left). The outer track represents the 26 chromosomes of the G. hirsutum genome. Tracks 1–7 represent NEC , EC , SE , WT , R0, R2 and R4, respectively. (d) Comparison of differentially methylated cytosines ( DMC s) in CG , CHG and CHH contexts in NEC , EC , SE and regenerated plants (R0, R2 and R4). Methylated cytosines observed in callus of three stages of somatic embryogenesis and all regenerated plant leaf are referred to as constitutive cytosines. Methylated cytosines observed in two or three stages/regenerated plants are referred to as varied cytosines. Methylated cytosines observed in only one stage/regenerated plant leaf are referred to as unique cytosines. (e) Percentages of unique si RNA s in the callus and leaf tissues. The 24‐nt unique si RNA s are shown in the green dotted box (Student's t ‐test * P
Figure Legend Snippet: Genomic landscape of DNA methylation in cotton somatic embryogenesis process and regenerated plants. (a) Percentages of methylated cytosines ( mC s) in the somatic embryogenesis and regenerated plants. (b) The relative content of mC s in CG , CHG and CHH contexts from seven samples. (c) Circos plot showing gene density, TE density, ratios of CG , CHG and CHH methylation in callus and leaves of regenerated plants using 1‐Mb sliding 200‐kb windows among A t ‐subgenome chromosomes (right) and D t ‐subgenome chromosomes (left). The outer track represents the 26 chromosomes of the G. hirsutum genome. Tracks 1–7 represent NEC , EC , SE , WT , R0, R2 and R4, respectively. (d) Comparison of differentially methylated cytosines ( DMC s) in CG , CHG and CHH contexts in NEC , EC , SE and regenerated plants (R0, R2 and R4). Methylated cytosines observed in callus of three stages of somatic embryogenesis and all regenerated plant leaf are referred to as constitutive cytosines. Methylated cytosines observed in two or three stages/regenerated plants are referred to as varied cytosines. Methylated cytosines observed in only one stage/regenerated plant leaf are referred to as unique cytosines. (e) Percentages of unique si RNA s in the callus and leaf tissues. The 24‐nt unique si RNA s are shown in the green dotted box (Student's t ‐test * P

Techniques Used: DNA Methylation Assay, Methylation

37) Product Images from "In vitro/vivo Mechanism of Action of MP1102 With Low/Nonresistance Against Streptococcus suis Type 2 Strain CVCC 3928"

Article Title: In vitro/vivo Mechanism of Action of MP1102 With Low/Nonresistance Against Streptococcus suis Type 2 Strain CVCC 3928

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2019.00048

In vitro binding of MP1102 to the S. suis strain CVCC 3928 genomic DNA. (A) Gel retardation analysis of the binding of MP1102 to genomic DNA. Line 1: DNA maker λ DNA/ Hind III; Lines 2–9: the mass ratios of MP1102 and genomic DNA were 0, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0, respectively. (B) CD spectra analysis of genomic DNA. The mass ratios of MP1102 and genomic DNA were 0, 0.5, and 10.0, respectively.
Figure Legend Snippet: In vitro binding of MP1102 to the S. suis strain CVCC 3928 genomic DNA. (A) Gel retardation analysis of the binding of MP1102 to genomic DNA. Line 1: DNA maker λ DNA/ Hind III; Lines 2–9: the mass ratios of MP1102 and genomic DNA were 0, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0, respectively. (B) CD spectra analysis of genomic DNA. The mass ratios of MP1102 and genomic DNA were 0, 0.5, and 10.0, respectively.

