Structured Review

tiangen biotech co genomic dna
Promoter methylation and histone H3 deacetylation were involved in <t>GPER</t> 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 <t>DNA</t> 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
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1) 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

2) 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

3) 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

4) 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

5) 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

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 "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

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 "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

10) 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

11) 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

12) 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

13) 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

14) 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

15) 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

16) 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

17) 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

18) 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

19) 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

20) 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

21) 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

22) 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

23) 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

24) 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

25) 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

26) 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

27) 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

28) 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

29) 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

30) 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

31) Product Images from "Chimerism in piglets developed from aggregated cloned embryos"

Article Title: Chimerism in piglets developed from aggregated cloned embryos

Journal: FEBS Open Bio

doi: 10.1002/2211-5463.12037

Genotype identification of chimeric piglets by PCR analysis. GAPDH was included as a loading control. (A) SRY gene amplification. Lanes 1, 3, 5, 7, 9, 11, 13, and 15: amplification of genomic DNA from the ears of piglets 2453, 2455, 2457, 2459, 2460, 2461, 2462, and 2463, respectively; lanes 2, 4, 6, 8, 10, 12, 14, and 16: amplification of tail genomic DNA from piglets 2453, 2455, 2457, 2459, 2460, 2461, 2462, and 2463, respectively; lanes 17, 18 and 19: amplification of genomic DNA from EGFP‐expressing cells, tdTomato‐transfected cells and ddH 2 O (negative control), respectively. (B) The EGFP gene and tdTomato gene were amplified from the genomic DNA of newborn piglets. Lanes 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21: the EGFP gene was detected in the ear genomic DNA from piglets 2453, 2455, 2457, 2459, 2460, 2461, 2462, and 2463; EGFP expressing cells; tdTomato‐transfected cells; and ddH 2 O (negative control), respectively; lanes 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22: the tdTomato gene was detected in the ear genomic DNA from piglets 2453, 2455, 2457, 2459, 2460, 2461, 2462, and 2463; EGFP‐expressing cells; tdTomato‐transfected cells; and ddH 2 O (negative control), respectively. (C) The EGFP gene and the tdTomato gene were amplified from genomic DNA from different tissues of Rature. Lanes 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19: the EGFP gene was detected in the genomic DNA from liver, lung, heart, kidney, spleen, skin, testis, EGFP‐expressing cells, tdTomato‐transfected cells and ddH 2 O (negative control), respectively; lanes 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20: the tdTomato gene was detected in the genomic DNA from liver, lung, heart, kidney, spleen, skin, testis, EGFP‐expressing cells, tdTomato‐transfected cells, and ddH 2 O (negative control), respectively.
Figure Legend Snippet: Genotype identification of chimeric piglets by PCR analysis. GAPDH was included as a loading control. (A) SRY gene amplification. Lanes 1, 3, 5, 7, 9, 11, 13, and 15: amplification of genomic DNA from the ears of piglets 2453, 2455, 2457, 2459, 2460, 2461, 2462, and 2463, respectively; lanes 2, 4, 6, 8, 10, 12, 14, and 16: amplification of tail genomic DNA from piglets 2453, 2455, 2457, 2459, 2460, 2461, 2462, and 2463, respectively; lanes 17, 18 and 19: amplification of genomic DNA from EGFP‐expressing cells, tdTomato‐transfected cells and ddH 2 O (negative control), respectively. (B) The EGFP gene and tdTomato gene were amplified from the genomic DNA of newborn piglets. Lanes 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21: the EGFP gene was detected in the ear genomic DNA from piglets 2453, 2455, 2457, 2459, 2460, 2461, 2462, and 2463; EGFP expressing cells; tdTomato‐transfected cells; and ddH 2 O (negative control), respectively; lanes 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22: the tdTomato gene was detected in the ear genomic DNA from piglets 2453, 2455, 2457, 2459, 2460, 2461, 2462, and 2463; EGFP‐expressing cells; tdTomato‐transfected cells; and ddH 2 O (negative control), respectively. (C) The EGFP gene and the tdTomato gene were amplified from genomic DNA from different tissues of Rature. Lanes 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19: the EGFP gene was detected in the genomic DNA from liver, lung, heart, kidney, spleen, skin, testis, EGFP‐expressing cells, tdTomato‐transfected cells and ddH 2 O (negative control), respectively; lanes 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20: the tdTomato gene was detected in the genomic DNA from liver, lung, heart, kidney, spleen, skin, testis, EGFP‐expressing cells, tdTomato‐transfected cells, and ddH 2 O (negative control), respectively.

Techniques Used: Polymerase Chain Reaction, Amplification, Expressing, Transfection, Negative Control

32) Product Images from "Over-Expression of a Tobacco Nitrate Reductase Gene in Wheat (Triticum aestivum L.) Increases Seed Protein Content and Weight without Augmenting Nitrogen Supplying"

Article Title: Over-Expression of a Tobacco Nitrate Reductase Gene in Wheat (Triticum aestivum L.) Increases Seed Protein Content and Weight without Augmenting Nitrogen Supplying

Journal: PLoS ONE

doi: 10.1371/journal.pone.0074678

PCR and Southern analysis of PCR products identification of T 0 transformants of wheat. PCR ( a, b ) and Southern analysis of PCR products( c, d ) detection of npt II + nos fragment in G418-resistant T 0 transformants of ND146 ( a, c ) and JM6358 ( b, d ). In a and b : M: Molecular weight DNA markers (λDNA/ Eco R I + Hin d III). Lanes 1-14: G418-resistant T 0 plants from independent transformation events. P: Vector plasmid. C: Control (untransformed plant). H: H 2 O (PCR mix without DNA). The arrow indicates the 735 bp fragment of npt II + nos . In c and d : Lanes 1-9: PCR-positive T 0 plants from independent transformation events. P: Vector plasmid. C: Control (untransformed plant).
Figure Legend Snippet: PCR and Southern analysis of PCR products identification of T 0 transformants of wheat. PCR ( a, b ) and Southern analysis of PCR products( c, d ) detection of npt II + nos fragment in G418-resistant T 0 transformants of ND146 ( a, c ) and JM6358 ( b, d ). In a and b : M: Molecular weight DNA markers (λDNA/ Eco R I + Hin d III). Lanes 1-14: G418-resistant T 0 plants from independent transformation events. P: Vector plasmid. C: Control (untransformed plant). H: H 2 O (PCR mix without DNA). The arrow indicates the 735 bp fragment of npt II + nos . In c and d : Lanes 1-9: PCR-positive T 0 plants from independent transformation events. P: Vector plasmid. C: Control (untransformed plant).

