multiplex pcr mix  (Qiagen)

 
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    Type it Microsatellite PCR Kit
    Description:
    For fast and reliable multiplex PCR analysis of microsatellite loci Kit contents Qiagen Type it Microsatellite PCR Kit 70 x 25μL rxns Genomic DNA Sample For Fast and Reliable Multiplex PCR Analysis of Microsatellite loci Ideal for Multiplex PCR Microsatellites STRs VNTRs SSRs Includes Type it Multiplex PCR Master Mix 1 x 0 85mL 5x Q Solution 1 x 2mL and RNAse free Water 1 x 1 9mL Benefits Reliable microsatellite analysis by multiplex PCR Microsatellite assay development without optimization Successful and specific coamplification of all fragments Optimized protocol for fast and reliable results Easy instructions for use with various downstream analysis platform
    Catalog Number:
    206241
    Price:
    100
    Category:
    Type it Microsatellite PCR Kit
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    Structured Review

    Qiagen multiplex pcr mix
    Type it Microsatellite PCR Kit
    For fast and reliable multiplex PCR analysis of microsatellite loci Kit contents Qiagen Type it Microsatellite PCR Kit 70 x 25μL rxns Genomic DNA Sample For Fast and Reliable Multiplex PCR Analysis of Microsatellite loci Ideal for Multiplex PCR Microsatellites STRs VNTRs SSRs Includes Type it Multiplex PCR Master Mix 1 x 0 85mL 5x Q Solution 1 x 2mL and RNAse free Water 1 x 1 9mL Benefits Reliable microsatellite analysis by multiplex PCR Microsatellite assay development without optimization Successful and specific coamplification of all fragments Optimized protocol for fast and reliable results Easy instructions for use with various downstream analysis platform
    https://www.bioz.com/result/multiplex pcr mix/product/Qiagen
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    Images

    1) Product Images from "Development of User-Friendly Method to Distinguish Subspecies of the Korean Medicinal Herb Perilla frutescens Using Multiplex-PCR"

    Article Title: Development of User-Friendly Method to Distinguish Subspecies of the Korean Medicinal Herb Perilla frutescens Using Multiplex-PCR

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

    doi: 10.3390/molecules22040665

    Multiplex PCR assay using three specific markers for the perilla subspecies in a single reaction. A mixture of three specific markers, PfLS , PfMyb - P1pro , and PfDFRpro , was used for PCR amplification. Lanes on 3% electrophoresis gel: M: 100 bp DNA ladder; 1–3: ‘Cham-Dlggae’, ‘Ip-Dlggae’, and ‘Bora-Dlggae’, respectively, which represent the three cultivar types of P . frutescens var. japonica ; 4: ‘Jureum-soyeop’, representing P . frutescens var. crispa ; 5: ‘Jasoyeop’, representing P . frutescens var. acuta ; 6: ‘Chungsoyeop’, representing P . frutescens f. viridis .
    Figure Legend Snippet: Multiplex PCR assay using three specific markers for the perilla subspecies in a single reaction. A mixture of three specific markers, PfLS , PfMyb - P1pro , and PfDFRpro , was used for PCR amplification. Lanes on 3% electrophoresis gel: M: 100 bp DNA ladder; 1–3: ‘Cham-Dlggae’, ‘Ip-Dlggae’, and ‘Bora-Dlggae’, respectively, which represent the three cultivar types of P . frutescens var. japonica ; 4: ‘Jureum-soyeop’, representing P . frutescens var. crispa ; 5: ‘Jasoyeop’, representing P . frutescens var. acuta ; 6: ‘Chungsoyeop’, representing P . frutescens f. viridis .

    Techniques Used: Multiplex Assay, Polymerase Chain Reaction, Amplification, Electrophoresis

    Multiplex PCR identification of 11 commercial dried leaves and twigs of Jasoyeop products. ( a ) Lanes on 3% electrophoresis gel: M: 100 bp DNA ladder; A–K: purchased commercial dried Jasoyeop products (see Table 3 for full details); ( b , c ) Sequence analysis of PCR products amplified using the PfMybpro and PfDFRpro marker primers, respectively, aligned using Clustal W2.
    Figure Legend Snippet: Multiplex PCR identification of 11 commercial dried leaves and twigs of Jasoyeop products. ( a ) Lanes on 3% electrophoresis gel: M: 100 bp DNA ladder; A–K: purchased commercial dried Jasoyeop products (see Table 3 for full details); ( b , c ) Sequence analysis of PCR products amplified using the PfMybpro and PfDFRpro marker primers, respectively, aligned using Clustal W2.

    Techniques Used: Multiplex Assay, Polymerase Chain Reaction, Electrophoresis, Sequencing, Amplification, Marker

    2) Product Images from "Advances in multiplex PCR: balancing primer efficiencies and improving detection success"

    Article Title: Advances in multiplex PCR: balancing primer efficiencies and improving detection success

    Journal: Methods in Ecology and Evolution

    doi: 10.1111/j.2041-210X.2012.00215.x

    Multiplex PCR conducted with standardised numbers of DNA templates and separated with QIAxcel (Qiagen) where an internal marker (15 and 3000 bp) is run with each sample. Description of Lanes: Pn, Pardosa nigra ; Nr, Nebria rufescens ; Oc, Oreonebria castanea ; Mg, Mitopus glacialis ; Nj, Nebria jockischii ; Ng, Nebria germari ; Col, Collembola; each with 10 000 double-stranded copies as template (tc); M1–M4 standardised DNA mixes. M1, 2100 tc per target; M2, 1000 tc per target; M3, 200 tc per target; M4, 100 tc per target; E, electropherogram of Lane M3. Note: when a single target was present at high concentrations, signal strength was not balanced (e.g. Oc and Ng resulted in stronger signals); however, this did not occur at lower concentrations.
    Figure Legend Snippet: Multiplex PCR conducted with standardised numbers of DNA templates and separated with QIAxcel (Qiagen) where an internal marker (15 and 3000 bp) is run with each sample. Description of Lanes: Pn, Pardosa nigra ; Nr, Nebria rufescens ; Oc, Oreonebria castanea ; Mg, Mitopus glacialis ; Nj, Nebria jockischii ; Ng, Nebria germari ; Col, Collembola; each with 10 000 double-stranded copies as template (tc); M1–M4 standardised DNA mixes. M1, 2100 tc per target; M2, 1000 tc per target; M3, 200 tc per target; M4, 100 tc per target; E, electropherogram of Lane M3. Note: when a single target was present at high concentrations, signal strength was not balanced (e.g. Oc and Ng resulted in stronger signals); however, this did not occur at lower concentrations.

    Techniques Used: Multiplex Assay, Polymerase Chain Reaction, Marker

    3) Product Images from "Genomic Sequencing and Comparative Analysis of Epstein-Barr Virus Genome Isolated from Primary Nasopharyngeal Carcinoma Biopsy"

    Article Title: Genomic Sequencing and Comparative Analysis of Epstein-Barr Virus Genome Isolated from Primary Nasopharyngeal Carcinoma Biopsy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0036939

    Circular representation of HKNPC1 genome. Numbered tracks represent the following: (1) PCR amplicons prepared for next-generation sequencing (NGS); (2) reverse open reading frames; (3) forward open reading frames; (4) repeat regions; (5) read depth of NGS reads, height of the track represents 10,000 reads; single nucleotide variations (SNVs) and indels of (6) HKNPC1, (7) GD1, (8) GD2, and (9) AG876, in comparison to reference EBV sequence. This figure was created using Circos software [37] .
    Figure Legend Snippet: Circular representation of HKNPC1 genome. Numbered tracks represent the following: (1) PCR amplicons prepared for next-generation sequencing (NGS); (2) reverse open reading frames; (3) forward open reading frames; (4) repeat regions; (5) read depth of NGS reads, height of the track represents 10,000 reads; single nucleotide variations (SNVs) and indels of (6) HKNPC1, (7) GD1, (8) GD2, and (9) AG876, in comparison to reference EBV sequence. This figure was created using Circos software [37] .

    Techniques Used: Polymerase Chain Reaction, Next-Generation Sequencing, Sequencing, Software

    4) Product Images from "Recovery of bisulfite-converted genomic sequences in the methylation-sensitive QPCR"

    Article Title: Recovery of bisulfite-converted genomic sequences in the methylation-sensitive QPCR

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm055

    Recovery of target sequence from bisulfite-treated genomic DNA. High molecular weight DNA was subjected to bisulfite treatment, matrix purification and amplification using the duplex QPCR. Serial dilution of the plasmid standards was used to construct a standard curve for recovery of genomic target DNA from a cell line (HK293) in which the target APC gene is completely unmethylated. Target DNA recovery is plotted as a function of initial DNA concentration receiving bisulfite treatment and matrix purification. That portion of the recovered volume that would represent 200 ng of DNA (assuming 100% recovery at these two steps) was subjected to PCR amplification. A separate PCR reaction was performed using the unconverted primer/probe system to obtain an experimental value for full recovery of the target. Each point is the average of 10 determinations of the ratio of the observed unmethylated copy number to the unconverted copy number Error bars indicate ± 1 S.D. ( A ) Analytical prediction for the recovery based on Equation (4) . This graph represents the plot of the equation with the following parameters: θ is a unit-less fraction equal to the ratio of target copies recovered to the total input target copies. L u = 7500 nt, L l = 75 nt, k b = 5.2 × 10 3 M −1 , f = 1/587 nt = 0.0017 nt −1 . DNA concentration is expressed as the molar concentration of nucleotides ([nt] M) in input genomic single-strands. The points on the graph correspond to 0, 200, 400, 800 and 1600 ng of treated DNA. ( B ) Analytical prediction for the recovery based on Equation (5) . This graph represents the plot of the equation with the following parameters: θ is a unit-less fraction equal to the ratio of target copies recovered to the total input target copies. L u = 7500 nt, L l = 75 nt, k = 0.625 h −1 M −1 , t = 16 h, k b = 6.00 × 10 3 M −1 , DNA concentration is expressed as the molar concentration of nucleotides ([nt] M) in input genomic single-strands. The points on the graph correspond to 0, 200, 400, 800 and 1600 ng of treated DNA.
    Figure Legend Snippet: Recovery of target sequence from bisulfite-treated genomic DNA. High molecular weight DNA was subjected to bisulfite treatment, matrix purification and amplification using the duplex QPCR. Serial dilution of the plasmid standards was used to construct a standard curve for recovery of genomic target DNA from a cell line (HK293) in which the target APC gene is completely unmethylated. Target DNA recovery is plotted as a function of initial DNA concentration receiving bisulfite treatment and matrix purification. That portion of the recovered volume that would represent 200 ng of DNA (assuming 100% recovery at these two steps) was subjected to PCR amplification. A separate PCR reaction was performed using the unconverted primer/probe system to obtain an experimental value for full recovery of the target. Each point is the average of 10 determinations of the ratio of the observed unmethylated copy number to the unconverted copy number Error bars indicate ± 1 S.D. ( A ) Analytical prediction for the recovery based on Equation (4) . This graph represents the plot of the equation with the following parameters: θ is a unit-less fraction equal to the ratio of target copies recovered to the total input target copies. L u = 7500 nt, L l = 75 nt, k b = 5.2 × 10 3 M −1 , f = 1/587 nt = 0.0017 nt −1 . DNA concentration is expressed as the molar concentration of nucleotides ([nt] M) in input genomic single-strands. The points on the graph correspond to 0, 200, 400, 800 and 1600 ng of treated DNA. ( B ) Analytical prediction for the recovery based on Equation (5) . This graph represents the plot of the equation with the following parameters: θ is a unit-less fraction equal to the ratio of target copies recovered to the total input target copies. L u = 7500 nt, L l = 75 nt, k = 0.625 h −1 M −1 , t = 16 h, k b = 6.00 × 10 3 M −1 , DNA concentration is expressed as the molar concentration of nucleotides ([nt] M) in input genomic single-strands. The points on the graph correspond to 0, 200, 400, 800 and 1600 ng of treated DNA.

    Techniques Used: Sequencing, Molecular Weight, Purification, Amplification, Real-time Polymerase Chain Reaction, Serial Dilution, Plasmid Preparation, Construct, Concentration Assay, Polymerase Chain Reaction

    5) Product Images from "Cathepsin F Cysteine Protease of the Human Liver Fluke, Opisthorchis viverrini"

    Article Title: Cathepsin F Cysteine Protease of the Human Liver Fluke, Opisthorchis viverrini

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0000398

    Transcription of Opisthorchis viverrini cysteine protease mRNA revealed by RT-PCR. Developmental stages examined: (lane 1), metacercariae (2), juvenile 1 week (3), juvenile 2 weeks (4), juvenile 3 weeks (5), adult worms (6) and O. viverrini cDNA library (7).
    Figure Legend Snippet: Transcription of Opisthorchis viverrini cysteine protease mRNA revealed by RT-PCR. Developmental stages examined: (lane 1), metacercariae (2), juvenile 1 week (3), juvenile 2 weeks (4), juvenile 3 weeks (5), adult worms (6) and O. viverrini cDNA library (7).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, cDNA Library Assay

    Neighbor joining tree. The tree revealed the phylogenetic relationship between the cathepsin F cysteine protease of Opisthorchis viverrini and homologous enzymes from ∼75 other informative species. Species names and GenBank accessions are provided on the branches. Bootstrap values of 1,000 replicates are provided at the nodes of the branches (bootstrap values less than 500 were omitted).
    Figure Legend Snippet: Neighbor joining tree. The tree revealed the phylogenetic relationship between the cathepsin F cysteine protease of Opisthorchis viverrini and homologous enzymes from ∼75 other informative species. Species names and GenBank accessions are provided on the branches. Bootstrap values of 1,000 replicates are provided at the nodes of the branches (bootstrap values less than 500 were omitted).