Techniques Used: In Vitro, Binding Assay, Electrophoretic Mobility Shift Assay

Mode of actions of MP1102 against S. suis in vitro and its action effects on mice challenged with S. suis . In vitro mechanism of MP1102 against S. suis is spatiotemporal. First, the cell membrane of the pathogen was slightly destroyed by MP1102, and the latter penetrated cell membrane and bound to genomic DNA of S. suis , resulting in DNA destruction, inhibition of DNA replication and cell cycle interference. As a result, severe damage of the membrane occurred, causing leakage of cell contents and cell lysis as an irreversibly comprehensive cyto-event. At the same time, in vivo mechanism of MP1102-protected mice challenged with S. suis could be accordingly deduced as inhibition of bacterial translocation, suppression of proinflammatory cytokines and alleviation of organ injury. These events ultimately contributed to improvement of mouse survival.
Figure Legend Snippet: Mode of actions of MP1102 against S. suis in vitro and its action effects on mice challenged with S. suis . In vitro mechanism of MP1102 against S. suis is spatiotemporal. First, the cell membrane of the pathogen was slightly destroyed by MP1102, and the latter penetrated cell membrane and bound to genomic DNA of S. suis , resulting in DNA destruction, inhibition of DNA replication and cell cycle interference. As a result, severe damage of the membrane occurred, causing leakage of cell contents and cell lysis as an irreversibly comprehensive cyto-event. At the same time, in vivo mechanism of MP1102-protected mice challenged with S. suis could be accordingly deduced as inhibition of bacterial translocation, suppression of proinflammatory cytokines and alleviation of organ injury. These events ultimately contributed to improvement of mouse survival.

Techniques Used: In Vitro, Mouse Assay, Inhibition, Lysis, In Vivo, Translocation Assay

38) Product Images from "The MarR Family Regulator BmrR Is Involved in Bile Tolerance of Bifidobacterium longum BBMN68 via Controlling the Expression of an ABC Transporter"

Article Title: The MarR Family Regulator BmrR Is Involved in Bile Tolerance of Bifidobacterium longum BBMN68 via Controlling the Expression of an ABC Transporter

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.02453-18

In silico analysis and RT-PCR assays to verify the cotranscription of bmrR to bmrB . (A) Linear map of bmrR , bmrA , and bmrB with the genomic DNA flanking these genes in BBMN68. (B) RT-PCR assays to verify the cotranscription of bmrR to bmrB . gDNA, genomic DNA of wild-type BBMN68; + and −, cDNA and RNA, respectively, used as the template for PCR amplification; M, DNA marker. (C) Sequence analysis of the promoter region upstream of the bmrR gene. The putative −35 and −10 sequences and the ribosome binding site (RBS) are marked in red. The putative binding site is shown in italics.
Figure Legend Snippet: In silico analysis and RT-PCR assays to verify the cotranscription of bmrR to bmrB . (A) Linear map of bmrR , bmrA , and bmrB with the genomic DNA flanking these genes in BBMN68. (B) RT-PCR assays to verify the cotranscription of bmrR to bmrB . gDNA, genomic DNA of wild-type BBMN68; + and −, cDNA and RNA, respectively, used as the template for PCR amplification; M, DNA marker. (C) Sequence analysis of the promoter region upstream of the bmrR gene. The putative −35 and −10 sequences and the ribosome binding site (RBS) are marked in red. The putative binding site is shown in italics.

Techniques Used: In Silico, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Marker, Sequencing, Binding Assay

39) Product Images from "The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis"