Techniques Used: Polymerase Chain Reaction, Molecular Weight, Transformation Assay, Plasmid Preparation

33) Product Images from "An Efficient CRISPR/Cas9 Platform for Rapidly Generating Simultaneous Mutagenesis of Multiple Gene Homoeologs in Allotetraploid Oilseed Rape"

Article Title: An Efficient CRISPR/Cas9 Platform for Rapidly Generating Simultaneous Mutagenesis of Multiple Gene Homoeologs in Allotetraploid Oilseed Rape

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2018.00442

High-throughput sequencing analysis of CRISPR/Cas9-Induced mutagenesis in three BnSPL3 homoeologs (A–C) , statistical analysis of editing frequency occurred in rapeseed SPL3A, SPL3B , and SPL3C genomic sites, respectively. Rapeseed sample 1#, 2#, 20#, 24#, 41# were from independent transgenic events. Excepting transgenic line 24#, all the rest samples display obvious heteroduplexed DNA bands in PAGE-based assays. (D–F) Sequences alignment of different mutation types identified from high-throughput sequencing analysis.
Figure Legend Snippet: High-throughput sequencing analysis of CRISPR/Cas9-Induced mutagenesis in three BnSPL3 homoeologs (A–C) , statistical analysis of editing frequency occurred in rapeseed SPL3A, SPL3B , and SPL3C genomic sites, respectively. Rapeseed sample 1#, 2#, 20#, 24#, 41# were from independent transgenic events. Excepting transgenic line 24#, all the rest samples display obvious heteroduplexed DNA bands in PAGE-based assays. (D–F) Sequences alignment of different mutation types identified from high-throughput sequencing analysis.

Techniques Used: Next-Generation Sequencing, CRISPR, Mutagenesis, Transgenic Assay, Polyacrylamide Gel Electrophoresis

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Clone Assay:

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Amplification:

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Synthesized:

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Construct:

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SYBR Green Assay:

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Incubation:

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Article Snippet: After incubation at 30°C for 18 to 22 h, transformants were analyzed by colony PCR. .. Putative pXO1-cured clones were selected, and genomic DNA was purified with TIANamp Bacteria DNA kit (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions for isolating genomic DNA from gram-positive bacteria.

Article Title: The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis
Article Snippet: Following overnight incubation at 30 °C, the bacteria were scraped off the filter, resuspended in 5 mL 10 mM MgSO4 , and spread on TSA containing erythromycin and kanamycin to select for transconjugants. .. Briefly, genomic DNA of the mutant strain RA1062 was extracted using the TIANamp Bacteria DNA kit (Tiangen), digested with the endonuclease Xba Ι, then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane.

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.

Article Title: Phytophthora methylomes are modulated by 6mA methyltransferases and associated with adaptive genome regions
Article Snippet: Genomic DNA of P. sojae and P. infestans were extracted using TIANGEN DNAsecure Plant kit. .. The membrane was blocked in 5% milk PBST for 1 h at room temperature and then incubated with 6mA antibody (sysy202003) in 5% milk PBST overnight at 4 °C.

Infection:

Article Title: The heat shock protein 90 of Toxoplasma gondii is essential for invasion of host cells and tachyzoite growth
Article Snippet: The noninvasive tachyzoites were removed after infection for 2 h and fresh medium was added to the cells. .. Next, 24, 48, 72, and 96 h post-infection (PI), the parasites were collected and genomic DNA was extracted using the TIANGEN genomic DNA isolation kit by following the manufacturer’s protocol (TIANGEN, Beijing).

Modification:

Article Title: Analysis of promoter methylation and epigenetic regulation of miR-32 in colorectal cancer cells
Article Snippet: Genomic DNA was extracted from the HT-29, HCT-116 and NCM460 cells using the TIANamp Genomic DNA kit (cat. no. DP304; Tiangen Biotech, Beijing, China). .. It was subjected to bisulfite modification using the EpiTect Bisulfite kit (cat. no. 59826; Qiagen GmBH, Hilden, Germany), according to the manufacturer's protocol.

Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
Article Snippet: Confirmation test using human cancer cell lines and clinical samples To further evaluate the detection efficiency of the modified PR-PCR in realistic samples, two breast cancer cell lines HCC1937(ATCC#CRL-2336) and MCF-7(ATCC#HTB-22), purchased from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China) and 60 clinical blood samples obtained from breast cancer patients were prepared. .. For the 60 blood samples, gDNA was extracted using the TIANamp Blood DNA Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions.

Non-Homologous End Joining:

Article Title: CRISPR-offinder: a CRISPR guide RNA design and off-target searching tool for user-defined protospacer adjacent motif
Article Snippet: T7 endonuclease I mutation detection assay and Sanger sequencing CRISPR/Cas9-induced lesions at the endogenous target site were quantified using the T7 endonuclease I (T7EN I) mutation detection assay to investigate the insertions/deletions (indels) mutation characteristics of nuclease-mediated non-homologous end joining (NHEJ). .. After transfection, cells were incubated for 48 h at 37 ℃ and genomic DNA was extracted using the TIANamp Genomic DNA Kit (Tiangen Biotech).

Hybridization:

Article Title: The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis
Article Snippet: Briefly, genomic DNA of the mutant strain RA1062 was extracted using the TIANamp Bacteria DNA kit (Tiangen), digested with the endonuclease Xba Ι, then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. .. The probe was generated and hybridization was conducted using the DIG DNA labeling and detection kit (Roche Diagnostics USA, Indianapolis, IN, USA), according to the manufacturer’s protocol.