    Techniques Used:

    Multiple sequence alignment of deduced amino acids of the cathepsin F from Opisthorchis viverrini Ov -CF-1 (AAV69023). The alignment includes other members of the Clan CA, family C1 peptidase family, including CsCF-6 from Clonorchis sinensis (ABK918111) and orthologues from Paragonimus westermani (AAY81944), Teladorsagia circumcincta (ABA01328) and Homo sapiens (NP_003784). (The T. circumcincta orthologue was included as a representative of a nematode helminth sequence, while human cathepsin F sequence was included to highlight the large prosegment with a cystatin domain, underlined, that exists in some cathepsins F.) Identical residues in more than 50% of sequences are presented in black boxes. Conserved substitutions are identified by grey boxes. The conserved active site cysteine, asparagine and histidine residues are highlighted (stars). The protease-susceptible region at the junction of the prosegment and mature domain is indicated by the thick black line. This region contains the cleavage sites for release of the prosegment from the mature domain and includes the FhCL1 cleavage site for Ov -CF-1, VT↓MDNSNFD, and the cleavage site in human cathepsin F, DL↓APPEWD, determined by X-ray crystallography [50] . The thin (green) arrow indicates the position of signal peptide cleavage site. The arrow heads indicate six cysteine residues likely involved in disulfide bridges. The box indicates a conserved, putative N -linked glycosylation site, NGS in the mature enzyme domain. The position of the ERFNAQ propeptide motif, conserved in cathepsin F, is indicated.
    Figure Legend Snippet: Multiple sequence alignment of deduced amino acids of the cathepsin F from Opisthorchis viverrini Ov -CF-1 (AAV69023). The alignment includes other members of the Clan CA, family C1 peptidase family, including CsCF-6 from Clonorchis sinensis (ABK918111) and orthologues from Paragonimus westermani (AAY81944), Teladorsagia circumcincta (ABA01328) and Homo sapiens (NP_003784). (The T. circumcincta orthologue was included as a representative of a nematode helminth sequence, while human cathepsin F sequence was included to highlight the large prosegment with a cystatin domain, underlined, that exists in some cathepsins F.) Identical residues in more than 50% of sequences are presented in black boxes. Conserved substitutions are identified by grey boxes. The conserved active site cysteine, asparagine and histidine residues are highlighted (stars). The protease-susceptible region at the junction of the prosegment and mature domain is indicated by the thick black line. This region contains the cleavage sites for release of the prosegment from the mature domain and includes the FhCL1 cleavage site for Ov -CF-1, VT↓MDNSNFD, and the cleavage site in human cathepsin F, DL↓APPEWD, determined by X-ray crystallography [50] . The thin (green) arrow indicates the position of signal peptide cleavage site. The arrow heads indicate six cysteine residues likely involved in disulfide bridges. The box indicates a conserved, putative N -linked glycosylation site, NGS in the mature enzyme domain. The position of the ERFNAQ propeptide motif, conserved in cathepsin F, is indicated.

    Techniques Used: Sequencing, Next-Generation Sequencing

    Immunolocalization of cathepsin F cysteine protease ( Ov -CF-1) in Opisthorchis viverrini infected hamster liver. Thin sections of paraffin embedded liver tissues were probed with rabbit antiserum. (A) Representative section of liver from an uninfected hamster, spanning a portal triad including a secondary bile duct, probed with rabbit anti- Ov -CF-1serum (negative control). Infected hamster liver in the vicinity of the secondary bile ducts too small in internal diameter to include an adult fluke, probed with the rabbit anti- Ov -CF-1 serum (B and C). Immunoperoxidase stain (brown) indicates the presence of Ov -CF-1 in bile ducts epithelial cells (B) and in sinusoidal Kupffer and mononuclear cells (C). Section through bile duct containing an adult O. viverrini , showing strong reactivity to organs and tissues of the fluke (including the gut), and to the epithelial cells lining the infected bile duct (panel D). Immunoperoxidase staining, original magnification, ×100.
    Figure Legend Snippet: Immunolocalization of cathepsin F cysteine protease ( Ov -CF-1) in Opisthorchis viverrini infected hamster liver. Thin sections of paraffin embedded liver tissues were probed with rabbit antiserum. (A) Representative section of liver from an uninfected hamster, spanning a portal triad including a secondary bile duct, probed with rabbit anti- Ov -CF-1serum (negative control). Infected hamster liver in the vicinity of the secondary bile ducts too small in internal diameter to include an adult fluke, probed with the rabbit anti- Ov -CF-1 serum (B and C). Immunoperoxidase stain (brown) indicates the presence of Ov -CF-1 in bile ducts epithelial cells (B) and in sinusoidal Kupffer and mononuclear cells (C). Section through bile duct containing an adult O. viverrini , showing strong reactivity to organs and tissues of the fluke (including the gut), and to the epithelial cells lining the infected bile duct (panel D). Immunoperoxidase staining, original magnification, ×100.

    Techniques Used: Infection, Negative Control, Staining, Immunoperoxidase Staining

    Genome structure of the cathepsin F gene of Opisthorchis viverrini . (A) Structure of the O. viverrini cathepsin F gene locus, as determined by nucleotide sequence analysis of a ∼3 kb PCR product of genomic DNA of O. viverrini . The top panel shows the size of the cDNA while the bottom panel shows a schematic of positions and relative sizes of the 7 exons and 6 introns (arrowed) that comprise the gene. The 5′-flanking region was generated by inverse PCR. (B) Schematic to compare the structure and exon numbers of the C1 family genes, including the cathepsin F genes of O. viverrini , Schistosoma mansoni , and Homo sapiens , and the cathepsin L gene of S. mansoni . Colored blocks represent exons, thin lines represent introns, and the asterisks identify the exon(s) that encodes the catalytic Cys residue. Database accession numbers are provided for the illustrated sequences. (C) Comparison of the exon/intron structures of O. viverrini cathepsin F and human cathepsin F. The two enzymes were aligned for maximal homology and the amino acid sequences corresponding to an exon for each protein was separated by red (human cathepsin F) or blue ( O. viverrini cathepsin F) arrows. Exon numbering is shown below the blocks of amino acid sequences. Positions of the active site triad of Cys, His and Asn residues are indicated with stars. The position of signal sequence cleavage site is indicated with gray arrows, and the position of FhCL1 trans -processing cleavage site between prosegment and mature enzyme indicated with the yellow arrow.
    Figure Legend Snippet: Genome structure of the cathepsin F gene of Opisthorchis viverrini . (A) Structure of the O. viverrini cathepsin F gene locus, as determined by nucleotide sequence analysis of a ∼3 kb PCR product of genomic DNA of O. viverrini . The top panel shows the size of the cDNA while the bottom panel shows a schematic of positions and relative sizes of the 7 exons and 6 introns (arrowed) that comprise the gene. The 5′-flanking region was generated by inverse PCR. (B) Schematic to compare the structure and exon numbers of the C1 family genes, including the cathepsin F genes of O. viverrini , Schistosoma mansoni , and Homo sapiens , and the cathepsin L gene of S. mansoni . Colored blocks represent exons, thin lines represent introns, and the asterisks identify the exon(s) that encodes the catalytic Cys residue. Database accession numbers are provided for the illustrated sequences. (C) Comparison of the exon/intron structures of O. viverrini cathepsin F and human cathepsin F. The two enzymes were aligned for maximal homology and the amino acid sequences corresponding to an exon for each protein was separated by red (human cathepsin F) or blue ( O. viverrini cathepsin F) arrows. Exon numbering is shown below the blocks of amino acid sequences. Positions of the active site triad of Cys, His and Asn residues are indicated with stars. The position of signal sequence cleavage site is indicated with gray arrows, and the position of FhCL1 trans -processing cleavage site between prosegment and mature enzyme indicated with the yellow arrow.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Generated, Inverse PCR

    Immunolocalization of cathepsin F cysteine protease in adult Opisthorchis viverrini using thin sections of paraffin embedded worms probed with rabbit antiserum. (A) Representative section spanning the gut, vitellaria, parenchyma and tegument, probed with pre-immunization serum (negative control). Sections of adults, probed with the rabbit anti-cathepsin F serum, the vicinity of the tegument and vitellaria (B), tegument and gut (C), and testis, seminal receptacle, parenchyma and eggs (D). Gut (g), vitelline glands (v), egg (e) and testis (T) all showed strong positive reactions whereas the sperm seminal receptacle (s) was negative. The tegument (t) was faintly positive but the tegumental cells were negative for the cathepsin F cysteine protease. Immunoperoxidase staining, original magnification, ×100.
    Figure Legend Snippet: Immunolocalization of cathepsin F cysteine protease in adult Opisthorchis viverrini using thin sections of paraffin embedded worms probed with rabbit antiserum. (A) Representative section spanning the gut, vitellaria, parenchyma and tegument, probed with pre-immunization serum (negative control). Sections of adults, probed with the rabbit anti-cathepsin F serum, the vicinity of the tegument and vitellaria (B), tegument and gut (C), and testis, seminal receptacle, parenchyma and eggs (D). Gut (g), vitelline glands (v), egg (e) and testis (T) all showed strong positive reactions whereas the sperm seminal receptacle (s) was negative. The tegument (t) was faintly positive but the tegumental cells were negative for the cathepsin F cysteine protease. Immunoperoxidase staining, original magnification, ×100.

    Techniques Used: Negative Control, Immunoperoxidase Staining

    6) Product Images from "Genetic Testing Confirmed the Early Diagnosis of X-Linked Hypophosphatemic Rickets in a 7-Month-Old Infant"

    Article Title: Genetic Testing Confirmed the Early Diagnosis of X-Linked Hypophosphatemic Rickets in a 7-Month-Old Infant

    Journal: Journal of Investigative Medicine High Impact Case Reports

    doi: 10.1177/2324709615598167

    (A) Pedigree of a Chinese family with XLHR. Arrow indicates the proband. II-1 is the affected son and II-2 is the pre-symptomatic affected son. (B-F) Electropherograms showing the aligned genomic sequence of the last 10 nucleotides of the 3′ splice acceptor site of intron 9 and the first 47 nucleotides of exon 10. Nucleotide position of c.1080-2A > C mutation is indicated by an arrow. The wild-type A nucleotide was detected in a normal control (B) and in the father (C). The mutation (A > C nucleotide change) was detected in heterozygous state in the mother (D) and in hemizygous state in the 2 male offspring (E and F).
    Figure Legend Snippet: (A) Pedigree of a Chinese family with XLHR. Arrow indicates the proband. II-1 is the affected son and II-2 is the pre-symptomatic affected son. (B-F) Electropherograms showing the aligned genomic sequence of the last 10 nucleotides of the 3′ splice acceptor site of intron 9 and the first 47 nucleotides of exon 10. Nucleotide position of c.1080-2A > C mutation is indicated by an arrow. The wild-type A nucleotide was detected in a normal control (B) and in the father (C). The mutation (A > C nucleotide change) was detected in heterozygous state in the mother (D) and in hemizygous state in the 2 male offspring (E and F).

    Techniques Used: Sequencing, Mutagenesis

    (A) Electrophoresis of RT-PCR products derived from the mother (proband), the 2 sons and the controls. Lane 1, mother; Lane 2, father; Lane 3, elder son; Lane 4, younger son; Lane 5, normal female control; Lane 6, normal male control; Lane 7, non-template control; Lane 8, DNA marker. Electropherograms showing the nucleotides of the RT-PCR products of 594 bp in (B) and 500 bp in (C) The 94-bp coding sequence of exon 10 was missing in the 500-bp RT-PCR product in (C). (D) Diagrammatic representation of the genomic region of the PHEX gene. Exons 1 to 22 are represented as numbered boxes while grayed boxes are intronic regions. (E) Schematic representation of aberrant mRNA transcripts. In the mutant allele with c.1080A > C mutation, the altered splice acceptor site at intron 9 causes the production of an aberrant mRNA transcript with skipped (loss of) exon 10. (F) Parts of the protein translation from the aberrant (upper) and normal (lower) mRNA transcripts are compared. Premature stop codon is introduced at codon 361 resulting in a truncated PHEX protein with 360 amino acid fragments.
    Figure Legend Snippet: (A) Electrophoresis of RT-PCR products derived from the mother (proband), the 2 sons and the controls. Lane 1, mother; Lane 2, father; Lane 3, elder son; Lane 4, younger son; Lane 5, normal female control; Lane 6, normal male control; Lane 7, non-template control; Lane 8, DNA marker. Electropherograms showing the nucleotides of the RT-PCR products of 594 bp in (B) and 500 bp in (C) The 94-bp coding sequence of exon 10 was missing in the 500-bp RT-PCR product in (C). (D) Diagrammatic representation of the genomic region of the PHEX gene. Exons 1 to 22 are represented as numbered boxes while grayed boxes are intronic regions. (E) Schematic representation of aberrant mRNA transcripts. In the mutant allele with c.1080A > C mutation, the altered splice acceptor site at intron 9 causes the production of an aberrant mRNA transcript with skipped (loss of) exon 10. (F) Parts of the protein translation from the aberrant (upper) and normal (lower) mRNA transcripts are compared. Premature stop codon is introduced at codon 361 resulting in a truncated PHEX protein with 360 amino acid fragments.

    Techniques Used: Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Marker, Sequencing, Mutagenesis

    7) Product Images from "Simplified strategy for rapid first-line screening of fragile X syndrome: closed-tube triplet-primed PCR and amplicon melt peak analysis"

    Article Title: Simplified strategy for rapid first-line screening of fragile X syndrome: closed-tube triplet-primed PCR and amplicon melt peak analysis

    Journal: Expert Reviews in Molecular Medicine

    doi: 10.1017/erm.2015.5

    Direct TP-PCR normalised melt curves (a, b) and the derivative melt peaks (c, d) of 13 CCR reference male and 16 CCR reference female samples, followed by the GeneScan electropherograms of representative samples (bottom). Grey melt curves and peaks indicate the MCA profiles of the internal reference controls. GeneScan electropherograms of samples marked with asterisk (*) are shown in Figure 1 .
    Figure Legend Snippet: Direct TP-PCR normalised melt curves (a, b) and the derivative melt peaks (c, d) of 13 CCR reference male and 16 CCR reference female samples, followed by the GeneScan electropherograms of representative samples (bottom). Grey melt curves and peaks indicate the MCA profiles of the internal reference controls. GeneScan electropherograms of samples marked with asterisk (*) are shown in Figure 1 .