Article Title: The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis

Journal: Veterinary Research

doi: 10.1186/s13567-018-0589-8

Identification of the mutant strain RA1062. A PCR amplification. M: Takara DL2000 marker; lanes 1–2: R. anatipestifer 16S rRNA was amplified from the WT strain CH3 (lane 1), the mutant strain RA1062 (lane 2), showing a 744-bp fragment of 16S rRNA; lanes 4–5: a 678-bp fragment of M949_RS01035 was amplified from the WT strain CH3 (lane 4), but not the mutant strain RA1062 (lane 5); lanes 7–8: the 644-bp fragment of erm gene was not amplified from the WT strain CH3 (lane 7), but amplified from the mutant strain RA1062 (lane 8); lanes 3, 6 and 9: the avian pathogenic E. coli strain (APEC, CVCC1547), as negative controls. B Southern blot analysis of the transposon Tn4351 insertion. Lane 1, 10 μg of pEP 4351 digested with Xba I (positive control); Lane 2, 10 μg of chromosomal DNA from mutant strain RA1062 digested with Xba I; Lane 3, 10 μg of chromosomal DNA from the WT strain CH3 digested with Xba I (negative control). The digested sample was resolved on a 0.7% agarose gel and Southern blot analysis was performed using a TnDIG-labeled probe. C Schematic chart of Tn4351 insertion in RA1062 chromosome at 318 bp of the gene, which is 678 nucleotides in length. D qPCR analysis. The expression of the mRNAs were expressed as fold change and calculated using the comparative C T (2 −∆∆CT ) method. Data were normalized to the housekeeping gene ldh and expressed as fold changes. The expression of M949_RS01035 in the mutant strain RA1062 was disrupted. However, no change was shown for its upstream M949_RS10475 gene and downstream M949_RS01030 gene. Error bars represent standard deviations from three replicates (*** p
Figure Legend Snippet: Identification of the mutant strain RA1062. A PCR amplification. M: Takara DL2000 marker; lanes 1–2: R. anatipestifer 16S rRNA was amplified from the WT strain CH3 (lane 1), the mutant strain RA1062 (lane 2), showing a 744-bp fragment of 16S rRNA; lanes 4–5: a 678-bp fragment of M949_RS01035 was amplified from the WT strain CH3 (lane 4), but not the mutant strain RA1062 (lane 5); lanes 7–8: the 644-bp fragment of erm gene was not amplified from the WT strain CH3 (lane 7), but amplified from the mutant strain RA1062 (lane 8); lanes 3, 6 and 9: the avian pathogenic E. coli strain (APEC, CVCC1547), as negative controls. B Southern blot analysis of the transposon Tn4351 insertion. Lane 1, 10 μg of pEP 4351 digested with Xba I (positive control); Lane 2, 10 μg of chromosomal DNA from mutant strain RA1062 digested with Xba I; Lane 3, 10 μg of chromosomal DNA from the WT strain CH3 digested with Xba I (negative control). The digested sample was resolved on a 0.7% agarose gel and Southern blot analysis was performed using a TnDIG-labeled probe. C Schematic chart of Tn4351 insertion in RA1062 chromosome at 318 bp of the gene, which is 678 nucleotides in length. D qPCR analysis. The expression of the mRNAs were expressed as fold change and calculated using the comparative C T (2 −∆∆CT ) method. Data were normalized to the housekeeping gene ldh and expressed as fold changes. The expression of M949_RS01035 in the mutant strain RA1062 was disrupted. However, no change was shown for its upstream M949_RS10475 gene and downstream M949_RS01030 gene. Error bars represent standard deviations from three replicates (*** p

Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, Marker, Southern Blot, Positive Control, Negative Control, Agarose Gel Electrophoresis, Labeling, Real-time Polymerase Chain Reaction, Expressing

40) Product Images from "Improvement and Evaluation of Loop-Mediated Isothermal Amplification for Rapid Detection of Toxoplasma gondii Infection in Human Blood Samples"

Article Title: Improvement and Evaluation of Loop-Mediated Isothermal Amplification for Rapid Detection of Toxoplasma gondii Infection in Human Blood Samples

Journal: PLoS ONE

doi: 10.1371/journal.pone.0169125

Optimization ofLAMP reactions. LAMP reaction were carried out using genomic DNA from tachyzoites under various conditions, as different reaction temperatures (A), reaction times (B), Mg 2+ concentrations (C) and betine concentrations (D). Lane (M), DNA ladder.
Figure Legend Snippet: Optimization ofLAMP reactions. LAMP reaction were carried out using genomic DNA from tachyzoites under various conditions, as different reaction temperatures (A), reaction times (B), Mg 2+ concentrations (C) and betine concentrations (D). Lane (M), DNA ladder.

Techniques Used:

Specificity of LAMP. LAMP reaction was monitored for DNA amplification of Eimeriatenella (lane 1) , Toxoplasma gondii (lane 2) , Trypanosoma evansi (lane 3) , Cryptosporidium parvum (lane 4) and Neosporacaninum (lane 5) by gel electrophoresis. Lane (M), DNA ladder.
Figure Legend Snippet: Specificity of LAMP. LAMP reaction was monitored for DNA amplification of Eimeriatenella (lane 1) , Toxoplasma gondii (lane 2) , Trypanosoma evansi (lane 3) , Cryptosporidium parvum (lane 4) and Neosporacaninum (lane 5) by gel electrophoresis. Lane (M), DNA ladder.