Countercurrent Chromatography:

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’.

Transfection:

Article Title: Apolipoprotein E deficiency accelerates atherosclerosis development in miniature pigs
Article Snippet: .. PFFs transfected with or without Cas9 -sgRNA targeting plasmids (as described above) were cultured for 48 h. Genomic DNA was extracted using a DNA extraction kit (TianGen, Beijing, China) and the genomic regions surrounding the CRISPR/Cas9 target site were PCR amplified. .. PCR primers used are as follows: 5′-CCTCAGGTGGTCTAGGTTGG-3′ and 5′-TTTTGCAATGGAGGAGTGGC-3′ for sgRNA1; 5′-CGCCTCTCTCTGTTCATTGC-3′ and 5′-TTTTGCAATGGAGGAGTGGC-3′ for sgRNA2.

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.

Southern Blot:

Article Title: Enhancement of grain number per spike by RNA interference of cytokinin oxidase 2 gene in bread wheat
Article Snippet: Paragraph title: Southern blotting analysis ... 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).

Article Title: The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis
Article Snippet: To confirm one insertion of the Tn4351 transposon in the mutant strain RA1062, Southern blot analysis was conducted as previously described [ ]. .. Briefly, genomic DNA of the mutant strain RA1062 was extracted using the TIANamp Bacteria DNA kit (Tiangen), digested with the endonuclease Xba Ι, then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane.

Cell Culture:

Article Title: Genome wide analyses uncover allele-specific RNA editing in human and mouse
Article Snippet: Paragraph title: Cell culture, DNA/RNA purification and sequencing from human U87MG cells ... Genomic DNA and total RNA were extracted together using the DNA/RNA Isolation Kit (TianGen) according to the manufacturer's protocol.

Article Title: Apolipoprotein E deficiency accelerates atherosclerosis development in miniature pigs
Article Snippet: .. PFFs transfected with or without Cas9 -sgRNA targeting plasmids (as described above) were cultured for 48 h. Genomic DNA was extracted using a DNA extraction kit (TianGen, Beijing, China) and the genomic regions surrounding the CRISPR/Cas9 target site were PCR amplified. .. PCR primers used are as follows: 5′-CCTCAGGTGGTCTAGGTTGG-3′ and 5′-TTTTGCAATGGAGGAGTGGC-3′ for sgRNA1; 5′-CGCCTCTCTCTGTTCATTGC-3′ and 5′-TTTTGCAATGGAGGAGTGGC-3′ for sgRNA2.

Generated:

Article Title: The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis
Article Snippet: Briefly, genomic DNA of the mutant strain RA1062 was extracted using the TIANamp Bacteria DNA kit (Tiangen), digested with the endonuclease Xba Ι, then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. .. The probe was generated and hybridization was conducted using the DIG DNA labeling and detection kit (Roche Diagnostics USA, Indianapolis, IN, USA), according to the manufacturer’s protocol.

DNA Sequencing:

Article Title: Genome wide analyses uncover allele-specific RNA editing in human and mouse
Article Snippet: Genomic DNA and total RNA were extracted together using the DNA/RNA Isolation Kit (TianGen) according to the manufacturer's protocol. .. The standard Illumina protocol was used to construct libraries for DNA-seq and RNA-seq on the Illumina HiSeq X ten platform.

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’.

DNA Labeling:

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). .. The fragments resulting from the digestion were separated by 0.8% agarose gel electrophoresis followed by blotting onto nitrocellulose membranes and hybridizing with the DIG high prime DNA labeling and detection starter kit II from Roche (Catalog NO. 11585614910).

Article Title: The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis
Article Snippet: Briefly, genomic DNA of the mutant strain RA1062 was extracted using the TIANamp Bacteria DNA kit (Tiangen), digested with the endonuclease Xba Ι, then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. .. The probe was generated and hybridization was conducted using the DIG DNA labeling and detection kit (Roche Diagnostics USA, Indianapolis, IN, USA), according to the manufacturer’s protocol.

Polymerase Chain Reaction:

Article Title: All-Trans Retinoic Acid Induces CD4+CD25+FOXP3+ Regulatory T Cells by Increasing FOXP3 Demethylation in Systemic Sclerosis CD4+ T Cells
Article Snippet: Genomic DNA was isolated from CD4+ T cells using the TIANamp Genomic DNA kit (Tiangen Biotech, Beijing, China). .. The 217 bp-long fragment of the FOXP3 promoter locus (−204 to +12) was amplified by the PCR assay using the designed primer sequences as follows: 5′-TATAATTAAGAAAAGGAGAAATATAGAGAG-3′ (forward) and 5′-TCAACCTAACTTATAAAAAACTATCAC-3′ (reverse).

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). .. The 882-bp PCR fragment of FAD2 intron was labeled with digoxin.

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: Analysis of promoter methylation and epigenetic regulation of miR-32 in colorectal cancer cells
Article Snippet: Genomic DNA was extracted from the HT-29, HCT-116 and NCM460 cells using the TIANamp Genomic DNA kit (cat. no. DP304; Tiangen Biotech, Beijing, China). .. The target region of the promoter of TMEM245, the host gene of miR-32, containing 94 CpG sites, located between −195 and +521 bp numbered from the start codon ATG, was amplified by PCR using DreamTaq Hot Start DNA Polymerase (Thermo Fisher Scientific, Inc.) and specific primers ( ; ).

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’.

Article Title: Characteristics of dihydroflavonol 4-reductase gene promoters from different leaf colored Malus crabapple cultivars
Article Snippet: Cloning and analysis of the McDFR1 promoters To analyze the differences in McDFR1 promoter sequences of the three different leaf colored crabapple cultivars, genomic DNA was isolated from ‘Royalty’, ‘Radiant’ and ‘Flame’ leaves using the Plant Genomic DNA Kit (TIANGEN BIOTECH CO., LTD, Beijing, China). .. The 5′-upstream sequences were amplified by hi-TAIL PCR the primers are shown in .

Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
Article Snippet: For the 60 blood samples, gDNA was extracted using the TIANamp Blood DNA Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. .. For the clinical samples, the modified PR-PCR was performed in a total volume of 25 μL reaction mixture including 20 ng of gDNA, 3.75 μL of (5×) PrimeSTAR buffer, 1.25 μL of (5×) Taq PCR buffer, 0.2 mM dNTPs, 0.2 U of PrimeSTAR HS DNA polymerase, 0.7 U of Taq DNA polymerase, 1 μL of DMSO and 0.3 μM each of reverse primer, fusion-blocked forward primer and adaptor.

Article Title: Curing of Plasmid pXO1 from Bacillus anthracis Using Plasmid Incompatibility
Article Snippet: Single clones were analyzed by colony PCR to determine the elimination of pXO1 from B. anthracis A16R using three pairs of primers (pag-F/R, lef-F/R, and cya-F/R) ( ). .. Putative pXO1-cured clones were selected, and genomic DNA was purified with TIANamp Bacteria DNA kit (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions for isolating genomic DNA from gram-positive bacteria.

Article Title: The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis
Article Snippet: Briefly, genomic DNA of the mutant strain RA1062 was extracted using the TIANamp Bacteria DNA kit (Tiangen), digested with the endonuclease Xba Ι, then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. .. PCR was performed to amplify the transposon-specific probe representing the 410-bp IS4351 fragment from the plasmid pEP4351 using the primer pair Tn4351-F/Tn4351-R (Table ).

Article Title: Apolipoprotein E deficiency accelerates atherosclerosis development in miniature pigs
Article Snippet: .. PFFs transfected with or without Cas9 -sgRNA targeting plasmids (as described above) were cultured for 48 h. Genomic DNA was extracted using a DNA extraction kit (TianGen, Beijing, China) and the genomic regions surrounding the CRISPR/Cas9 target site were PCR amplified. .. PCR primers used are as follows: 5′-CCTCAGGTGGTCTAGGTTGG-3′ and 5′-TTTTGCAATGGAGGAGTGGC-3′ for sgRNA1; 5′-CGCCTCTCTCTGTTCATTGC-3′ and 5′-TTTTGCAATGGAGGAGTGGC-3′ for sgRNA2.

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.

Cleavage Assay:

Article Title: Apolipoprotein E deficiency accelerates atherosclerosis development in miniature pigs
Article Snippet: Paragraph title: T7E1 cleavage assay ... PFFs transfected with or without Cas9 -sgRNA targeting plasmids (as described above) were cultured for 48 h. Genomic DNA was extracted using a DNA extraction kit (TianGen, Beijing, China) and the genomic regions surrounding the CRISPR/Cas9 target site were PCR amplified.

DNA Extraction:

Article Title: All-Trans Retinoic Acid Induces CD4+CD25+FOXP3+ Regulatory T Cells by Increasing FOXP3 Demethylation in Systemic Sclerosis CD4+ T Cells
Article Snippet: Paragraph title: 2.6. Genomic DNA Extraction and Bisulfite Sequencing ... Genomic DNA was isolated from CD4+ T cells using the TIANamp Genomic DNA kit (Tiangen Biotech, Beijing, China).

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.

Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
Article Snippet: Genomic DNA (gDNA) was extracted from both cell lines using the MiniBEST Universal Genomic DNA Extraction Kit (TaKaRa, Dalian, China) following the manufacturer’s instructions. .. For the 60 blood samples, gDNA was extracted using the TIANamp Blood DNA Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions.

Article Title: The heat shock protein 90 of Toxoplasma gondii is essential for invasion of host cells and tachyzoite growth
Article Snippet: .. Next, 24, 48, 72, and 96 h post-infection (PI), the parasites were collected and genomic DNA was extracted using the TIANGEN genomic DNA isolation kit by following the manufacturer’s protocol (TIANGEN, Beijing). .. The standard curve was obtained by the known concentration of the RHΔku80 parasites via SYBR-green real-time PCR using B1 gene primers, and the parasite numbers were calculated by interpolation from this standard curve [ ].

Article Title: Apolipoprotein E deficiency accelerates atherosclerosis development in miniature pigs
Article Snippet: .. PFFs transfected with or without Cas9 -sgRNA targeting plasmids (as described above) were cultured for 48 h. Genomic DNA was extracted using a DNA extraction kit (TianGen, Beijing, China) and the genomic regions surrounding the CRISPR/Cas9 target site were PCR amplified. .. PCR primers used are as follows: 5′-CCTCAGGTGGTCTAGGTTGG-3′ and 5′-TTTTGCAATGGAGGAGTGGC-3′ for sgRNA1; 5′-CGCCTCTCTCTGTTCATTGC-3′ and 5′-TTTTGCAATGGAGGAGTGGC-3′ for sgRNA2.

RNA Sequencing Assay:

Article Title: Genome wide analyses uncover allele-specific RNA editing in human and mouse
Article Snippet: Genomic DNA and total RNA were extracted together using the DNA/RNA Isolation Kit (TianGen) according to the manufacturer's protocol. .. The standard Illumina protocol was used to construct libraries for DNA-seq and RNA-seq on the Illumina HiSeq X ten platform.

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).

Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
Article Snippet: HCC1937 that carries a homozygous TP53 mutation in codon 306 (p.R306X, c.916C > T) was used as the mutant sample, while MCF-7 that does not carry this mutation was used as the wild-type sample [ ]. .. For the 60 blood samples, gDNA was extracted using the TIANamp Blood DNA Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions.

Article Title: The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis
Article Snippet: .. Briefly, genomic DNA of the mutant strain RA1062 was extracted using the TIANamp Bacteria DNA kit (Tiangen), digested with the endonuclease Xba Ι, then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. ..

Article Title: CRISPR-offinder: a CRISPR guide RNA design and off-target searching tool for user-defined protospacer adjacent motif
Article Snippet: Paragraph title: T7 endonuclease I mutation detection assay and Sanger sequencing ... After transfection, cells were incubated for 48 h at 37 ℃ and genomic DNA was extracted using the TIANamp Genomic DNA Kit (Tiangen Biotech).