    Techniques Used: Polymerase Chain Reaction

    dTP-PCR MCA profiles (left) and GeneScan electropherograms (right) of eight FMR1 genotype-known reference male and female DNA samples. Coriell IDs and CGG repeat sizes of the samples are indicated on the left and the melt peak temperatures ( T m ) are indicated on the MCA profile of each sample. The −d F /d T values are shown on the y -axis and the temperatures (°C) are shown on the x -axis. Distribution pattern of AGG interruptions within the CGG repeat region are shown on the top right corner of each dTP-PCR GeneScan electropherogram, where a ‘+’ sign represents an AGG interruption. Number of CGG repeats is indicated by numbered black and grey arrows. Red arrowheads in the inset panels indicate the base-pair (bp) size, and the red peaks in the main panel are from a ROX-labelled internal size calibrator, whose bp sizes are indicated at the bottom of the electropherogram panel. rpts: total number of CGG repeats including AGG interruptions.
    Figure Legend Snippet: dTP-PCR MCA profiles (left) and GeneScan electropherograms (right) of eight FMR1 genotype-known reference male and female DNA samples. Coriell IDs and CGG repeat sizes of the samples are indicated on the left and the melt peak temperatures ( T m ) are indicated on the MCA profile of each sample. The −d F /d T values are shown on the y -axis and the temperatures (°C) are shown on the x -axis. Distribution pattern of AGG interruptions within the CGG repeat region are shown on the top right corner of each dTP-PCR GeneScan electropherogram, where a ‘+’ sign represents an AGG interruption. Number of CGG repeats is indicated by numbered black and grey arrows. Red arrowheads in the inset panels indicate the base-pair (bp) size, and the red peaks in the main panel are from a ROX-labelled internal size calibrator, whose bp sizes are indicated at the bottom of the electropherogram panel. rpts: total number of CGG repeats including AGG interruptions.

    Techniques Used: Polymerase Chain Reaction

    Direct TP-PCR normalised melt curves (a, b) and the derivative melt peaks (c, d) of 107 archived patient DNA samples, followed by the GeneScan electropherograms (bottom) of selected samples. MCA profiles of samples carrying NL and expanded FMR1 alleles are clustered to the left and right of the ‘Indeterminate Zones’ (highlighted in grey), respectively.
    Figure Legend Snippet: Direct TP-PCR normalised melt curves (a, b) and the derivative melt peaks (c, d) of 107 archived patient DNA samples, followed by the GeneScan electropherograms (bottom) of selected samples. MCA profiles of samples carrying NL and expanded FMR1 alleles are clustered to the left and right of the ‘Indeterminate Zones’ (highlighted in grey), respectively.

    Techniques Used: Polymerase Chain Reaction

    Direct TP-PCR melt peaks (left) and GeneScan electropherograms (right) of NL/FM DNA mixtures. Presence of FM allele in the NL/FM DNA mixtures was confirmed by the identification of MCA peaks with higher T m in the melting domains highlighted in pink.
    Figure Legend Snippet: Direct TP-PCR melt peaks (left) and GeneScan electropherograms (right) of NL/FM DNA mixtures. Presence of FM allele in the NL/FM DNA mixtures was confirmed by the identification of MCA peaks with higher T m in the melting domains highlighted in pink.

    Techniques Used: Polymerase Chain Reaction

    8) Product Images from "Nanopore sequencing as a scalable, cost-effective platform for analyzing polyclonal vector integration sites following clinical T cell therapy"

    Article Title: Nanopore sequencing as a scalable, cost-effective platform for analyzing polyclonal vector integration sites following clinical T cell therapy

    Journal: Journal for Immunotherapy of Cancer

    doi: 10.1136/jitc-2019-000299

    Schematic and agarose gel electrophoresis of PCR amplification products. (A) Schematic representations of PCR amplicons from inverse PCR (upper) and cassette ligation PCR (lower). The theoretical minimum lengths including the tailing sequences (22 bp x 2) are 929 bp and 406 bp, respectively. (B) 1% agarose gel electrophoresis of inverse PCR (left) and cassette ligation PCR (right) products. Lane 0: non-transduced genomic DNA (negative control). A small proportion of bands (especially sample 5A) were smaller than the theoretical minimum lengths. (C) 2% agarose gel electrophoresis of PCR products after SmaI digestion. Arrows point to the expected specific bands.
    Figure Legend Snippet: Schematic and agarose gel electrophoresis of PCR amplification products. (A) Schematic representations of PCR amplicons from inverse PCR (upper) and cassette ligation PCR (lower). The theoretical minimum lengths including the tailing sequences (22 bp x 2) are 929 bp and 406 bp, respectively. (B) 1% agarose gel electrophoresis of inverse PCR (left) and cassette ligation PCR (right) products. Lane 0: non-transduced genomic DNA (negative control). A small proportion of bands (especially sample 5A) were smaller than the theoretical minimum lengths. (C) 2% agarose gel electrophoresis of PCR products after SmaI digestion. Arrows point to the expected specific bands.

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Inverse PCR, Ligation, Negative Control

    Read alignment and clustering. (A) Read-length distribution for the polyclonal clinical samples by inverse PCR (top row) and cassette ligation PCR (bottom row) relative to their respective minimum theoretical lengths of 885 and 335 bp (after cassette trimming). Shown are reads that were below the minimum theoretical length (black), reads that were above the minimum theoretical length but did not have the expected flanking sequences (orange), and reads with the expected flanking sequences that were aligned (green) or unaligned (blue) to genomic DNA. (B) Schematic representation of the dominant short inverse PCR amplicons in samples 4A (i and ii) and 5A (iii). Note that the junctions between the 3′LTR and transgene were formed by circularization of DNA fragments and were not the native junction. (C) Schematic representation of the formation of short amplicons from DNA fragments. (D) Distribution of number of reads per cluster. Shown are data for all 12 samples. (E) Span of 3′LTR-genome junction for clusters that contained two to four reads. ‘0’ indicates identical 3′LTR-genome junction for all reads within the cluster. (F) Span of 3′LTR-genome junction that includes 80% of reads in clusters that include five or more reads. (G) Number of clusters per clone after merging clusters with overlapping read alignment.
    Figure Legend Snippet: Read alignment and clustering. (A) Read-length distribution for the polyclonal clinical samples by inverse PCR (top row) and cassette ligation PCR (bottom row) relative to their respective minimum theoretical lengths of 885 and 335 bp (after cassette trimming). Shown are reads that were below the minimum theoretical length (black), reads that were above the minimum theoretical length but did not have the expected flanking sequences (orange), and reads with the expected flanking sequences that were aligned (green) or unaligned (blue) to genomic DNA. (B) Schematic representation of the dominant short inverse PCR amplicons in samples 4A (i and ii) and 5A (iii). Note that the junctions between the 3′LTR and transgene were formed by circularization of DNA fragments and were not the native junction. (C) Schematic representation of the formation of short amplicons from DNA fragments. (D) Distribution of number of reads per cluster. Shown are data for all 12 samples. (E) Span of 3′LTR-genome junction for clusters that contained two to four reads. ‘0’ indicates identical 3′LTR-genome junction for all reads within the cluster. (F) Span of 3′LTR-genome junction that includes 80% of reads in clusters that include five or more reads. (G) Number of clusters per clone after merging clusters with overlapping read alignment.

    Techniques Used: Inverse PCR, Ligation, Polymerase Chain Reaction

    Schematic for PCR amplification of flanking genomic sequences. (A) Genomic DNA is digested with two 6-cutter restriction enzymes, NcoI and BspHI , which together are anticipated to cut at approximately 2 kb intervals. There are four restriction sites within the transgene sequence, the most distal of which is 1185 bp from the 3′LTR / genomic junction. NcoI and BspHI generate identical 4-nucleotide 5′ overhangs: 5′-CATG-3′, which can be circularized for inverse PCR or ligated to linker cassettes. (B) Inverse PCR begins with circularization with T4 DNA ligase, followed by PCR amplification of the unknown flanking genomic sequences using primers targeting the 3′LTR and the 3′LTR/distal transgene junction, indicated by continuous arrows. This is followed by nested PCR, indicated by dotted arrows, which incorporates tailing sequences for subsequent barcoding. The combined lengths of the dotted lines in the inner circle indicate the minimum theoretical length prior to the addition of tailing sequences and barcodes. (C) The ligation cassette comprises two partially complementary strands: a 27-nucleotide strand and a 14-nucleotide strand, the latter with a mismatched A at the 3′ end and a 5′overhang (5′-ATG-3′). Before cassette ligation, the genomic DNA fragments are filled with a single ddCTP to prevent elongation or ligation at the recessed 3′ end. Cassette ligation results in a nick on this strand, indicated by ‘X’. During the first cycle of PCR, fragments containing flanking genomic DNA are amplified by a primer spanning the transgene/3′LTR. The longer cassette strand does not prime because its complementary shorter strand has not ligated; whereas the shorter cassette strand does not prime because only 10 nucleotides are complementary to the longer cassette strand, resulting in a low annealing temperature. This cassette design limits the amplification of non-flanking genomic DNA and reduces PCR blocking by the shorter cassette strand. Subsequent cycles are primed by both the transgene/3′LTR primer and the longer cassette strand.
    Figure Legend Snippet: Schematic for PCR amplification of flanking genomic sequences. (A) Genomic DNA is digested with two 6-cutter restriction enzymes, NcoI and BspHI , which together are anticipated to cut at approximately 2 kb intervals. There are four restriction sites within the transgene sequence, the most distal of which is 1185 bp from the 3′LTR / genomic junction. NcoI and BspHI generate identical 4-nucleotide 5′ overhangs: 5′-CATG-3′, which can be circularized for inverse PCR or ligated to linker cassettes. (B) Inverse PCR begins with circularization with T4 DNA ligase, followed by PCR amplification of the unknown flanking genomic sequences using primers targeting the 3′LTR and the 3′LTR/distal transgene junction, indicated by continuous arrows. This is followed by nested PCR, indicated by dotted arrows, which incorporates tailing sequences for subsequent barcoding. The combined lengths of the dotted lines in the inner circle indicate the minimum theoretical length prior to the addition of tailing sequences and barcodes. (C) The ligation cassette comprises two partially complementary strands: a 27-nucleotide strand and a 14-nucleotide strand, the latter with a mismatched A at the 3′ end and a 5′overhang (5′-ATG-3′). Before cassette ligation, the genomic DNA fragments are filled with a single ddCTP to prevent elongation or ligation at the recessed 3′ end. Cassette ligation results in a nick on this strand, indicated by ‘X’. During the first cycle of PCR, fragments containing flanking genomic DNA are amplified by a primer spanning the transgene/3′LTR. The longer cassette strand does not prime because its complementary shorter strand has not ligated; whereas the shorter cassette strand does not prime because only 10 nucleotides are complementary to the longer cassette strand, resulting in a low annealing temperature. This cassette design limits the amplification of non-flanking genomic DNA and reduces PCR blocking by the shorter cassette strand. Subsequent cycles are primed by both the transgene/3′LTR primer and the longer cassette strand.

    Techniques Used: Polymerase Chain Reaction, Amplification, Genomic Sequencing, Sequencing, Inverse PCR, Nested PCR, Ligation, Blocking Assay

    9) Product Images from "Viruses and atypical bacteria in the respiratory tract of immunocompromised and immunocompetent patients with airway infection"

    Article Title: Viruses and atypical bacteria in the respiratory tract of immunocompromised and immunocompetent patients with airway infection

    Journal: European Journal of Clinical Microbiology & Infectious Diseases

    doi: 10.1007/s10096-020-03878-9

    Comparison of EBV-DNA copies/ml in respiratory specimens from immunocompromised and immunocompetent patients. Data are presented in a logarithmic scale. The median EBV concentration is significantly higher in immunocompromised patients ( p = 0.030, Mann-Whitney U test)
    Figure Legend Snippet: Comparison of EBV-DNA copies/ml in respiratory specimens from immunocompromised and immunocompetent patients. Data are presented in a logarithmic scale. The median EBV concentration is significantly higher in immunocompromised patients ( p = 0.030, Mann-Whitney U test)

    Techniques Used: Concentration Assay, MANN-WHITNEY

    10) Product Images from "Na/K-ATPase signaling mediates miR-29b-3p regulation and cardiac fibrosis formation in mice with chronic kidney disease"

    Article Title: Na/K-ATPase signaling mediates miR-29b-3p regulation and cardiac fibrosis formation in mice with chronic kidney disease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0197688

    The effect of NFκB inhibitor on ouabain-induced miR-29b-3p regulation in cardiac fibroblasts isolated from WT and α1+/- mice. Isolated primary cultures of mouse cardiac fibroblasts were pretreated with 1μM of the NFκB inhibitor BAY 11–7082 for 15 min followed by ouabain (10 or 100nM) treatment for 24h. Non-treated or ouabain treatment alone without BAY 11–7082 was used as control. After treatment, cell lysates were collected. Expression of miR-29b-3p was measured using RT-qPCR in RNA isolated from cells from WT animals ( A ) and α1 +/- animals ( B ). Fibroblasts were were obtained from 4 animals in each group. Data was analyzed using One-Way ANOVA with GraphPad software 7.0. * indicates p
    Figure Legend Snippet: The effect of NFκB inhibitor on ouabain-induced miR-29b-3p regulation in cardiac fibroblasts isolated from WT and α1+/- mice. Isolated primary cultures of mouse cardiac fibroblasts were pretreated with 1μM of the NFκB inhibitor BAY 11–7082 for 15 min followed by ouabain (10 or 100nM) treatment for 24h. Non-treated or ouabain treatment alone without BAY 11–7082 was used as control. After treatment, cell lysates were collected. Expression of miR-29b-3p was measured using RT-qPCR in RNA isolated from cells from WT animals ( A ) and α1 +/- animals ( B ). Fibroblasts were were obtained from 4 animals in each group. Data was analyzed using One-Way ANOVA with GraphPad software 7.0. * indicates p