Techniques Used: Amplification, Nucleic Acid Electrophoresis

Sensitivity of the LAMP method. LAMP reactions were carried out using genomic DNA from various tachyzoite(s)mixed with 200 μL fresh human blood sample (A) and genomic DNA from single tachyzoite in PBS (B) as the template.
Figure Legend Snippet: Sensitivity of the LAMP method. LAMP reactions were carried out using genomic DNA from various tachyzoite(s)mixed with 200 μL fresh human blood sample (A) and genomic DNA from single tachyzoite in PBS (B) as the template.

Techniques Used:

The sensitivity of LAMP was checked. (A) LAMP reaction with different copies of the recombinant plasmid as the template.Lane (M), DNA ladder. (B) LAMP reaction with different copies of the recombinant plasmid was monitoredon the LoopampRealtimeTuribidimeter.
Figure Legend Snippet: The sensitivity of LAMP was checked. (A) LAMP reaction with different copies of the recombinant plasmid as the template.Lane (M), DNA ladder. (B) LAMP reaction with different copies of the recombinant plasmid was monitoredon the LoopampRealtimeTuribidimeter.

Techniques Used: Recombinant, Plasmid Preparation

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DNA Extraction:

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Article Snippet: .. DNA extraction and PCR analysis When the engineered hairy roots grew to about 5 cm, genomic DNA was isolated from hairy root samples by using DNA pure Plant Kit (Tiangen Biotech Co., Ltd, Beijing, China), which was used in PCR analysis for detecting the presence of AaPMT and/or AaTRI in transgenic hairy root cultures (Table ). .. RNA extraction and gene expression analysis by RT-PCR and real-time fluorescence quantitative analysis The transgenic hairy roots identified by PCR analysis were chosen and inoculated into 150 mL 1/2MS liquid nutrient media (pH 5.8) in 250 mL conical flasks on a gyratory shaker operating at 100 rpm at 27°C in darkness for RT-PCR and real-time fluorescence quantitative after 60 days' culture.

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Transfection:

Article Title: CRISPR-offinder: a CRISPR guide RNA design and off-target searching tool for user-defined protospacer adjacent motif
Article Snippet: .. After transfection, cells were incubated for 48 h at 37 ℃ and genomic DNA was extracted using the TIANamp Genomic DNA Kit (Tiangen Biotech). .. The target locus was amplified by 32 cycles of PCR with the TaKaRa LA Taq kit (TaKaRa) using primers specific to each locus.

Methylation:

Article Title: Epigenetic down regulation of G protein-coupled estrogen receptor (GPER) functions as a tumor suppressor in colorectal cancer
Article Snippet: .. Bisulfite genomic DNA sequencing To analyze the methylation of GPER promoter, genomic DNA of CRC cells was prepared using TIANamp Genomic DNA kit (TIANGEN), followed by the treatment of sodium bisulfite using the Epitect Bisulfite DNA kit (QIAGEN, cat: 59824). .. Products were amplified by PCR primer pairs used to recognize the bisulfite-modified regions (-781 to -461) of the GPER promoter as: forward 5’- TTG AAG TTT TTT TTT GAG GAA-3’, reverse 5’- TAA TAA CCT CTT CCC CACC-3’.

Mutagenesis:

Article Title: Deletion of CGLD1 Impairs PSII and Increases Singlet Oxygen Tolerance of Green Alga Chlamydomonas reinhardtii
Article Snippet: .. For DNA blot analysis, genomic DNA was isolated from wild-type (CC400) and x32 mutant using the Plant Genomic DNA Kit by following the manufacturer’s instructions (Tiangen Biotech; Beijing, China). .. About 10 μg of genomic DNA was digested overnight with the restriction endonucleases Kpn I and Hind III (New England Biolabs).