Isolation:

Article Title: Genome wide analyses uncover allele-specific RNA editing in human and mouse
Article Snippet: .. Genomic DNA and total RNA were extracted together using the DNA/RNA Isolation Kit (TianGen) according to the manufacturer's protocol. .. The standard Illumina protocol was used to construct libraries for DNA-seq and RNA-seq on the Illumina HiSeq X ten platform.

Article Title: All-Trans Retinoic Acid Induces CD4+CD25+FOXP3+ Regulatory T Cells by Increasing FOXP3 Demethylation in Systemic Sclerosis CD4+ T Cells
Article Snippet: .. Genomic DNA was isolated from CD4+ T cells using the TIANamp Genomic DNA kit (Tiangen Biotech, Beijing, China). .. Bisulfite conversion was performed using the EpiTect Bisulfite Kit (Qiagen, Germany).

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).

Article Title: Characteristics of dihydroflavonol 4-reductase gene promoters from different leaf colored Malus crabapple cultivars
Article Snippet: .. Cloning and analysis of the McDFR1 promoters To analyze the differences in McDFR1 promoter sequences of the three different leaf colored crabapple cultivars, genomic DNA was isolated from ‘Royalty’, ‘Radiant’ and ‘Flame’ leaves using the Plant Genomic DNA Kit (TIANGEN BIOTECH CO., LTD, Beijing, China). .. The 5′-upstream sequences were amplified by hi-TAIL PCR the primers are shown in .

Article Title: Apolipoprotein E deficiency accelerates atherosclerosis development in miniature pigs
Article Snippet: PFFs were isolated from Chinese Landrace fetuses at day 35 of gestation using 200 U/ml collagenase (Invitrogen) and 25 kU/ml DNaseI (Invitrogen), and cultured as previously described ( ). .. PFFs transfected with or without Cas9 -sgRNA targeting plasmids (as described above) were cultured for 48 h. Genomic DNA was extracted using a DNA extraction kit (TianGen, Beijing, China) and the genomic regions surrounding the CRISPR/Cas9 target site were PCR amplified.

Detection Assay:

Article Title: CRISPR-offinder: a CRISPR guide RNA design and off-target searching tool for user-defined protospacer adjacent motif
Article Snippet: Paragraph title: T7 endonuclease I mutation detection assay and Sanger sequencing ... After transfection, cells were incubated for 48 h at 37 ℃ and genomic DNA was extracted using the TIANamp Genomic DNA Kit (Tiangen Biotech).

Labeling:

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). .. The 882-bp PCR fragment of FAD2 intron was labeled with digoxin.

Purification:

Article Title: Genome wide analyses uncover allele-specific RNA editing in human and mouse
Article Snippet: Paragraph title: Cell culture, DNA/RNA purification and sequencing from human U87MG cells ... Genomic DNA and total RNA were extracted together using the DNA/RNA Isolation Kit (TianGen) according to the manufacturer's protocol.

Article Title: Curing of Plasmid pXO1 from Bacillus anthracis Using Plasmid Incompatibility
Article Snippet: .. Putative pXO1-cured clones were selected, and genomic DNA was purified with TIANamp Bacteria DNA kit (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions for isolating genomic DNA from gram-positive bacteria. .. To ensure the elimination of pXO1 from the A16R strain, 14 different pXO1 genes were analyzed by PCR using the total genomic DNA from strain A16R and the putative pXO1-cured clones as templates ( ).

Article Title: Apolipoprotein E deficiency accelerates atherosclerosis development in miniature pigs
Article Snippet: PFFs transfected with or without Cas9 -sgRNA targeting plasmids (as described above) were cultured for 48 h. Genomic DNA was extracted using a DNA extraction kit (TianGen, Beijing, China) and the genomic regions surrounding the CRISPR/Cas9 target site were PCR amplified. .. The PCR conditions were as follows: 95°C for 5 min, followed by 30 cycles of 95°C for 10 s, 60°C for 30 s, and 72°C for 40 s, and a final 72°C for 7 min. A total of 250 ng of the purified PCR product was mixed with NEB Buffer 2, denatured, and annealed to allow formation of heteroduplexes using the following conditions: 95°C for 5 min, 95°C to 85°C ramping at −2°C/s, 85°C to 25°C at −0.1°C/s, and 4°C hold.

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). .. Purified PCR product was denatured and reannealed using a thermocycler.

Dot Blot:

Article Title: Phytophthora methylomes are modulated by 6mA methyltransferases and associated with adaptive genome regions
Article Snippet: Paragraph title: Dot blot assay ... Genomic DNA of P. sojae and P. infestans were extracted using TIANGEN DNAsecure Plant kit.

Sequencing:

Article Title: Genome wide analyses uncover allele-specific RNA editing in human and mouse
Article Snippet: Paragraph title: Cell culture, DNA/RNA purification and sequencing from human U87MG cells ... Genomic DNA and total RNA were extracted together using the DNA/RNA Isolation Kit (TianGen) according to the manufacturer's protocol.

Article Title: Characteristics of dihydroflavonol 4-reductase gene promoters from different leaf colored Malus crabapple cultivars
Article Snippet: Cloning and analysis of the McDFR1 promoters To analyze the differences in McDFR1 promoter sequences of the three different leaf colored crabapple cultivars, genomic DNA was isolated from ‘Royalty’, ‘Radiant’ and ‘Flame’ leaves using the Plant Genomic DNA Kit (TIANGEN BIOTECH CO., LTD, Beijing, China). .. The primers for hi-TAIL PCR (high-efficient thermal asymmetric interlaced PCR) were designed based on the McDFRs cDNA sequence (GenBank Accession: FJ817487, AF117268).