    Techniques Used: Isolation, Mouse Assay, Expressing, Quantitative RT-PCR, Software

    PNx-induced miR-29b-3p expression change in WT and α1+/- mice. WT and α1+/- mice were subjected to sham or 5/6 th partial nephrectomy (PNx) surgery and left ventricle tissue was collected at the end of 16 th week for RNA extraction, RT-qPCR, and Western blot analyses. A): Western blot of Na/K-ATPase α1 subunit expression in left ventricle tissue from different group of mice. !! indicates p
    Figure Legend Snippet: PNx-induced miR-29b-3p expression change in WT and α1+/- mice. WT and α1+/- mice were subjected to sham or 5/6 th partial nephrectomy (PNx) surgery and left ventricle tissue was collected at the end of 16 th week for RNA extraction, RT-qPCR, and Western blot analyses. A): Western blot of Na/K-ATPase α1 subunit expression in left ventricle tissue from different group of mice. !! indicates p

    Techniques Used: Expressing, Mouse Assay, RNA Extraction, Quantitative RT-PCR, Western Blot

    Injection of pNaKtide diminishes PNx-induced decrease in miR-29b-3p expression. Total RNA obtained from left ventricle tissue were used for RT-qPCR analyses. Expression of miR-29b-3p was presented as fold regulation relevant to WT sham animals. Data were obtained from 5 animals in each group and was analyzed using Two-Way ANOVA with GraphPad software 7.0. * indicates p
    Figure Legend Snippet: Injection of pNaKtide diminishes PNx-induced decrease in miR-29b-3p expression. Total RNA obtained from left ventricle tissue were used for RT-qPCR analyses. Expression of miR-29b-3p was presented as fold regulation relevant to WT sham animals. Data were obtained from 5 animals in each group and was analyzed using Two-Way ANOVA with GraphPad software 7.0. * indicates p

    Techniques Used: Injection, Expressing, Quantitative RT-PCR, Software

    11) Product Images from "LCP1 preferentially binds clasped αMβ2 integrin and attenuates leukocyte adhesion under flow"

    Article Title: LCP1 preferentially binds clasped αMβ2 integrin and attenuates leukocyte adhesion under flow

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.218214

    Interactome analysis of individual, lipid-incorporated α and β integrin TMcyto domains. (A) Pull-down of talin and kindlin-2 using recombinant His-tagged α5–TMcyto or β1–TMcyto proteins with or without bicelle
    Figure Legend Snippet: Interactome analysis of individual, lipid-incorporated α and β integrin TMcyto domains. (A) Pull-down of talin and kindlin-2 using recombinant His-tagged α5–TMcyto or β1–TMcyto proteins with or without bicelle

    Techniques Used: Recombinant

    12) Product Images from "Fragile X CGG Repeat Variation in Tamil Nadu, South India: A Comparison of Radioactive and Methylation-Specific Polymerase Chain Reaction in CGG Repeat Sizing"

    Article Title: Fragile X CGG Repeat Variation in Tamil Nadu, South India: A Comparison of Radioactive and Methylation-Specific Polymerase Chain Reaction in CGG Repeat Sizing

    Journal: Genetic Testing and Molecular Biomarkers

    doi: 10.1089/gtmb.2011.0102

    Representative GeneScan profiles of four fluorescent Ms-PCR samples used in validation-II. The mTP, met, and nonmet-PCR products appear as black, blue (#), and green (*) peaks, respectively. FMR1 alleles are sized by using Rox-labeled internal size calibrator.
    Figure Legend Snippet: Representative GeneScan profiles of four fluorescent Ms-PCR samples used in validation-II. The mTP, met, and nonmet-PCR products appear as black, blue (#), and green (*) peaks, respectively. FMR1 alleles are sized by using Rox-labeled internal size calibrator.

    Techniques Used: Mass Spectrometry, Polymerase Chain Reaction, Labeling

    13) Product Images from "Lymphocytic infiltration leads to degradation of lacrimal gland extracellular matrix structures in NOD mice exhibiting a Sj?gren's syndrome-like exocrinopathy"

    Article Title: Lymphocytic infiltration leads to degradation of lacrimal gland extracellular matrix structures in NOD mice exhibiting a Sj?gren's syndrome-like exocrinopathy

    Journal: Experimental eye research

    doi: 10.1016/j.exer.2009.10.008

    Immunofluorescence images of LG tissues from 18 week old BALB/c ( A, D, G, J, M ), NOD ( B, E, H, K, N ) and NOD- scid ( C, F, I, L, O ) mice showing F-actin staining ( red ) as well as positive staining ( green ) for: tropoelastin ( A–C ); fibrillin 1 ( D–F
    Figure Legend Snippet: Immunofluorescence images of LG tissues from 18 week old BALB/c ( A, D, G, J, M ), NOD ( B, E, H, K, N ) and NOD- scid ( C, F, I, L, O ) mice showing F-actin staining ( red ) as well as positive staining ( green ) for: tropoelastin ( A–C ); fibrillin 1 ( D–F

    Techniques Used: Immunofluorescence, Mouse Assay, Staining

    14) Product Images from "Detection of mismatched 5-hydroxymethyluracil in DNA by selective chemical labeling"

    Article Title: Detection of mismatched 5-hydroxymethyluracil in DNA by selective chemical labeling

    Journal: Methods (San Diego, Calif.)

    doi: 10.1016/j.ymeth.2014.11.007

    Overview of the chemical labeling and enrichment strategy for mismatched 5hmU. Genomic DNA is first treated with recombinant mTet1 to convert all 5mC and 5hmC to 5caC. Then βGT is utilized to selectively label mismatched 5hmU with N 3 -glucose.
    Figure Legend Snippet: Overview of the chemical labeling and enrichment strategy for mismatched 5hmU. Genomic DNA is first treated with recombinant mTet1 to convert all 5mC and 5hmC to 5caC. Then βGT is utilized to selectively label mismatched 5hmU with N 3 -glucose.

    Techniques Used: Labeling, Recombinant

    15) Product Images from "Major Intercontinentally Distributed Sequence Types of Kingella kingae and Development of a Rapid Molecular Typing Tool"

    Article Title: Major Intercontinentally Distributed Sequence Types of Kingella kingae and Development of a Rapid Molecular Typing Tool

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.01609-14

    Dendrogram, using Pearson's correlation and constructed by using the UPGMA method, of PCR targeting the DNA uptake sequence on 7 to 8 randomly chosen Kingella kingae strains for each of the 9 main sequence type complexes (STcs). Fingerprint profiles are
    Figure Legend Snippet: Dendrogram, using Pearson's correlation and constructed by using the UPGMA method, of PCR targeting the DNA uptake sequence on 7 to 8 randomly chosen Kingella kingae strains for each of the 9 main sequence type complexes (STcs). Fingerprint profiles are

    Techniques Used: Construct, Polymerase Chain Reaction, Sequencing

    Gel electrophoresis of the PCR targeting the Kingella kingae DNA uptake sequence. (A) One K. kingae strain was used for each of 9 studied sequence type complexes (STcs). Lane 1, STc-1; lane 2, STc-3; lane 3, STc-35; lane 4, STc-6; lane 5, STc-14; lane
    Figure Legend Snippet: Gel electrophoresis of the PCR targeting the Kingella kingae DNA uptake sequence. (A) One K. kingae strain was used for each of 9 studied sequence type complexes (STcs). Lane 1, STc-1; lane 2, STc-3; lane 3, STc-35; lane 4, STc-6; lane 5, STc-14; lane

    Techniques Used: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Sequencing

    16) Product Images from "Impact of Promoter Polymorphisms on the Transcriptional Regulation of the Organic Cation Transporter OCT1 (SLC22A1)"

    Article Title: Impact of Promoter Polymorphisms on the Transcriptional Regulation of the Organic Cation Transporter OCT1 (SLC22A1)

    Journal: Journal of Personalized Medicine

    doi: 10.3390/jpm8040042

    Clinical effects of the −1795G > A SNP. Genotyping was performed using a custom TaqMan ® SNP genotyping assay (Thermo Fisher Scientific). ( A ) Renal clearance of metformin in 51 healthy volunteers. ( B ) Efficacy of tropisetron in 45 cancer patients suffering from vomiting. ( C – E ) Shown are the area under the time-concentration curves of fenoterol, sumatriptan and the ratio of cycloguanil to proguanil in dependence of the −1795G > A genotype of the volunteers. As comparison, the effects of amino acid mutations resulting in complete loss of OCT1 activity are shown on the right of each chart as red dots. p -values are based on non-parametric comparison using the Mann–Whitney-U Test.
    Figure Legend Snippet: Clinical effects of the −1795G > A SNP. Genotyping was performed using a custom TaqMan ® SNP genotyping assay (Thermo Fisher Scientific). ( A ) Renal clearance of metformin in 51 healthy volunteers. ( B ) Efficacy of tropisetron in 45 cancer patients suffering from vomiting. ( C – E ) Shown are the area under the time-concentration curves of fenoterol, sumatriptan and the ratio of cycloguanil to proguanil in dependence of the −1795G > A genotype of the volunteers. As comparison, the effects of amino acid mutations resulting in complete loss of OCT1 activity are shown on the right of each chart as red dots. p -values are based on non-parametric comparison using the Mann–Whitney-U Test.

    Techniques Used: TaqMan SNP Genotyping Assay, Concentration Assay, Activity Assay, MANN-WHITNEY

    In vitro effects of the −1795G > A SNP on the binding of transcription factor NF-Y and OCT1 promoter activity. ( A ) Model for the allele-specific binding of the transcription factor NF-Y to a CCAAT-box built by the variant A- but not by the wild-type G-allele of −1795 G > A. ( B ) Sequences of the annealed oligonucleotides used as EMSA probes. The specific sequences are given in upper case, and the unspecific sequences used in the radioactive labeling of the EMSA probes are given in lower case letters. The SNP and mutated bases are shown in red. ( C ) A 32 P-labeled probe containing either a −1795G or −1795A probe was incubated with nuclear extracts from HepG2 (left) and Hep3B cells (right), respectively, in the absence or presence of unlabeled probes (cold competition) or antibodies (supershift). The unlabeled probes were given in 5- and 15-fold molar excess of the 32 P-labeled probe. ( D ) Luciferase reporter gene assay with the native −1736 and −1853 OCT1 promoter cloned in front of the luciferase gene in the pGL3-basic vector. The −1853 constructs include either the −1795G or the −1795 A-allele. The coordinates are given in base pairs related to the distance to the translational start site of OCT1 . The data represent means and standard deviations of at least three independent experiments conducted in duplicate.
    Figure Legend Snippet: In vitro effects of the −1795G > A SNP on the binding of transcription factor NF-Y and OCT1 promoter activity. ( A ) Model for the allele-specific binding of the transcription factor NF-Y to a CCAAT-box built by the variant A- but not by the wild-type G-allele of −1795 G > A. ( B ) Sequences of the annealed oligonucleotides used as EMSA probes. The specific sequences are given in upper case, and the unspecific sequences used in the radioactive labeling of the EMSA probes are given in lower case letters. The SNP and mutated bases are shown in red. ( C ) A 32 P-labeled probe containing either a −1795G or −1795A probe was incubated with nuclear extracts from HepG2 (left) and Hep3B cells (right), respectively, in the absence or presence of unlabeled probes (cold competition) or antibodies (supershift). The unlabeled probes were given in 5- and 15-fold molar excess of the 32 P-labeled probe. ( D ) Luciferase reporter gene assay with the native −1736 and −1853 OCT1 promoter cloned in front of the luciferase gene in the pGL3-basic vector. The −1853 constructs include either the −1795G or the −1795 A-allele. The coordinates are given in base pairs related to the distance to the translational start site of OCT1 . The data represent means and standard deviations of at least three independent experiments conducted in duplicate.

    Techniques Used: In Vitro, Binding Assay, Activity Assay, Variant Assay, Labeling, Incubation, Luciferase, Reporter Gene Assay, Clone Assay, Plasmid Preparation, Construct

    17) Product Images from "Structural and functional insights into the B30.2/SPRY domain"

    Article Title: Structural and functional insights into the B30.2/SPRY domain

    Journal: The EMBO Journal

    doi: 10.1038/sj.emboj.7600994

    Identification of the GUS-binding region of VASA. ( A ) (His) 6 -tag pull-down experiment. Each of the indicated (His) 6 -tagged MBP-fused VASA deletion mutants and GUS without a tag were mixed and incubated with Ni-NTA agarose resin (QIAGEN). The resin was vigorously washed and subjected to denaturing gel electrophoresis. ( B ) ITC analysis of the interaction between GUS and VASA. The ITC experiments were carried out by titrating 5 μl of 0.15 mM of GUS into the solution of full-length VASA (5 μM) or titrating 5 μl of 0.2 mM of the 30 amino-acid VASA peptide into the solution of GUS (9 μM) or GUS in complex with ElonginBC (9 μM). K D values were deduced from curve fittings of the integrated heat per mol of added ligand and presented in the table. The titration curves obtained for the formation of the indicated complexes are shown in the insets.
    Figure Legend Snippet: Identification of the GUS-binding region of VASA. ( A ) (His) 6 -tag pull-down experiment. Each of the indicated (His) 6 -tagged MBP-fused VASA deletion mutants and GUS without a tag were mixed and incubated with Ni-NTA agarose resin (QIAGEN). The resin was vigorously washed and subjected to denaturing gel electrophoresis. ( B ) ITC analysis of the interaction between GUS and VASA. The ITC experiments were carried out by titrating 5 μl of 0.15 mM of GUS into the solution of full-length VASA (5 μM) or titrating 5 μl of 0.2 mM of the 30 amino-acid VASA peptide into the solution of GUS (9 μM) or GUS in complex with ElonginBC (9 μM). K D values were deduced from curve fittings of the integrated heat per mol of added ligand and presented in the table. The titration curves obtained for the formation of the indicated complexes are shown in the insets.