Isolation:

Article Title: Genome wide analyses uncover allele-specific RNA editing in human and mouse
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Article Title: Co-expression of AaPMT and AaTRI effectively enhances the yields of tropane alkaloids in Anisodus acutangulus hairy roots
Article Snippet: .. DNA extraction and PCR analysis When the engineered hairy roots grew to about 5 cm, genomic DNA was isolated from hairy root samples by using DNA pure Plant Kit (Tiangen Biotech Co., Ltd, Beijing, China), which was used in PCR analysis for detecting the presence of AaPMT and/or AaTRI in transgenic hairy root cultures (Table ). .. RNA extraction and gene expression analysis by RT-PCR and real-time fluorescence quantitative analysis The transgenic hairy roots identified by PCR analysis were chosen and inoculated into 150 mL 1/2MS liquid nutrient media (pH 5.8) in 250 mL conical flasks on a gyratory shaker operating at 100 rpm at 27°C in darkness for RT-PCR and real-time fluorescence quantitative after 60 days' culture.

Article Title: Deletion of CGLD1 Impairs PSII and Increases Singlet Oxygen Tolerance of Green Alga Chlamydomonas reinhardtii
Article Snippet: .. For DNA blot analysis, genomic DNA was isolated from wild-type (CC400) and x32 mutant using the Plant Genomic DNA Kit by following the manufacturer’s instructions (Tiangen Biotech; Beijing, China). .. About 10 μg of genomic DNA was digested overnight with the restriction endonucleases Kpn I and Hind III (New England Biolabs).

Genomic Sequencing:

Article Title: Evolutionary forces on A-to-I RNA editing revealed by sequencing individual honeybee drones
Article Snippet: .. Genomic sequencing of four drone individuals To efficiently exclude the SNPs from RNA editing sites, we extracted genomic DNA from abdomen tissues from each drone individual mentioned above with the Genomic DNA Extraction Kit (TIANGEN) separately, following the manufacturer’s instructions. .. The library preparation and sequencing were performed in Biomedical Pioneering Innovation Center, Peking University (Illumina HiSeq-2500 sequencer; run type: paired-end; read length: 100 nt).

Transgenic Assay:

Article Title: Enhancement of grain number per spike by RNA interference of cytokinin oxidase 2 gene in bread wheat
Article Snippet: .. Genomic DNA was extracted from leaves of T3 transgenic plants with single copies selected preliminarily by Kana and non-transgenic plants using plant genomic DNA Extraction Kit (Tiangen Biotech, Beijing, China). .. About 20 μg of DNA was successfully digested with 5 U of EcoRV and incubated at 37 °C for 24 h. The digested genomic DNA fragments were separated on a 0.8% ( w / v ) agarose gel, and transferred onto Zeta-Probe GT nylon membrane (Bio-Rad, Hercules, CA, USA).

Article Title: Co-expression of AaPMT and AaTRI effectively enhances the yields of tropane alkaloids in Anisodus acutangulus hairy roots
Article Snippet: .. DNA extraction and PCR analysis When the engineered hairy roots grew to about 5 cm, genomic DNA was isolated from hairy root samples by using DNA pure Plant Kit (Tiangen Biotech Co., Ltd, Beijing, China), which was used in PCR analysis for detecting the presence of AaPMT and/or AaTRI in transgenic hairy root cultures (Table ). .. RNA extraction and gene expression analysis by RT-PCR and real-time fluorescence quantitative analysis The transgenic hairy roots identified by PCR analysis were chosen and inoculated into 150 mL 1/2MS liquid nutrient media (pH 5.8) in 250 mL conical flasks on a gyratory shaker operating at 100 rpm at 27°C in darkness for RT-PCR and real-time fluorescence quantitative after 60 days' culture.

Incubation:

Article Title: CRISPR-offinder: a CRISPR guide RNA design and off-target searching tool for user-defined protospacer adjacent motif
Article Snippet: .. After transfection, cells were incubated for 48 h at 37 ℃ and genomic DNA was extracted using the TIANamp Genomic DNA Kit (Tiangen Biotech). .. The target locus was amplified by 32 cycles of PCR with the TaKaRa LA Taq kit (TaKaRa) using primers specific to each locus.