Article Title: CRISPR-offinder: a CRISPR guide RNA design and off-target searching tool for user-defined protospacer adjacent motif
Article Snippet: Paragraph title: T7 endonuclease I mutation detection assay and Sanger sequencing ... After transfection, cells were incubated for 48 h at 37 ℃ and genomic DNA was extracted using the TIANamp Genomic DNA Kit (Tiangen Biotech).

Polyacrylamide Gel Electrophoresis:

Article Title: The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis
Article Snippet: .. Briefly, genomic DNA of the mutant strain RA1062 was extracted using the TIANamp Bacteria DNA kit (Tiangen), digested with the endonuclease Xba Ι, then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. ..

CRISPR:

Article Title: Apolipoprotein E deficiency accelerates atherosclerosis development in miniature pigs
Article Snippet: .. PFFs transfected with or without Cas9 -sgRNA targeting plasmids (as described above) were cultured for 48 h. Genomic DNA was extracted using a DNA extraction kit (TianGen, Beijing, China) and the genomic regions surrounding the CRISPR/Cas9 target site were PCR amplified. .. PCR primers used are as follows: 5′-CCTCAGGTGGTCTAGGTTGG-3′ and 5′-TTTTGCAATGGAGGAGTGGC-3′ for sgRNA1; 5′-CGCCTCTCTCTGTTCATTGC-3′ and 5′-TTTTGCAATGGAGGAGTGGC-3′ for sgRNA2.

Article Title: CRISPR-offinder: a CRISPR guide RNA design and off-target searching tool for user-defined protospacer adjacent motif
Article Snippet: T7 endonuclease I mutation detection assay and Sanger sequencing CRISPR/Cas9-induced lesions at the endogenous target site were quantified using the T7 endonuclease I (T7EN I) mutation detection assay to investigate the insertions/deletions (indels) mutation characteristics of nuclease-mediated non-homologous end joining (NHEJ). .. After transfection, cells were incubated for 48 h at 37 ℃ and genomic DNA was extracted using the TIANamp Genomic DNA Kit (Tiangen Biotech).

SDS Page:

Article Title: The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis
Article Snippet: .. Briefly, genomic DNA of the mutant strain RA1062 was extracted using the TIANamp Bacteria DNA kit (Tiangen), digested with the endonuclease Xba Ι, then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. ..

Plasmid Preparation:

Article Title: All-Trans Retinoic Acid Induces CD4+CD25+FOXP3+ Regulatory T Cells by Increasing FOXP3 Demethylation in Systemic Sclerosis CD4+ T Cells
Article Snippet: Genomic DNA was isolated from CD4+ T cells using the TIANamp Genomic DNA kit (Tiangen Biotech, Beijing, China). .. The amplified products were then cloned into the pGEM-T easy vector (Promega, USA).

Article Title: Curing of Plasmid pXO1 from Bacillus anthracis Using Plasmid Incompatibility
Article Snippet: Paragraph title: Curing of Plasmid pXO1 from B. anthracis A16R Strain ... Putative pXO1-cured clones were selected, and genomic DNA was purified with TIANamp Bacteria DNA kit (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions for isolating genomic DNA from gram-positive bacteria.

Article Title: The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis
Article Snippet: The E. coli BW19851 with the plasmid pEP4351 was used as the donor strain and R. anatipestifer CH3 as the recipient. .. Briefly, genomic DNA of the mutant strain RA1062 was extracted using the TIANamp Bacteria DNA kit (Tiangen), digested with the endonuclease Xba Ι, then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane.

Real-time Polymerase Chain Reaction:

Article Title: The heat shock protein 90 of Toxoplasma gondii is essential for invasion of host cells and tachyzoite growth
Article Snippet: Next, 24, 48, 72, and 96 h post-infection (PI), the parasites were collected and genomic DNA was extracted using the TIANGEN genomic DNA isolation kit by following the manufacturer’s protocol (TIANGEN, Beijing). .. The standard curve was obtained by the known concentration of the RHΔku80 parasites via SYBR-green real-time PCR using B1 gene primers, and the parasite numbers were calculated by interpolation from this standard curve [ ].

Agarose Gel Electrophoresis:

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: 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). .. The fragments resulting from the digestion were separated by 0.8% agarose gel electrophoresis followed by blotting onto nitrocellulose membranes and hybridizing with the DIG high prime DNA labeling and detection starter kit II from Roche (Catalog NO. 11585614910).

Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
Article Snippet: For the 60 blood samples, gDNA was extracted using the TIANamp Blood DNA Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. .. The reactions were carried out under cycling conditions of pre-denaturation at 94°C for 2 min; 40 cycles of denaturation at 98°C for 10 s, annealing at 59°C for 20 s and extension at 72°C for 18 s. The PCR products were visualized via 1.5% agarose gel electrophoresis.

Article Title: Apolipoprotein E deficiency accelerates atherosclerosis development in miniature pigs
Article Snippet: PFFs transfected with or without Cas9 -sgRNA targeting plasmids (as described above) were cultured for 48 h. Genomic DNA was extracted using a DNA extraction kit (TianGen, Beijing, China) and the genomic regions surrounding the CRISPR/Cas9 target site were PCR amplified. .. After reannealing, the products were digested with 1 μl of T7 endonuclease I (NEB, Beverly, MA, USA) at 37°C for 15 min and then run on a 2% agarose gel stained with ethidium bromide.

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). .. Hybridized PCR products were digested with T7EN I (NEB, M0302L) for 15 min and separated by 2 % agarose gel.

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.

Spectrophotometry:

Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
Article Snippet: For the 60 blood samples, gDNA was extracted using the TIANamp Blood DNA Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. .. The extracted gDNA was eluted into 70 μL of (1×) TE buffer (pH 8.0) and then quantified using a Nanodrop-1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE).

Concentration Assay:

Article Title: The heat shock protein 90 of Toxoplasma gondii is essential for invasion of host cells and tachyzoite growth
Article Snippet: Next, 24, 48, 72, and 96 h post-infection (PI), the parasites were collected and genomic DNA was extracted using the TIANGEN genomic DNA isolation kit by following the manufacturer’s protocol (TIANGEN, Beijing). .. The standard curve was obtained by the known concentration of the RHΔku80 parasites via SYBR-green real-time PCR using B1 gene primers, and the parasite numbers were calculated by interpolation from this standard curve [ ].