    Techniques Used: Binding Assay, Incubation, Nucleic Acid Electrophoresis, Titration

    18) Product Images from "Quantitative threefold allele-specific PCR (QuanTAS-PCR) for highly sensitive JAK2 V617F mutant allele detection"

    Article Title: Quantitative threefold allele-specific PCR (QuanTAS-PCR) for highly sensitive JAK2 V617F mutant allele detection

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-13-206

    Increasing the assay specificity of the  JAK2  V617F mutation-specific PCR.  Samples tested: 100% mutant control DNA (MUT 100%) analysed in triplicate, 100% wild-type control DNA (WT 100%) analysed in 10 replicates, non-template control (NTC) analysed in triplicate.  A . Mutant allele-specific PCR. The reactions contained two oligonucleotides: the mutant allele-specific forward primer and the reverse primer. The graph shows a significant number of false-positive amplifications. We observed a Cq value difference of 20 cycles between the MUT 100% and the first false-positive amplification.  B . Mutant allele-specific PCR with the introduction of the wild-type specific 3′ dideoxy blocker. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele specific blocker and the reverse primer. The graph still shows the presence of a number of false positives. We observed a Cq value difference of 23 cycles between the MUT 100% and the first false-positive amplification.  C . Mutant allele-specific PCR with the introduction of 1X Q-Solution. The reactions contained two oligonucleotides: the mutant allele-specific forward and the reverse primers. The graph shows a significant reduction of false-positive amplifications to a single false positive. We observed a Cq value difference of 16 cycles between the MUT 100% and the first false-positive amplification.  D . Mutant allele-specific PCR with the introduction of both the 3′ dideoxy blocker and 1X Q-Solution. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele-specific blocker and the reverse primer. One false-positive amplification was observed, at a very late Cq value. We observed a Cq value of 23 cycles difference between the MUT 100% and the first false-positive amplification.
    Figure Legend Snippet: Increasing the assay specificity of the JAK2 V617F mutation-specific PCR. Samples tested: 100% mutant control DNA (MUT 100%) analysed in triplicate, 100% wild-type control DNA (WT 100%) analysed in 10 replicates, non-template control (NTC) analysed in triplicate. A . Mutant allele-specific PCR. The reactions contained two oligonucleotides: the mutant allele-specific forward primer and the reverse primer. The graph shows a significant number of false-positive amplifications. We observed a Cq value difference of 20 cycles between the MUT 100% and the first false-positive amplification. B . Mutant allele-specific PCR with the introduction of the wild-type specific 3′ dideoxy blocker. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele specific blocker and the reverse primer. The graph still shows the presence of a number of false positives. We observed a Cq value difference of 23 cycles between the MUT 100% and the first false-positive amplification. C . Mutant allele-specific PCR with the introduction of 1X Q-Solution. The reactions contained two oligonucleotides: the mutant allele-specific forward and the reverse primers. The graph shows a significant reduction of false-positive amplifications to a single false positive. We observed a Cq value difference of 16 cycles between the MUT 100% and the first false-positive amplification. D . Mutant allele-specific PCR with the introduction of both the 3′ dideoxy blocker and 1X Q-Solution. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele-specific blocker and the reverse primer. One false-positive amplification was observed, at a very late Cq value. We observed a Cq value of 23 cycles difference between the MUT 100% and the first false-positive amplification.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification

    19) Product Images from "Rapid Generation of MicroRNA Sponges for MicroRNA Inhibition"

    Article Title: Rapid Generation of MicroRNA Sponges for MicroRNA Inhibition

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029275

    Confirmation of miR-19 binding to miR-19 sponge constructs. ( A ) Luciferase reporter assays in HEK293 cells reveal that repression of Renilla activity is more prominent in reporter vectors that contain perfect MBS sequences as compared to reporter vectors that encode bulged MBS sequences and is in both cases dependent on the number of MBS. ( B ) Release of miR-19 specific repression of Renilla luciferase activity by anti-miR-19a/b oligos confirms that miR-19 binds to the miR-19 MBS sequences. No release of luciferase activity is observed with a control anti-miR-16 oligo. Open bars: Mock, grey bars: miR-16 inhibitor and black bar: miR-19 inhibitor mix. For each graph the number of MBS per reporter vector is indicated on the x-axis and on the y-axis the ratio of Renilla (R) over Firefly (F) luciferase is depicted. * = p-value
    Figure Legend Snippet: Confirmation of miR-19 binding to miR-19 sponge constructs. ( A ) Luciferase reporter assays in HEK293 cells reveal that repression of Renilla activity is more prominent in reporter vectors that contain perfect MBS sequences as compared to reporter vectors that encode bulged MBS sequences and is in both cases dependent on the number of MBS. ( B ) Release of miR-19 specific repression of Renilla luciferase activity by anti-miR-19a/b oligos confirms that miR-19 binds to the miR-19 MBS sequences. No release of luciferase activity is observed with a control anti-miR-16 oligo. Open bars: Mock, grey bars: miR-16 inhibitor and black bar: miR-19 inhibitor mix. For each graph the number of MBS per reporter vector is indicated on the x-axis and on the y-axis the ratio of Renilla (R) over Firefly (F) luciferase is depicted. * = p-value

    Techniques Used: Binding Assay, Construct, Luciferase, Activity Assay, Plasmid Preparation

    20) Product Images from "A genetically distinct hybrid zone occurs for two globally invasive mosquito fish species with striking phenotypic resemblance), A genetically distinct hybrid zone occurs for two globally invasive mosquito fish species with striking phenotypic resemblance"

    Article Title: A genetically distinct hybrid zone occurs for two globally invasive mosquito fish species with striking phenotypic resemblance), A genetically distinct hybrid zone occurs for two globally invasive mosquito fish species with striking phenotypic resemblance

    Journal: Ecology and Evolution

    doi: 10.1002/ece3.2562

    Microsatellite data from this study (Wilk and Horth), historical allozyme (Wooten et al., 1988 ) and mt DNA (Scribner Avise, 1993 ) patterns for mosquito fish. Bright blue, red, and green pies represent Wilk and Horth's microsatellite alleles (western, hybrid, and eastern, respectively). Pale blue, green, and black pies represent Wooten et al. ( 1988 ) allozymes. For Wooten et al.'s ( 1988 ) allozymes, the left half of each pie represents the frequency of Glycerol‐3‐phosphate dehydrogenase (G‐3) alleles; the right half, M‐aspartate amino transferase (M‐A) alleles. Alleles are color‐coded for the most common allele by geographic region. G‐3 117 is represented by blue coloration on the left half of the pie, since it was common in the west. G‐3 100 is represented by green coloration on the left half of the pie since it was common in the east. M‐A 108 is blue on the right half of the pie since it was common in the west. M‐A 100 is green and was common in the east. Rare alleles are black. Pale blue and green squares represent Scribner and Avise's ( 1993 ) mt DNA for G. affinis and G. holbrooki, respectively
    Figure Legend Snippet: Microsatellite data from this study (Wilk and Horth), historical allozyme (Wooten et al., 1988 ) and mt DNA (Scribner Avise, 1993 ) patterns for mosquito fish. Bright blue, red, and green pies represent Wilk and Horth's microsatellite alleles (western, hybrid, and eastern, respectively). Pale blue, green, and black pies represent Wooten et al. ( 1988 ) allozymes. For Wooten et al.'s ( 1988 ) allozymes, the left half of each pie represents the frequency of Glycerol‐3‐phosphate dehydrogenase (G‐3) alleles; the right half, M‐aspartate amino transferase (M‐A) alleles. Alleles are color‐coded for the most common allele by geographic region. G‐3 117 is represented by blue coloration on the left half of the pie, since it was common in the west. G‐3 100 is represented by green coloration on the left half of the pie since it was common in the east. M‐A 108 is blue on the right half of the pie since it was common in the west. M‐A 100 is green and was common in the east. Rare alleles are black. Pale blue and green squares represent Scribner and Avise's ( 1993 ) mt DNA for G. affinis and G. holbrooki, respectively

    Techniques Used: Fluorescence In Situ Hybridization, Western Blot

    21) Product Images from "Studies of Wilms' Tumor (WT1) Gene Expression in Adult Acute Leukemias in Singapore"

    Article Title: Studies of Wilms' Tumor (WT1) Gene Expression in Adult Acute Leukemias in Singapore

    Journal: Biomarker Insights

    doi:

    Nested PCR for WT1 expression.
    Figure Legend Snippet: Nested PCR for WT1 expression.

    Techniques Used: Nested PCR, Expressing

    22) Product Images from "RpoHII Activates Oxidative-Stress Defense Systems and Is Controlled by RpoE in the Singlet Oxygen-Dependent Response in Rhodobacter sphaeroides ▿ ▿ †"

    Article Title: RpoHII Activates Oxidative-Stress Defense Systems and Is Controlled by RpoE in the Singlet Oxygen-Dependent Response in Rhodobacter sphaeroides ▿ ▿ †

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00925-08

    Separation of 5′ RACE products obtained from RNA extracts of wild-type and rpoH II mutant cultures after 10 min of photooxidative stress. PCR products obtained after the second PCR (nested) were separated on a 10% polyacrylamide gel and stained with ethidium bromide. Upstream of the 5′ ends of the sequences corresponding to the depicted DNA bands, RpoH II target sequences were found and are depicted as aligned sequences below the gel image. DNA marker lanes, 100-bp ladder. In the alignment, transcription start sites are underlined and putative −35 and −10 regions are printed in bold letters. The dnaK P1 promoter sequence is shown for comparison and is recognized only by RpoH I ).
    Figure Legend Snippet: Separation of 5′ RACE products obtained from RNA extracts of wild-type and rpoH II mutant cultures after 10 min of photooxidative stress. PCR products obtained after the second PCR (nested) were separated on a 10% polyacrylamide gel and stained with ethidium bromide. Upstream of the 5′ ends of the sequences corresponding to the depicted DNA bands, RpoH II target sequences were found and are depicted as aligned sequences below the gel image. DNA marker lanes, 100-bp ladder. In the alignment, transcription start sites are underlined and putative −35 and −10 regions are printed in bold letters. The dnaK P1 promoter sequence is shown for comparison and is recognized only by RpoH I ).

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Staining, Marker, Sequencing

    23) Product Images from "RNA editing generates cellular subsets with diverse sequence within populations"

    Article Title: RNA editing generates cellular subsets with diverse sequence within populations

    Journal: Nature Communications

    doi: 10.1038/ncomms12145

    Validation of model predictions using targeted amplification of editable sites from single cells. ( a ) Wiggle plots showing coverage in 3′-untranslated regions for B2m, Anxa5 and Cybb in the 24 bone marrow-derived macrophages profiled. ( b – d ) Sequence alignments from targeted RT–PCR amplification and Sanger sequencing of bacterial colonies for ( b ) B2m, ( c ) Anxa5 and ( d ) Cybb transcripts from gDNA and cDNA from a bulk sample (amplified using standard PCR), and cDNA of single cells (amplified using a modified OneStep RT–PCR protocol, per Supplementary Fig. 4 ). Alignments, showing the sequence space surrounding a particular editable site, are clustered by sample. Alignments are colour-coded to indicate whether the sequence aligned contained (red) or lacked (grey) editing in the length of the amplicon. Though a C-to-U change may not be shown in the narrow window illustrated, a red sequence would indicate that the amplicon sequence contained at least one C-to-U edit elsewhere (red). Lack of editing in the gDNA indicates that the C-to-U transitions observed are bona fide APOBEC1-mediated RNA editing events.
    Figure Legend Snippet: Validation of model predictions using targeted amplification of editable sites from single cells. ( a ) Wiggle plots showing coverage in 3′-untranslated regions for B2m, Anxa5 and Cybb in the 24 bone marrow-derived macrophages profiled. ( b – d ) Sequence alignments from targeted RT–PCR amplification and Sanger sequencing of bacterial colonies for ( b ) B2m, ( c ) Anxa5 and ( d ) Cybb transcripts from gDNA and cDNA from a bulk sample (amplified using standard PCR), and cDNA of single cells (amplified using a modified OneStep RT–PCR protocol, per Supplementary Fig. 4 ). Alignments, showing the sequence space surrounding a particular editable site, are clustered by sample. Alignments are colour-coded to indicate whether the sequence aligned contained (red) or lacked (grey) editing in the length of the amplicon. Though a C-to-U change may not be shown in the narrow window illustrated, a red sequence would indicate that the amplicon sequence contained at least one C-to-U edit elsewhere (red). Lack of editing in the gDNA indicates that the C-to-U transitions observed are bona fide APOBEC1-mediated RNA editing events.