DNA Sequencing:

Article Title: Epigenetic down regulation of G protein-coupled estrogen receptor (GPER) functions as a tumor suppressor in colorectal cancer
Article Snippet: .. Bisulfite genomic DNA sequencing To analyze the methylation of GPER promoter, genomic DNA of CRC cells was prepared using TIANamp Genomic DNA kit (TIANGEN), followed by the treatment of sodium bisulfite using the Epitect Bisulfite DNA kit (QIAGEN, cat: 59824). .. Products were amplified by PCR primer pairs used to recognize the bisulfite-modified regions (-781 to -461) of the GPER promoter as: forward 5’- TTG AAG TTT TTT TTT GAG GAA-3’, reverse 5’- TAA TAA CCT CTT CCC CACC-3’.

Polymerase Chain Reaction:

Article Title: Co-expression of AaPMT and AaTRI effectively enhances the yields of tropane alkaloids in Anisodus acutangulus hairy roots
Article Snippet: .. DNA extraction and PCR analysis When the engineered hairy roots grew to about 5 cm, genomic DNA was isolated from hairy root samples by using DNA pure Plant Kit (Tiangen Biotech Co., Ltd, Beijing, China), which was used in PCR analysis for detecting the presence of AaPMT and/or AaTRI in transgenic hairy root cultures (Table ). .. RNA extraction and gene expression analysis by RT-PCR and real-time fluorescence quantitative analysis The transgenic hairy roots identified by PCR analysis were chosen and inoculated into 150 mL 1/2MS liquid nutrient media (pH 5.8) in 250 mL conical flasks on a gyratory shaker operating at 100 rpm at 27°C in darkness for RT-PCR and real-time fluorescence quantitative after 60 days' culture.

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  • 91
    tiangen biotech co direct sequencing genomic dna
    Detection of BRAF gene mutation at exons 11 and 15. (A) Gel electrophoresis of the <t>PCR</t> products of exon 11 and 15 of the BRAF gene. The PCR product size of exon 11 is 356 bp, and that of exon 15 is 249 bp. Part of a sequence chromatogram from the Sanger sequencing of BRAF from (B) a patient and (C) the positive control. The red arrow indicates a BRAF mutation (T1799A) of exon 15 in the BCPAP papillary thyroid cancer cell line. BRAF, B-Raf proto-oncogene, serine/threonine kinase; PCR, polymerase chain reaction; M, <t>DNA</t> marker; NE, normal endometrium; EU, eutopic endometrium of endometriosis; EC, ectopic endometrium of endometriosis.
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    tiangen biotech co bacterial dna bacteria genomic dna
    The effect of <t>SML</t> on zeta potential distribution of L. monocytogenes . ( a ) L. monocytogenes , without exposure to SML for 24 h; ( b ) L. monocytogenes , with exposure to SML for 24 h. Interaction of <t>DNA</t> of L. monocytogents with increasing amounts of SML; ( c ) Ultraviolet spectroscopic measurements.
    Bacterial Dna Bacteria Genomic Dna, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co dna genomic dnas
    Profiling of N 6 -adenine methylation in adipocyte differentiation of 3T3-L1 cells. ( a ) Staining of cellular lipid of preadipocytes on Day(0), differentiating adipocytes on Day(+4), and mature adipocytes on Day(+8). Lipid staining was performed with Bodipy 493/503 (Green) and Oil red O (Red), nuclei were co-stained with Hoechst 33342 (Blue). ( b ) Expression profiles of Cebpa and Pparg were determined by qRT-PCR, in relative to the expression of β-actin . ( c ) Workflow to quantify 6 mA abundance of genome <t>DNA</t> sample. ( d ) Dot blot assay of N 6 -adenine methylation. NC, a DNA oligo without N 6 -adenine methylation. 6mA-oligo, a DNA oligo containing one N 6 -adenine methylation. Genome DNA, genome <t>DNAs</t> extracted from 3T3-L1 preadipocytes. ( e,f ) Abundances of 6 mA and 5mC methylation were quantified using HPLC-MS/MS analysis. Molar ratios of 6 mA to dA were calculated, and molar ratios of 5mC to dC were calculated. Data are presented as mean ± s.d, n > 3 independent assay. *P
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    tiangen biotech co genomic dna
    Numbers of different types of <t>DNA-RNA</t> differences detected in heads, thoraxes, or abdomens of honeybees.
    Genomic Dna, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 94/100, based on 916 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Detection of BRAF gene mutation at exons 11 and 15. (A) Gel electrophoresis of the PCR products of exon 11 and 15 of the BRAF gene. The PCR product size of exon 11 is 356 bp, and that of exon 15 is 249 bp. Part of a sequence chromatogram from the Sanger sequencing of BRAF from (B) a patient and (C) the positive control. The red arrow indicates a BRAF mutation (T1799A) of exon 15 in the BCPAP papillary thyroid cancer cell line. BRAF, B-Raf proto-oncogene, serine/threonine kinase; PCR, polymerase chain reaction; M, DNA marker; NE, normal endometrium; EU, eutopic endometrium of endometriosis; EC, ectopic endometrium of endometriosis.