Staining:

Article Title: Apolipoprotein E deficiency accelerates atherosclerosis development in miniature pigs
Article Snippet: PFFs transfected with or without Cas9 -sgRNA targeting plasmids (as described above) were cultured for 48 h. Genomic DNA was extracted using a DNA extraction kit (TianGen, Beijing, China) and the genomic regions surrounding the CRISPR/Cas9 target site were PCR amplified. .. After reannealing, the products were digested with 1 μl of T7 endonuclease I (NEB, Beverly, MA, USA) at 37°C for 15 min and then run on a 2% agarose gel stained with ethidium bromide.

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    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.
    Direct Sequencing Genomic Dna, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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: 79/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co pou3f4 genomic dna
    Pedigree, clinical phenotypes and mutation analysis in family 2741. a Pedigree of family 2741 with congenital mixed hearing impairment and sensorineural hearing impairment cases (Affected subjects are denoted in black. Arrow indicates the proband. Mutation carrier are denoted with dot within a symbol); b Audiograms of both ears for the proband, who exhibited typical audiometric features of mixed hearing impairment; c Temporal bone CT images of the proband demonstrating dilation of the bottom of the IAM and a deficit in the bony plate, which separates the basal turn of the cochlea and the IAM (arrow); d Audiograms of both ears from the uncle of the proband, who shows profound sensorineural hearing impairment; e Temporal bone CT images of the uncle of the proband demonstrating dilation of the lateral end of the IAM and bone deficiency between the basal turn of the cochlea and the IAM (arrow). f Wild-type sequence of <t>POU3F4</t> including position 973; g A heterozygous c.973delT mutation was found in the female carriers; h A hemizygotic c.973delT mutation was detected in the affected males; i Amino acid change caused by changes in the <t>DNA</t> sequence leading to a predicted frameshift mutation and truncation of the POU3F4 protein; j Panel 1 marks the position of the c.973delT (p.Trp325Glyfs*12) mutation and panel 2 marks the position of the c.927delCTC (p.Ser310del) mutation. The POU homeodomain (from Gly276 to Arg335) is highly conserved in different species
    Pou3f4 Genomic Dna, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co plant genomic dna kit
    High-throughput sequencing analysis of CRISPR/Cas9-Induced mutagenesis in three BnSPL3 homoeologs (A–C) , statistical analysis of editing frequency occurred in rapeseed SPL3A, SPL3B , and SPL3C genomic sites, respectively. Rapeseed sample 1#, 2#, 20#, 24#, 41# were from independent <t>transgenic</t> events. Excepting transgenic line 24#, all the rest samples display obvious heteroduplexed <t>DNA</t> bands in PAGE-based assays. (D–F) Sequences alignment of different mutation types identified from high-throughput sequencing analysis.
    Plant Genomic Dna Kit, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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:

    Pedigree, clinical phenotypes and mutation analysis in family 2741. a Pedigree of family 2741 with congenital mixed hearing impairment and sensorineural hearing impairment cases (Affected subjects are denoted in black. Arrow indicates the proband. Mutation carrier are denoted with dot within a symbol); b Audiograms of both ears for the proband, who exhibited typical audiometric features of mixed hearing impairment; c Temporal bone CT images of the proband demonstrating dilation of the bottom of the IAM and a deficit in the bony plate, which separates the basal turn of the cochlea and the IAM (arrow); d Audiograms of both ears from the uncle of the proband, who shows profound sensorineural hearing impairment; e Temporal bone CT images of the uncle of the proband demonstrating dilation of the lateral end of the IAM and bone deficiency between the basal turn of the cochlea and the IAM (arrow). f Wild-type sequence of POU3F4 including position 973; g A heterozygous c.973delT mutation was found in the female carriers; h A hemizygotic c.973delT mutation was detected in the affected males; i Amino acid change caused by changes in the DNA sequence leading to a predicted frameshift mutation and truncation of the POU3F4 protein; j Panel 1 marks the position of the c.973delT (p.Trp325Glyfs*12) mutation and panel 2 marks the position of the c.927delCTC (p.Ser310del) mutation. The POU homeodomain (from Gly276 to Arg335) is highly conserved in different species

    Journal: BMC Medical Genetics

    Article Title: Clinical and molecular characterization of POU3F4 mutations in multiple DFNX2 Chinese families

    doi: 10.1186/s12881-018-0630-9

    Figure Lengend Snippet: Pedigree, clinical phenotypes and mutation analysis in family 2741. a Pedigree of family 2741 with congenital mixed hearing impairment and sensorineural hearing impairment cases (Affected subjects are denoted in black. Arrow indicates the proband. Mutation carrier are denoted with dot within a symbol); b Audiograms of both ears for the proband, who exhibited typical audiometric features of mixed hearing impairment; c Temporal bone CT images of the proband demonstrating dilation of the bottom of the IAM and a deficit in the bony plate, which separates the basal turn of the cochlea and the IAM (arrow); d Audiograms of both ears from the uncle of the proband, who shows profound sensorineural hearing impairment; e Temporal bone CT images of the uncle of the proband demonstrating dilation of the lateral end of the IAM and bone deficiency between the basal turn of the cochlea and the IAM (arrow). f Wild-type sequence of POU3F4 including position 973; g A heterozygous c.973delT mutation was found in the female carriers; h A hemizygotic c.973delT mutation was detected in the affected males; i Amino acid change caused by changes in the DNA sequence leading to a predicted frameshift mutation and truncation of the POU3F4 protein; j Panel 1 marks the position of the c.973delT (p.Trp325Glyfs*12) mutation and panel 2 marks the position of the c.927delCTC (p.Ser310del) mutation. The POU homeodomain (from Gly276 to Arg335) is highly conserved in different species

    Article Snippet: Sequencing analysis of POU3F4 Genomic DNA was extracted from blood using a DNA Extraction Kit (Tiangen Biotech).