    Techniques Used: Amplification, Derivative Assay, Sequencing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Modification

    24) Product Images from "The protein kinase C inhibitor, Ro-31-7459, is a potent activator of ERK and JNK MAP kinases in HUVECs and yet inhibits cyclic AMP-stimulated SOCS-3 gene induction through inactivation of the transcription factor c-Jun"

    Article Title: The protein kinase C inhibitor, Ro-31-7459, is a potent activator of ERK and JNK MAP kinases in HUVECs and yet inhibits cyclic AMP-stimulated SOCS-3 gene induction through inactivation of the transcription factor c-Jun

    Journal: Cellular Signalling

    doi: 10.1016/j.cellsig.2012.04.016

    Protein kinase C inhibitors block human SOCS-3 gene induction in HUVECs. A). HUVECs were stimulated for 5 h with MG132 (10 μM) in the presence or absence of either a combination of 10 μM forskolin plus 10 μM rolipram (F/R; upper panel ) or 10 μM PMA ( lower panel ) plus the indicated concentrations of the protein kinase C (PKC) inhibitors Ro-31-7549 or GF-109203X. Cell extracts were then prepared and immunoblotted with antibodies to SOCS-3 or β-tubulin as indicated. B). HUVECs were stimulated for 5 h with MG132 (10 μM) in the presence or absence of either F/R ( upper panel ) or 10 μM PMA ( lower panel ) plus the indicated concentrations of the PKC inhibitors Ro-31-7549 or Gö-6983. Cell extracts were then prepared and immunoblotted with antibodies to SOCS-3 or β-tubulin as indicated. C). HUVECs were stimulated for 5 h in the presence or absence of F/R ( upper panel ) or 10 μM PMA ( lower panel ) plus Ro-31-7549 (5 μM), Gö-6983 (25 μM) or GF-109203X (25 μM). Total RNA was then extracted from cells and subjected to one-step RT-PCR, with specific primers towards SOCS-3 or actin, as described in Materials and methods . Amplified DNA fragments were visualised by agarose gel electrophoresis.
    Figure Legend Snippet: Protein kinase C inhibitors block human SOCS-3 gene induction in HUVECs. A). HUVECs were stimulated for 5 h with MG132 (10 μM) in the presence or absence of either a combination of 10 μM forskolin plus 10 μM rolipram (F/R; upper panel ) or 10 μM PMA ( lower panel ) plus the indicated concentrations of the protein kinase C (PKC) inhibitors Ro-31-7549 or GF-109203X. Cell extracts were then prepared and immunoblotted with antibodies to SOCS-3 or β-tubulin as indicated. B). HUVECs were stimulated for 5 h with MG132 (10 μM) in the presence or absence of either F/R ( upper panel ) or 10 μM PMA ( lower panel ) plus the indicated concentrations of the PKC inhibitors Ro-31-7549 or Gö-6983. Cell extracts were then prepared and immunoblotted with antibodies to SOCS-3 or β-tubulin as indicated. C). HUVECs were stimulated for 5 h in the presence or absence of F/R ( upper panel ) or 10 μM PMA ( lower panel ) plus Ro-31-7549 (5 μM), Gö-6983 (25 μM) or GF-109203X (25 μM). Total RNA was then extracted from cells and subjected to one-step RT-PCR, with specific primers towards SOCS-3 or actin, as described in Materials and methods . Amplified DNA fragments were visualised by agarose gel electrophoresis.

    Techniques Used: Blocking Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis

    25) Product Images from "A peroxisomally localized acyl-activating enzyme is required for volatile benzenoid formation in a Petunia×hybrida cv. 'Mitchell Diploid' flower"

    Article Title: A peroxisomally localized acyl-activating enzyme is required for volatile benzenoid formation in a Petunia×hybrida cv. 'Mitchell Diploid' flower

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/ers153

    PhAAE comparative transcript accumulation analysis between MD and two independent, homozygous T 2 ir-PhAAE lines (15.15 and 24.8). 50ng total RNA was used per reaction in all cases for one-step qRT-PCR with RNA isolated from stage 8 flowers at 16.00h. Histograms are representative of multiple experiments and multiple biological replicates, and analyzed by the ∆∆Ct method with PhFBP1 and Ph18S as the internal references. The individual petunia transcript analyzed is PhAAE (mean±SE; n =3).
    Figure Legend Snippet: PhAAE comparative transcript accumulation analysis between MD and two independent, homozygous T 2 ir-PhAAE lines (15.15 and 24.8). 50ng total RNA was used per reaction in all cases for one-step qRT-PCR with RNA isolated from stage 8 flowers at 16.00h. Histograms are representative of multiple experiments and multiple biological replicates, and analyzed by the ∆∆Ct method with PhFBP1 and Ph18S as the internal references. The individual petunia transcript analyzed is PhAAE (mean±SE; n =3).

    Techniques Used: Quantitative RT-PCR, Isolation

    PhAAE transcript accumulation analysis (one-step qRT-PCR). Spatial analysis used root, stem, stigma, anther, leaf, petal tube, petal limb, and sepal tissues of MD harvested at 16.00h (A). Floral developmental analysis used MD flowers from 11 sequential stages collected on one day at 16.00h (B). Ethylene treatment (2 µl l –1 analysis used excised MD and 44 568 whole flowers treated for 0, 1, 2, 4, and 8h (C, D). 50ng total RNA was used per reaction in all cases. Histograms are representative of multiple experiments and multiple biological replicates, and analyzed by the ∆∆Ct method with PhFBP1 and Ph18S as the internal references (mean±SE; n =3).
    Figure Legend Snippet: PhAAE transcript accumulation analysis (one-step qRT-PCR). Spatial analysis used root, stem, stigma, anther, leaf, petal tube, petal limb, and sepal tissues of MD harvested at 16.00h (A). Floral developmental analysis used MD flowers from 11 sequential stages collected on one day at 16.00h (B). Ethylene treatment (2 µl l –1 analysis used excised MD and 44 568 whole flowers treated for 0, 1, 2, 4, and 8h (C, D). 50ng total RNA was used per reaction in all cases. Histograms are representative of multiple experiments and multiple biological replicates, and analyzed by the ∆∆Ct method with PhFBP1 and Ph18S as the internal references (mean±SE; n =3).

    Techniques Used: Quantitative RT-PCR

    PhAAE comparative transcript accumulation analysis between MD and two independent, homozygous T 2 ir-PhAAE lines (15.15 and 24.8). 50ng total RNA was used per reaction in all cases for one-step qRT-PCR with RNA isolated from stage 8 flowers at 16.00h. Histograms are representative of multiple experiments and multiple biological replicates, and analyzed by the ∆∆Ct method with PhFBP1 and Ph18S as the internal references. The individual petunia transcripts analyzed are PhBSMT , PhBPBT , PhCFAT , PhIGS1 , PhPAAS , PhKAT1 , PhCM1 , PhPAL1 , PhPAL2 , PhODO1 , PhC4H1 , PhC4H2 , and PhMYB4 (mean±SE; n =3).
    Figure Legend Snippet: PhAAE comparative transcript accumulation analysis between MD and two independent, homozygous T 2 ir-PhAAE lines (15.15 and 24.8). 50ng total RNA was used per reaction in all cases for one-step qRT-PCR with RNA isolated from stage 8 flowers at 16.00h. Histograms are representative of multiple experiments and multiple biological replicates, and analyzed by the ∆∆Ct method with PhFBP1 and Ph18S as the internal references. The individual petunia transcripts analyzed are PhBSMT , PhBPBT , PhCFAT , PhIGS1 , PhPAAS , PhKAT1 , PhCM1 , PhPAL1 , PhPAL2 , PhODO1 , PhC4H1 , PhC4H2 , and PhMYB4 (mean±SE; n =3).

    Techniques Used: Quantitative RT-PCR, Isolation

    26) Product Images from "EOBII Controls Flower Opening by Functioning as a General Transcriptomic Switch 1 Controls Flower Opening by Functioning as a General Transcriptomic Switch 1 [C] Controls Flower Opening by Functioning as a General Transcriptomic Switch 1 [C] [W]"

    Article Title: EOBII Controls Flower Opening by Functioning as a General Transcriptomic Switch 1 Controls Flower Opening by Functioning as a General Transcriptomic Switch 1 [C] Controls Flower Opening by Functioning as a General Transcriptomic Switch 1 [C] [W]

    Journal: Plant Physiology

    doi: 10.1104/pp.111.176248

    qRT-PCR transcript accumulation analysis of PhEOBII from MD plants. A, Spatial analysis used total RNA from root, stem, stigma, anther, leaf, petal (P.) tube, petal (P.) limb, and sepal tissues collected at 4 pm (mean ± se ; n = 3). B, Floral developmental
    Figure Legend Snippet: qRT-PCR transcript accumulation analysis of PhEOBII from MD plants. A, Spatial analysis used total RNA from root, stem, stigma, anther, leaf, petal (P.) tube, petal (P.) limb, and sepal tissues collected at 4 pm (mean ± se ; n = 3). B, Floral developmental

    Techniques Used: Quantitative RT-PCR

    27) Product Images from "Kin5 Knockdown in Tetrahymena thermophila Using RNAi Blocks Cargo Transport of Gef1"

    Article Title: Kin5 Knockdown in Tetrahymena thermophila Using RNAi Blocks Cargo Transport of Gef1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0004873

    Optimization of KIN5 sh RNA. A. Degradation of KIN5 message after sh RNA induction in KO cells using 0–0.5 µg/ml Cd 2+ . Above: RT-PCR products resolved on a 1% agarose gel. At Cd 2+ concentrations lower than 0.5 µg/ml, KIN5 mRNA is stable for 24 h. After 24 h in 0.5 µg/ml Cd 2+ , KIN5 mRNA is dramatically decreased, while PGM1 is unaffected. B. Effect of 0.5 µg/ml Cd 2+ on KIN5 and PGM1 messages in Inv2 cells. KIN5 and PGM1 mRNA levels remain unaffected after 24 h. DNA markers shown: lines indicate 600 and 300 bp. C. Effect of 0.5 µg/ml Cd 2+ on Kin5 protein levels in KO and Inv2 cells. Corresponding KO (left) and Inv2 (right) cell homogenates 12 h post-induction at either 0 or 0.5 µg/ml Cd 2+ and blotted with K5T1 Ab to Kin5. While the Kin5 protein is severely knocked down in the KO cells upon sh RNA induction, Kin5 levels remain unaffected in Inv2 cells under similar conditions.
    Figure Legend Snippet: Optimization of KIN5 sh RNA. A. Degradation of KIN5 message after sh RNA induction in KO cells using 0–0.5 µg/ml Cd 2+ . Above: RT-PCR products resolved on a 1% agarose gel. At Cd 2+ concentrations lower than 0.5 µg/ml, KIN5 mRNA is stable for 24 h. After 24 h in 0.5 µg/ml Cd 2+ , KIN5 mRNA is dramatically decreased, while PGM1 is unaffected. B. Effect of 0.5 µg/ml Cd 2+ on KIN5 and PGM1 messages in Inv2 cells. KIN5 and PGM1 mRNA levels remain unaffected after 24 h. DNA markers shown: lines indicate 600 and 300 bp. C. Effect of 0.5 µg/ml Cd 2+ on Kin5 protein levels in KO and Inv2 cells. Corresponding KO (left) and Inv2 (right) cell homogenates 12 h post-induction at either 0 or 0.5 µg/ml Cd 2+ and blotted with K5T1 Ab to Kin5. While the Kin5 protein is severely knocked down in the KO cells upon sh RNA induction, Kin5 levels remain unaffected in Inv2 cells under similar conditions.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Stability of KIN5 and PGM1 messages. A. CU522 cells grown in starvation conditions+5.0 µg/ml Cd 2+ prior to transformation showing comparable relative stabilities of the KIN5 and PGM1 mRNA. B. Time course of degradation of KIN5 message after sh RNA induction in K5KOAs.40 cells using 5.0 µg/ml Cd 2+ . RT-PCR products resolved on a 1% agarose gel. Left lane: DNA markers. The KIN5 message decreases at 45 min post-induction and is eliminated at 60 min. The PGM1 message remains constant.
    Figure Legend Snippet: Stability of KIN5 and PGM1 messages. A. CU522 cells grown in starvation conditions+5.0 µg/ml Cd 2+ prior to transformation showing comparable relative stabilities of the KIN5 and PGM1 mRNA. B. Time course of degradation of KIN5 message after sh RNA induction in K5KOAs.40 cells using 5.0 µg/ml Cd 2+ . RT-PCR products resolved on a 1% agarose gel. Left lane: DNA markers. The KIN5 message decreases at 45 min post-induction and is eliminated at 60 min. The PGM1 message remains constant.

    Techniques Used: Transformation Assay, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis

    28) Product Images from "PIPKI?90 negatively regulates LFA-1 mediated adhesion and activation in antigen-induced CD4+ T cells !"

    Article Title: PIPKI?90 negatively regulates LFA-1 mediated adhesion and activation in antigen-induced CD4+ T cells !

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1001445

    T cells from PIPKIγ90 −/− mice are deficient in the PIPKIγ90 but not the PIPKIγ87 isoform A) Schematic showing the two isoforms of PIPK expressed in T cells. PIPKIγ87 and PIPKIγ90 differ by the presence of a talin binding 26 amino acid C-terminal domain. B) RT-PCR indicates the presence of PIPKIγ87 but not PIPKIγ90 in knockout T cells C) Immunoblotting shows loss of PIPKIγ90 expression in knockout T cells.
    Figure Legend Snippet: T cells from PIPKIγ90 −/− mice are deficient in the PIPKIγ90 but not the PIPKIγ87 isoform A) Schematic showing the two isoforms of PIPK expressed in T cells. PIPKIγ87 and PIPKIγ90 differ by the presence of a talin binding 26 amino acid C-terminal domain. B) RT-PCR indicates the presence of PIPKIγ87 but not PIPKIγ90 in knockout T cells C) Immunoblotting shows loss of PIPKIγ90 expression in knockout T cells.