    Journal: International Journal of Molecular Medicine

    Article Title: Analysis of the oncogene BRAF mutation and the correlation of the expression of wild-type BRAF and CREB1 in endometriosis

    doi: 10.3892/ijmm.2017.3342

    Figure Lengend Snippet: Detection of BRAF gene mutation at exons 11 and 15. (A) Gel electrophoresis of the PCR products of exon 11 and 15 of the BRAF gene. The PCR product size of exon 11 is 356 bp, and that of exon 15 is 249 bp. Part of a sequence chromatogram from the Sanger sequencing of BRAF from (B) a patient and (C) the positive control. The red arrow indicates a BRAF mutation (T1799A) of exon 15 in the BCPAP papillary thyroid cancer cell line. BRAF, B-Raf proto-oncogene, serine/threonine kinase; PCR, polymerase chain reaction; M, DNA marker; NE, normal endometrium; EU, eutopic endometrium of endometriosis; EC, ectopic endometrium of endometriosis.

    Article Snippet: Genomic DNA isolation, PCR and direct sequencing Genomic DNA was extracted from freshly frozen tissue (100 mg) using the TIANamp Genomic DNA kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer's protocol.

    Techniques: Mutagenesis, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Sequencing, Positive Control, Marker

    The effect of SML on zeta potential distribution of L. monocytogenes . ( a ) L. monocytogenes , without exposure to SML for 24 h; ( b ) L. monocytogenes , with exposure to SML for 24 h. Interaction of DNA of L. monocytogents with increasing amounts of SML; ( c ) Ultraviolet spectroscopic measurements.

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Lipase-Catalyzed Synthesis of Sucrose Monolaurate and Its Antibacterial Property and Mode of Action against Four Pathogenic Bacteria

    doi: 10.3390/molecules23051118

    Figure Lengend Snippet: The effect of SML on zeta potential distribution of L. monocytogenes . ( a ) L. monocytogenes , without exposure to SML for 24 h; ( b ) L. monocytogenes , with exposure to SML for 24 h. Interaction of DNA of L. monocytogents with increasing amounts of SML; ( c ) Ultraviolet spectroscopic measurements.

    Article Snippet: Effect of SML on Bacterial DNA Bacteria genomic DNA was extracted by using TIANamp Bacteria DNA Kit (Tiangen Biotech, Co., Ltd., Beijing, China).

    Techniques:

    Profiling of N 6 -adenine methylation in adipocyte differentiation of 3T3-L1 cells. ( a ) Staining of cellular lipid of preadipocytes on Day(0), differentiating adipocytes on Day(+4), and mature adipocytes on Day(+8). Lipid staining was performed with Bodipy 493/503 (Green) and Oil red O (Red), nuclei were co-stained with Hoechst 33342 (Blue). ( b ) Expression profiles of Cebpa and Pparg were determined by qRT-PCR, in relative to the expression of β-actin . ( c ) Workflow to quantify 6 mA abundance of genome DNA sample. ( d ) Dot blot assay of N 6 -adenine methylation. NC, a DNA oligo without N 6 -adenine methylation. 6mA-oligo, a DNA oligo containing one N 6 -adenine methylation. Genome DNA, genome DNAs extracted from 3T3-L1 preadipocytes. ( e,f ) Abundances of 6 mA and 5mC methylation were quantified using HPLC-MS/MS analysis. Molar ratios of 6 mA to dA were calculated, and molar ratios of 5mC to dC were calculated. Data are presented as mean ± s.d, n > 3 independent assay. *P