    Techniques: Mutagenesis, Sequencing

    Pedigree, clinical phenotypes and mutation analysis in family ZSJ. a Audiograms of both ears from the proband exhibited profound sensorineural hearing impairment; b Temporal bone CT images of the proband demonstrating dilation of the lateral end of the IAM and a deficit in the basal turn of the cochlea in the right ear (arrow) in addition to dilation of the lateral end of the IAM and an incompletely developed cochlea in the left ear (arrow); c Wild-type sequence of POU3F4 , including site 669; d A hemizygotic c.669 T > A mutation was detected in the affected boy; e Pedigree of family ZSJ; f Stop codon caused by changes in the DNA sequence; g Molecular modeling of wild-type and mutant POU3F4 proteins. The c.669 T > A mutant creates a new stop codon and is predicted to result in a truncated protein lacking normal POU3F4 transcription factor function

    Journal: BMC Medical Genetics

    Article Title: Clinical and molecular characterization of POU3F4 mutations in multiple DFNX2 Chinese families

    doi: 10.1186/s12881-018-0630-9

    Figure Lengend Snippet: Pedigree, clinical phenotypes and mutation analysis in family ZSJ. a Audiograms of both ears from the proband exhibited profound sensorineural hearing impairment; b Temporal bone CT images of the proband demonstrating dilation of the lateral end of the IAM and a deficit in the basal turn of the cochlea in the right ear (arrow) in addition to dilation of the lateral end of the IAM and an incompletely developed cochlea in the left ear (arrow); c Wild-type sequence of POU3F4 , including site 669; d A hemizygotic c.669 T > A mutation was detected in the affected boy; e Pedigree of family ZSJ; f Stop codon caused by changes in the DNA sequence; g Molecular modeling of wild-type and mutant POU3F4 proteins. The c.669 T > A mutant creates a new stop codon and is predicted to result in a truncated protein lacking normal POU3F4 transcription factor function

    Article Snippet: Sequencing analysis of POU3F4 Genomic DNA was extracted from blood using a DNA Extraction Kit (Tiangen Biotech).

    Techniques: Mutagenesis, Sequencing

    Pedigree, clinical phenotypes, and mutation analysis in family 1486. a Temporal bone computed tomography (CT) images of the proband of family 1486 demonstrating dilation of the lateral end of the internal acoustic meatus (IAM) and a malformed cochlea; the basal turn of the cochlea was incompletely separated from the IAM (arrow); b Pedigree of Family 1486 with multiple congenital profound sensorineural hearing impairment cases (Affected subjects are denoted in black. Arrow indicates the proband. Mutation carrier are denoted with dot within a symbol); c Wild-type sequence of POU3F4 including sites 927–929; d A heterozygous c.927delCTC mutation was found in the female carriers; e A hemizygotic c.927delCTC mutation was detected in the affected males; f Amino acid changes caused by changes in the DNA sequence. A three-nucleotide deletion (from position 927 to 929) in the coding region of POU3F4 results in the deletion of serine at position 310

    Journal: BMC Medical Genetics

    Article Title: Clinical and molecular characterization of POU3F4 mutations in multiple DFNX2 Chinese families

    doi: 10.1186/s12881-018-0630-9

    Figure Lengend Snippet: Pedigree, clinical phenotypes, and mutation analysis in family 1486. a Temporal bone computed tomography (CT) images of the proband of family 1486 demonstrating dilation of the lateral end of the internal acoustic meatus (IAM) and a malformed cochlea; the basal turn of the cochlea was incompletely separated from the IAM (arrow); b Pedigree of Family 1486 with multiple congenital profound sensorineural hearing impairment cases (Affected subjects are denoted in black. Arrow indicates the proband. Mutation carrier are denoted with dot within a symbol); c Wild-type sequence of POU3F4 including sites 927–929; d A heterozygous c.927delCTC mutation was found in the female carriers; e A hemizygotic c.927delCTC mutation was detected in the affected males; f Amino acid changes caused by changes in the DNA sequence. A three-nucleotide deletion (from position 927 to 929) in the coding region of POU3F4 results in the deletion of serine at position 310

    Article Snippet: Sequencing analysis of POU3F4 Genomic DNA was extracted from blood using a DNA Extraction Kit (Tiangen Biotech).

    Techniques: Mutagenesis, Computed Tomography, Sequencing

    High-throughput sequencing analysis of CRISPR/Cas9-Induced mutagenesis in three BnSPL3 homoeologs (A–C) , statistical analysis of editing frequency occurred in rapeseed SPL3A, SPL3B , and SPL3C genomic sites, respectively. Rapeseed sample 1#, 2#, 20#, 24#, 41# were from independent transgenic events. Excepting transgenic line 24#, all the rest samples display obvious heteroduplexed DNA bands in PAGE-based assays. (D–F) Sequences alignment of different mutation types identified from high-throughput sequencing analysis.

    Journal: Frontiers in Plant Science

    Article Title: An Efficient CRISPR/Cas9 Platform for Rapidly Generating Simultaneous Mutagenesis of Multiple Gene Homoeologs in Allotetraploid Oilseed Rape

    doi: 10.3389/fpls.2018.00442

    Figure Lengend Snippet: High-throughput sequencing analysis of CRISPR/Cas9-Induced mutagenesis in three BnSPL3 homoeologs (A–C) , statistical analysis of editing frequency occurred in rapeseed SPL3A, SPL3B , and SPL3C genomic sites, respectively. Rapeseed sample 1#, 2#, 20#, 24#, 41# were from independent transgenic events. Excepting transgenic line 24#, all the rest samples display obvious heteroduplexed DNA bands in PAGE-based assays. (D–F) Sequences alignment of different mutation types identified from high-throughput sequencing analysis.

    Article Snippet: Genomic DNA was extracted from transgenic rapeseed plants using the Plant Genomic DNA Kit (TIANGEN, China).

    Techniques: Next-Generation Sequencing, CRISPR, Mutagenesis, Transgenic Assay, Polyacrylamide Gel Electrophoresis