    Techniques Used: Mouse Assay, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Knock-Out, Expressing

    29) Product Images from "The hub protein loquacious connects the microRNA and short interfering RNA pathways in mosquitoes"

    Article Title: The hub protein loquacious connects the microRNA and short interfering RNA pathways in mosquitoes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv152

    Characterization of dsRBP gene structure, expression and localization. ( A ) Structures of loqs-ra, loqs-rb , and l oqs-rc splice variants and r2d2 mRNA. Solid boxes represent ORFs, unfilled boxes represent UTRs, and gray bars represent predicted DRMs. Primer locations used for RT-PCR and cDNA sequencing are marked by block arrows; 3′ RACE primers indicated by open arrows. ( B ) One-step RT-PCR using head (H), thorax (T), midgut (M), sugar-fed ovaries (SFO), blood-fed ovaries (BFO), male pupae (MP), female pupae (FP) and L4 larvae (L4) total RNA as templates to detect dsRBP transcripts. ( C ) Localization of overexpressed HA or FLAG-tagged dsRBPs in Aag2 cells. HA-EGFP and HA-R2D2 were expressed via dsSINV; HA-Loqs-PA and HA-Loqs-PB were expressed via plasmid transfection. ( D ) Localization of mosquito Dcr and Ago proteins in uninfected and infected Aag2 cell fractions: cytoplasm (CP), membrane (M), nucleus (N), and cytoskeleton (CS). Antibodies recognizing β-actin (cytoplasmic) and heterochromatin protein 1 (HP1, nuclear) were used to verify the success of each fractionation experiment.
    Figure Legend Snippet: Characterization of dsRBP gene structure, expression and localization. ( A ) Structures of loqs-ra, loqs-rb , and l oqs-rc splice variants and r2d2 mRNA. Solid boxes represent ORFs, unfilled boxes represent UTRs, and gray bars represent predicted DRMs. Primer locations used for RT-PCR and cDNA sequencing are marked by block arrows; 3′ RACE primers indicated by open arrows. ( B ) One-step RT-PCR using head (H), thorax (T), midgut (M), sugar-fed ovaries (SFO), blood-fed ovaries (BFO), male pupae (MP), female pupae (FP) and L4 larvae (L4) total RNA as templates to detect dsRBP transcripts. ( C ) Localization of overexpressed HA or FLAG-tagged dsRBPs in Aag2 cells. HA-EGFP and HA-R2D2 were expressed via dsSINV; HA-Loqs-PA and HA-Loqs-PB were expressed via plasmid transfection. ( D ) Localization of mosquito Dcr and Ago proteins in uninfected and infected Aag2 cell fractions: cytoplasm (CP), membrane (M), nucleus (N), and cytoskeleton (CS). Antibodies recognizing β-actin (cytoplasmic) and heterochromatin protein 1 (HP1, nuclear) were used to verify the success of each fractionation experiment.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Sequencing, Blocking Assay, Plasmid Preparation, Transfection, Infection, Fractionation

    30) Product Images from "Functional organization of a single nif cluster in the mesophilic archaeon Methanosarcina mazei strain G?1"

    Article Title: Functional organization of a single nif cluster in the mesophilic archaeon Methanosarcina mazei strain G?1

    Journal: Archaea

    doi:

    Transcriptional analysis of the M. mazei nif gene cluster. (A) Northern blot analysis of total RNA isolated from M. mazei cells grown under conditions of nitrogen limitation (N 2 ) and nitrogen sufficiency ( NH 4 + ) using probes for nifH , nifK and nifN . Each lane was loaded with 0.25 µg total RNA from cells grown under nitrogen limitation (-) or nitrogen sufficiency (+); numbers on the left are molecular sizes in kilobases. (B) RT-PCR analysis. Reverse transcription was carried out on 0.1 µg RNA isolated from cells grown under conditions of nitrogen limitation (-) or nitrogen sufficiency (+) using the OneStep RT-PCR Kit from Qiagen and primers as described in Materials and methods. Control PCR reactions with RNA in the absence of reverse transcriptase showed that the isolated RNA preparations were free of genomic DNA. As a control, a 16S rDNA-specific RT-PCR was carried out on 10 ng of RNA from cells from each growth condition. Products of the expected size (450 bp ( nifH ), 417 bp ( nifK ), 438 bp ( nifN ), 415 bp ( glnK 1 ) and 420 bp (16S rDNA)) were separated in 1.5% agarose gels and visualized by ethidium bromide staining.
    Figure Legend Snippet: Transcriptional analysis of the M. mazei nif gene cluster. (A) Northern blot analysis of total RNA isolated from M. mazei cells grown under conditions of nitrogen limitation (N 2 ) and nitrogen sufficiency ( NH 4 + ) using probes for nifH , nifK and nifN . Each lane was loaded with 0.25 µg total RNA from cells grown under nitrogen limitation (-) or nitrogen sufficiency (+); numbers on the left are molecular sizes in kilobases. (B) RT-PCR analysis. Reverse transcription was carried out on 0.1 µg RNA isolated from cells grown under conditions of nitrogen limitation (-) or nitrogen sufficiency (+) using the OneStep RT-PCR Kit from Qiagen and primers as described in Materials and methods. Control PCR reactions with RNA in the absence of reverse transcriptase showed that the isolated RNA preparations were free of genomic DNA. As a control, a 16S rDNA-specific RT-PCR was carried out on 10 ng of RNA from cells from each growth condition. Products of the expected size (450 bp ( nifH ), 417 bp ( nifK ), 438 bp ( nifN ), 415 bp ( glnK 1 ) and 420 bp (16S rDNA)) were separated in 1.5% agarose gels and visualized by ethidium bromide staining.

    Techniques Used: Northern Blot, Isolation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Staining

    31) Product Images from "Dynamic glucoregulation and mammalian-like responses to metabolic and developmental disruption in zebrafish"

    Article Title: Dynamic glucoregulation and mammalian-like responses to metabolic and developmental disruption in zebrafish

    Journal: General and comparative endocrinology

    doi: 10.1016/j.ygcen.2010.10.010

    Temporal and spatial expression of pck1 during zebrafish development. (A) Phylogenetic analysis distinguishes Pck1- from Pck2-related proteins. (B) Semiquantitative RT-PCR showing onset of pck1 and pck2 gene expression. Note that a small amount of maternally provided pck1 is present in 16 cell embryos. Low-level zygotic expression of pck2 is first detectable at 6 hpf. (C) RNA:RNA in situ hybridization demonstrates pck1 expression in the 11 hpf YSL, brain, and tail. In 24 hpf embryos, pck1 is expressed in the eye and tail. By 48 hpf pck1 is expressed at the margin between the yolk extension and the embryo proper (red arrow). Expression in discrete YSL clusters (black arrows) as well as fin buds and pharyngeal arches is also seen. At 72 and 96 hpf, pck1 expression is seen in the liver, YSL, and cranial neuromasts. b, brain; e, eye; fb, fin bud; nm, neuromasts; pa, pharyngeal arch; tb, and tail bud.
    Figure Legend Snippet: Temporal and spatial expression of pck1 during zebrafish development. (A) Phylogenetic analysis distinguishes Pck1- from Pck2-related proteins. (B) Semiquantitative RT-PCR showing onset of pck1 and pck2 gene expression. Note that a small amount of maternally provided pck1 is present in 16 cell embryos. Low-level zygotic expression of pck2 is first detectable at 6 hpf. (C) RNA:RNA in situ hybridization demonstrates pck1 expression in the 11 hpf YSL, brain, and tail. In 24 hpf embryos, pck1 is expressed in the eye and tail. By 48 hpf pck1 is expressed at the margin between the yolk extension and the embryo proper (red arrow). Expression in discrete YSL clusters (black arrows) as well as fin buds and pharyngeal arches is also seen. At 72 and 96 hpf, pck1 expression is seen in the liver, YSL, and cranial neuromasts. b, brain; e, eye; fb, fin bud; nm, neuromasts; pa, pharyngeal arch; tb, and tail bud.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, RNA In Situ Hybridization

    Gene expression during zebrafish development. (A) Relative, quantitative expression of insa (open bars) and insb (black bars) during development. (B). Relative, quantitative expression of pck1 during development. (C) Non-quantitative RT-PCR demonstrates early expression of insulin receptors a and b .
    Figure Legend Snippet: Gene expression during zebrafish development. (A) Relative, quantitative expression of insa (open bars) and insb (black bars) during development. (B). Relative, quantitative expression of pck1 during development. (C) Non-quantitative RT-PCR demonstrates early expression of insulin receptors a and b .

    Techniques Used: Expressing, Quantitative RT-PCR

    32) Product Images from "The Arabidopsis homolog of human minor spliceosomal protein U11-48K plays a crucial role in U12 intron splicing and plant development"

    Article Title: The Arabidopsis homolog of human minor spliceosomal protein U11-48K plays a crucial role in U12 intron splicing and plant development

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/erw158

    Domain structure and cellular localization of the Arabidopsis homolog of human U11-48K protein and generation of artificial miRNA-mediated knockdown plants. (A) Schematic representation of the domain structure of the Arabidopsis homolog of human U11-48K. The conserved CHHC-type zinc finger (ZF) motif and arginine (Arg)-rich region are shown. (B) GFP signals from the 48K–GFP-expressing tobacco plant were observed using a confocal microscope. DAPI was used to stain the nucleus. Scale bar=10 μm. (C) Position of the artificial miRNA1 (amiR1) target site and the sequences of amiR1, along with its target, U11-48K (48K). Exons and introns are represented as gray boxes and thick lines, respectively, and the untranslated regions are represented as white boxes. (D) Confirmation of mature amiR1 generation. Total RNA extracted from each transgenic line (amiR1-1, amiR1-2, and amiR1-3) was separated via denaturing 12% PAGE, and the expression of 21 nucleotide long mature amiR1 in each line was confirmed by northern blotting. (E, F) Down-regulation of U11-48K in the transgenic plants. The levels of U11-48K in each transgenic plant were confirmed by (E) RT–PCR and (F) real-time RT–PCR analysis. The numbers 1, 2, and 3 in (F) indicate amiR1-1, amiR1-2, and amiR1-3, respectively. Values are means ±SE obtained from three independent biological replicates. (This figure is available in colour at JXB online.)
    Figure Legend Snippet: Domain structure and cellular localization of the Arabidopsis homolog of human U11-48K protein and generation of artificial miRNA-mediated knockdown plants. (A) Schematic representation of the domain structure of the Arabidopsis homolog of human U11-48K. The conserved CHHC-type zinc finger (ZF) motif and arginine (Arg)-rich region are shown. (B) GFP signals from the 48K–GFP-expressing tobacco plant were observed using a confocal microscope. DAPI was used to stain the nucleus. Scale bar=10 μm. (C) Position of the artificial miRNA1 (amiR1) target site and the sequences of amiR1, along with its target, U11-48K (48K). Exons and introns are represented as gray boxes and thick lines, respectively, and the untranslated regions are represented as white boxes. (D) Confirmation of mature amiR1 generation. Total RNA extracted from each transgenic line (amiR1-1, amiR1-2, and amiR1-3) was separated via denaturing 12% PAGE, and the expression of 21 nucleotide long mature amiR1 in each line was confirmed by northern blotting. (E, F) Down-regulation of U11-48K in the transgenic plants. The levels of U11-48K in each transgenic plant were confirmed by (E) RT–PCR and (F) real-time RT–PCR analysis. The numbers 1, 2, and 3 in (F) indicate amiR1-1, amiR1-2, and amiR1-3, respectively. Values are means ±SE obtained from three independent biological replicates. (This figure is available in colour at JXB online.)

    Techniques Used: Expressing, Microscopy, Staining, Transgenic Assay, Polyacrylamide Gel Electrophoresis, Northern Blot, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

    33) Product Images from "Cooperative effect of the VP1 amino acids 98E, 145A and 169F in the productive infection of mouse cell lines by enterovirus 71 (BS strain)"

    Article Title: Cooperative effect of the VP1 amino acids 98E, 145A and 169F in the productive infection of mouse cell lines by enterovirus 71 (BS strain)

    Journal: Emerging Microbes & Infections

    doi: 10.1038/emi.2016.56

    Assessing the role of the mSCARB2 protein in CDV:BS M-P1 and CDV:BS-VP1 K98E,E145A,L169F infection of murine cells. ( A – D ) Pre-incubation of 10 6 CCID 50 CDVs with the mSCARB2 protein for in vitro uncoating ( A, B ) or for cellular infection studies of NIH/3T3 cells ( C , D ). The viral RNA in the samples was extracted and quantified by RT–PCR (qRT–PCR). ( E–H ) Blocking viral entry by incubating NIH/3T3 cells with anti-mSCARB2 sera prior to inoculation with virus at an MOI of 10. Infection was assessed by determining viral titers in culture supernatant with dilutions of 10 -1 to 10 −10 in Vero cells at three days p.i. following chloroform viral disaggregation ( E , F ), and relative quantitation of EV71 RNA in extracted total cellular RNA by the ΔΔC T method using β-actin as an internal control ( G , H ). Tests were separately performed for CDV:BS M-P1 ( A , C , E , H ) and CDV:BS-VP1 K98E,E145A,L169F ( B , D , F , G ). For A – H , a t -test with Welch's correction for unequal variance was used to compare mean values ( n =4). Error bars represent the s.d.; * P
    Figure Legend Snippet: Assessing the role of the mSCARB2 protein in CDV:BS M-P1 and CDV:BS-VP1 K98E,E145A,L169F infection of murine cells. ( A – D ) Pre-incubation of 10 6 CCID 50 CDVs with the mSCARB2 protein for in vitro uncoating ( A, B ) or for cellular infection studies of NIH/3T3 cells ( C , D ). The viral RNA in the samples was extracted and quantified by RT–PCR (qRT–PCR). ( E–H ) Blocking viral entry by incubating NIH/3T3 cells with anti-mSCARB2 sera prior to inoculation with virus at an MOI of 10. Infection was assessed by determining viral titers in culture supernatant with dilutions of 10 -1 to 10 −10 in Vero cells at three days p.i. following chloroform viral disaggregation ( E , F ), and relative quantitation of EV71 RNA in extracted total cellular RNA by the ΔΔC T method using β-actin as an internal control ( G , H ). Tests were separately performed for CDV:BS M-P1 ( A , C , E , H ) and CDV:BS-VP1 K98E,E145A,L169F ( B , D , F , G ). For A – H , a t -test with Welch's correction for unequal variance was used to compare mean values ( n =4). Error bars represent the s.d.; * P

    Techniques Used: Infection, Incubation, In Vitro, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Blocking Assay, Quantitation Assay

    34) Product Images from "Sensitive Genotyping of Foodborne-Associated Human Noroviruses and Hepatitis A Virus Using an Array-Based Platform"

    Article Title: Sensitive Genotyping of Foodborne-Associated Human Noroviruses and Hepatitis A Virus Using an Array-Based Platform