    Journal: Scientific Reports

    Article Title: Regulation of Adipocyte Differentiation by METTL4, a 6 mA Methylase

    doi: 10.1038/s41598-020-64873-w

    Figure Lengend Snippet: Profiling of N 6 -adenine methylation in adipocyte differentiation of 3T3-L1 cells. ( a ) Staining of cellular lipid of preadipocytes on Day(0), differentiating adipocytes on Day(+4), and mature adipocytes on Day(+8). Lipid staining was performed with Bodipy 493/503 (Green) and Oil red O (Red), nuclei were co-stained with Hoechst 33342 (Blue). ( b ) Expression profiles of Cebpa and Pparg were determined by qRT-PCR, in relative to the expression of β-actin . ( c ) Workflow to quantify 6 mA abundance of genome DNA sample. ( d ) Dot blot assay of N 6 -adenine methylation. NC, a DNA oligo without N 6 -adenine methylation. 6mA-oligo, a DNA oligo containing one N 6 -adenine methylation. Genome DNA, genome DNAs extracted from 3T3-L1 preadipocytes. ( e,f ) Abundances of 6 mA and 5mC methylation were quantified using HPLC-MS/MS analysis. Molar ratios of 6 mA to dA were calculated, and molar ratios of 5mC to dC were calculated. Data are presented as mean ± s.d, n > 3 independent assay. *P

    Article Snippet: Enzymatic hydrolysis of genomic DNA Genomic DNAs were isolated and treated first by RNase A for 12 hr at 50 °C, purified using TIANquick Maxi Purification Kit (TIANGEN).

    Techniques: Methylation, Staining, Expressing, Quantitative RT-PCR, Dot Blot, High Performance Liquid Chromatography, Tandem Mass Spectroscopy

    Numbers of different types of DNA-RNA differences detected in heads, thoraxes, or abdomens of honeybees.

    Journal: bioRxiv

    Article Title: Evolutionary forces on A-to-I RNA editing revealed by sequencing individual honeybee drones

    doi: 10.1101/2020.01.15.907287

    Figure Lengend Snippet: Numbers of different types of DNA-RNA differences detected in heads, thoraxes, or abdomens of honeybees.

    Article Snippet: Genomic sequencing of four drone individuals To efficiently exclude the SNPs from RNA editing sites, we extracted genomic DNA from abdomen tissues from each drone individual mentioned above with the Genomic DNA Extraction Kit (TIANGEN) separately, following the manufacturer’s instructions.

    Techniques:

    Identification and annotation of the A-to-I RNA editing sites in the honeybee. (A) Workflow of sample collection, dissection and library construction. (B) Schematic diagram illustrating the identification of A-to-I RNA editing sites. (C) Numbers of different types of DNA-RNA differences detected in honeybee. (D) A summary of A-to-I editing sites according to their functional annotations.

    Journal: bioRxiv

    Article Title: Evolutionary forces on A-to-I RNA editing revealed by sequencing individual honeybee drones

    doi: 10.1101/2020.01.15.907287

    Figure Lengend Snippet: Identification and annotation of the A-to-I RNA editing sites in the honeybee. (A) Workflow of sample collection, dissection and library construction. (B) Schematic diagram illustrating the identification of A-to-I RNA editing sites. (C) Numbers of different types of DNA-RNA differences detected in honeybee. (D) A summary of A-to-I editing sites according to their functional annotations.

    Article Snippet: Genomic sequencing of four drone individuals To efficiently exclude the SNPs from RNA editing sites, we extracted genomic DNA from abdomen tissues from each drone individual mentioned above with the Genomic DNA Extraction Kit (TIANGEN) separately, following the manufacturer’s instructions.

    Techniques: Dissection, Functional Assay