    Journal: Sensors (Basel, Switzerland)

    doi: 10.3390/s17092157

    Steps of the array-based method for detecting distinct genotypes of NoV or HAV. The starting material was an RNA sample subjected to RT-PCR, purified, and enzymatically digested to remove the non-complementary strand. The hybridization steps was followed by the microarray labeling and signal amplification and quantification steps. Sample-to-result time is below 8 h.
    Figure Legend Snippet: Steps of the array-based method for detecting distinct genotypes of NoV or HAV. The starting material was an RNA sample subjected to RT-PCR, purified, and enzymatically digested to remove the non-complementary strand. The hybridization steps was followed by the microarray labeling and signal amplification and quantification steps. Sample-to-result time is below 8 h.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Purification, Hybridization, Microarray, Labeling, Amplification

    35) Product Images from "A Novel Calcium Uptake Transporter of Uncharacterized P-Type ATPase Family Supplies Calcium for Cell Surface Integrity in Mycobacterium smegmatis"

    Article Title: A Novel Calcium Uptake Transporter of Uncharacterized P-Type ATPase Family Supplies Calcium for Cell Surface Integrity in Mycobacterium smegmatis

    Journal: mBio

    doi: 10.1128/mBio.01388-17

    Ca 2+ negatively regulates expression of ctpE . Role of Ca 2+ on expression of ctpE in Mycobacterium smegmatis mc 2 155 strains. Strains were grown to mid-log phase in Sauton’s medium without any supplementation or supplemented with 1.0 mM EGTA or CaCl 2 and used for uptake assays. (A and B) Uptake of 45 Ca 2+ in M. smegmatis mc 2 155 (WT) (A) and in the mutant (MHK1+pSMT3) and mutant complemented with M. smegmatis ctpE (MHK1+pRHK2) (B). (C) Semiquantitative reverse transcriptase PCR analysis of the M. smegmatis mc 2 155 ctpE and echA . M. smegmatis mc 2 155 was grown to mid-log phase in Sauton’s medium and treated with EGTA or CaCl 2 for 2 h. RNA was isolated, and RT-PCR was carried out for ctpE , echA , and sigA . (D) Transcription profile of ctpE and echA . Quantification of amplified PCR product ( Fig. 6C ) was done by densitometry. sigA was taken as an endogenous control. The experiment was performed three times independently; values are averages, and standard deviations are shown as error bars. Values that are significantly different are indicated by asterisks as follows: *, P
    Figure Legend Snippet: Ca 2+ negatively regulates expression of ctpE . Role of Ca 2+ on expression of ctpE in Mycobacterium smegmatis mc 2 155 strains. Strains were grown to mid-log phase in Sauton’s medium without any supplementation or supplemented with 1.0 mM EGTA or CaCl 2 and used for uptake assays. (A and B) Uptake of 45 Ca 2+ in M. smegmatis mc 2 155 (WT) (A) and in the mutant (MHK1+pSMT3) and mutant complemented with M. smegmatis ctpE (MHK1+pRHK2) (B). (C) Semiquantitative reverse transcriptase PCR analysis of the M. smegmatis mc 2 155 ctpE and echA . M. smegmatis mc 2 155 was grown to mid-log phase in Sauton’s medium and treated with EGTA or CaCl 2 for 2 h. RNA was isolated, and RT-PCR was carried out for ctpE , echA , and sigA . (D) Transcription profile of ctpE and echA . Quantification of amplified PCR product ( Fig. 6C ) was done by densitometry. sigA was taken as an endogenous control. The experiment was performed three times independently; values are averages, and standard deviations are shown as error bars. Values that are significantly different are indicated by asterisks as follows: *, P

    Techniques Used: Expressing, Mutagenesis, Polymerase Chain Reaction, Isolation, Reverse Transcription Polymerase Chain Reaction, Amplification

    36) Product Images from "Paralemmin-1 is over-expressed in estrogen-receptor positive breast cancers"

    Article Title: Paralemmin-1 is over-expressed in estrogen-receptor positive breast cancers

    Journal: Cancer Cell International

    doi: 10.1186/1475-2867-12-17

    Tumor tissues and breast cell lines express a higher proportion of the Δ exon 8 splice variant of paralemmin-1 than do reduction mammoplasty tissues. RNA was isolated and amplified with RT-PCR analysis using a primer set to detect Δ exon 8 splice variant. RT-PCR products were separated on a 2% low melting agarose gel and visualized by ethidium bromide. The full abbreviations of the cell lines are in Table 1 . Tumor tissue samples are represented by the prefix T and reduction mammoplasty tissue samples are represented by the prefix R. Numbers on the left of the figures represent the full length product (275 bp) and the Δ exon 8 splice variant (143 bp).
    Figure Legend Snippet: Tumor tissues and breast cell lines express a higher proportion of the Δ exon 8 splice variant of paralemmin-1 than do reduction mammoplasty tissues. RNA was isolated and amplified with RT-PCR analysis using a primer set to detect Δ exon 8 splice variant. RT-PCR products were separated on a 2% low melting agarose gel and visualized by ethidium bromide. The full abbreviations of the cell lines are in Table 1 . Tumor tissue samples are represented by the prefix T and reduction mammoplasty tissue samples are represented by the prefix R. Numbers on the left of the figures represent the full length product (275 bp) and the Δ exon 8 splice variant (143 bp).

    Techniques Used: Variant Assay, Isolation, Amplification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Paralemmin-1 is differentially expressed in breast epithelial cell lines. RNA and protein lysates were isolated from tumorigenic and non-tumorigenic breast cell lines. Top: Real time qRT-PCR shows mRNA expression of paralemmin-1; means and S.E. from three separate cell cultures are presented; ER-positive cell lines, (hatched bars), ER-negative cell lines (solid bars). Bottom: Protein lysates (15 μg) were probed for paralemmin-1 expression by Western immunoblotting. Image is a representative of at least three separate experiments with different biological samples.
    Figure Legend Snippet: Paralemmin-1 is differentially expressed in breast epithelial cell lines. RNA and protein lysates were isolated from tumorigenic and non-tumorigenic breast cell lines. Top: Real time qRT-PCR shows mRNA expression of paralemmin-1; means and S.E. from three separate cell cultures are presented; ER-positive cell lines, (hatched bars), ER-negative cell lines (solid bars). Bottom: Protein lysates (15 μg) were probed for paralemmin-1 expression by Western immunoblotting. Image is a representative of at least three separate experiments with different biological samples.

    Techniques Used: Isolation, Quantitative RT-PCR, Expressing, Western Blot

    37) Product Images from "Heterochromatin and RNAi regulate centromeres by protecting CENP-A from ubiquitin-mediated degradation"

    Article Title: Heterochromatin and RNAi regulate centromeres by protecting CENP-A from ubiquitin-mediated degradation

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007572

    Cse4 is subject to efficient ubiquitin-dependent degradation in the fission yeast. A, RT-PCR analysis of cells expressing Cnp1-GFP or Cse4-GFP. Total RNA extracted from cells overexpressing Cnp1-GFP or Cse4-GFP was used. Cnp1-GFP or Cse4-GFP transcripts were analyzed with primers specific for GFP. Actin was used as an internal control. B, Lysates from cells collected at indicated time points (hrs) following cycloheximide treatment were analyzed by western blotting with an anti-GFP antibody. C, Cse4 level is enhanced after proteasome inactivation in fission yeast. Cells overexpressing Cse4-GFP in wild type or mts2-1 background were incubated at 37°C for 4 hours, and were subject to western blot analysis using an anti-GFP antibody. Tubulin was used as a loading control. D, Extracts from cells expressing indicated proteins were split, and subject to TUBE pull-down and reverse pull-down assays, respectively. For TUBE pull-down assays, extracts were immunoprecipitated with tandem ubiquitin-binding entities (+TUBE), or control Argarose beads (-TUBE), followed by western blot analysis using an anti-GFP antibody. For reverse pull-down assays (right panel), extracts were immunoprecipitated with an anti-GFP antibody, then analyzed by western blotting using a pan ubiquitin antibody. Induction time: 20 hours for Cnp1-GFP; 24 hours for Cse4-GFP.
    Figure Legend Snippet: Cse4 is subject to efficient ubiquitin-dependent degradation in the fission yeast. A, RT-PCR analysis of cells expressing Cnp1-GFP or Cse4-GFP. Total RNA extracted from cells overexpressing Cnp1-GFP or Cse4-GFP was used. Cnp1-GFP or Cse4-GFP transcripts were analyzed with primers specific for GFP. Actin was used as an internal control. B, Lysates from cells collected at indicated time points (hrs) following cycloheximide treatment were analyzed by western blotting with an anti-GFP antibody. C, Cse4 level is enhanced after proteasome inactivation in fission yeast. Cells overexpressing Cse4-GFP in wild type or mts2-1 background were incubated at 37°C for 4 hours, and were subject to western blot analysis using an anti-GFP antibody. Tubulin was used as a loading control. D, Extracts from cells expressing indicated proteins were split, and subject to TUBE pull-down and reverse pull-down assays, respectively. For TUBE pull-down assays, extracts were immunoprecipitated with tandem ubiquitin-binding entities (+TUBE), or control Argarose beads (-TUBE), followed by western blot analysis using an anti-GFP antibody. For reverse pull-down assays (right panel), extracts were immunoprecipitated with an anti-GFP antibody, then analyzed by western blotting using a pan ubiquitin antibody. Induction time: 20 hours for Cnp1-GFP; 24 hours for Cse4-GFP.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Incubation, Immunoprecipitation, Binding Assay

    38) Product Images from "Breakpoint Analysis of Transcriptional and Genomic Profiles Uncovers Novel Gene Fusions Spanning Multiple Human Cancer Types"

    Article Title: Breakpoint Analysis of Transcriptional and Genomic Profiles Uncovers Novel Gene Fusions Spanning Multiple Human Cancer Types

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003464

    Identification and characterization of FAM133B/CDK6 in J.RT3-T3.5. ( A ) Heatmap depicting rearrangement of CDK6 in J.RT3-T3.5 (Jurkat derivative). ( B ) Discovery of the FAM133B/CDK6 rearrangement by paired-end RNA-seq. The fusion junction was confirmed by RT-PCR (not shown) and Sanger sequencing. ( C ) Gene expression profiling reveals high-level expression of CDK6 in J.RT3-T3.5 compared to other leukemia cell lines. Note that array probes mapped to the portion of CDK6 retained in the fusion. ( D ) Jurkat demonstrates marked sensitivity to the CDK4/6 inhibitor PD0332991 (IC 50 = 0.27 µM). K562, which expresses only wildtype CDK6, is used as a negative control cell line and shows minimal sensitivity to PD0332991 (IC 50 = 5.9 µM).
    Figure Legend Snippet: Identification and characterization of FAM133B/CDK6 in J.RT3-T3.5. ( A ) Heatmap depicting rearrangement of CDK6 in J.RT3-T3.5 (Jurkat derivative). ( B ) Discovery of the FAM133B/CDK6 rearrangement by paired-end RNA-seq. The fusion junction was confirmed by RT-PCR (not shown) and Sanger sequencing. ( C ) Gene expression profiling reveals high-level expression of CDK6 in J.RT3-T3.5 compared to other leukemia cell lines. Note that array probes mapped to the portion of CDK6 retained in the fusion. ( D ) Jurkat demonstrates marked sensitivity to the CDK4/6 inhibitor PD0332991 (IC 50 = 0.27 µM). K562, which expresses only wildtype CDK6, is used as a negative control cell line and shows minimal sensitivity to PD0332991 (IC 50 = 5.9 µM).

    Techniques Used: RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Sequencing, Expressing, Negative Control

    DBA discovery of recurrent rearrangements of CLTC and VMP1 across diverse cancer types. ( A ) Heatmap depicting focal deletions between CLTC and VMP1 in the breast cancer cell lines BT-549 and HCC1954. ( B ) Discovery of the recurrent CLTC/VMP1 rearrangement in BT-549 ( left panel) and HCC1954 ( right panel) by paired-end RNA-seq. ( C ) RT-PCR verification of CLTC/VMP1 fusion in BT-549 and HCC1954. ( D ) Heatmap depicting focal deletions disrupting CLTC , PTRH2 and/or VMP1 in various cancer types (see legend). ( E ) A renal cell carcinoma line, RXF393, was also profiled by exon microarray where an expression breakpoint was evident within CLTC . *** P
    Figure Legend Snippet: DBA discovery of recurrent rearrangements of CLTC and VMP1 across diverse cancer types. ( A ) Heatmap depicting focal deletions between CLTC and VMP1 in the breast cancer cell lines BT-549 and HCC1954. ( B ) Discovery of the recurrent CLTC/VMP1 rearrangement in BT-549 ( left panel) and HCC1954 ( right panel) by paired-end RNA-seq. ( C ) RT-PCR verification of CLTC/VMP1 fusion in BT-549 and HCC1954. ( D ) Heatmap depicting focal deletions disrupting CLTC , PTRH2 and/or VMP1 in various cancer types (see legend). ( E ) A renal cell carcinoma line, RXF393, was also profiled by exon microarray where an expression breakpoint was evident within CLTC . *** P

    Techniques Used: RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Microarray, Expressing

    Discovery of new cell line models for the known rearrangements, EGFRvIII and FIP1L1/PDGFRA . ( A ) Heatmap depicting genomic breakpoints within EGFR in the glioblastoma cell lines, CAS-1 and DKMG. ( B ) Identification of EGFRvIII in DKMG cells by paired-end RNA-seq. Paired-end reads supporting the rearrangement are depicted. ( C ) Verification of EGFRvIII expression by RT-PCR (top panel) and Western blotting (bottom panel) in DKMG. RT-PCR was done using primers flanking the exon 1/exon 8 junction of EGFRvIII , and Western blotting was done using an antibody specific to the EGFRvIII isoform. Control samples include U87 glioblastoma cells without EGFR rearrangement, U87-vIII cells engineered to express exogenous EGFRvIII , and A431 epidermoid carcinoma cells with EGFR amplification. ( D ) RBA identification of expression-level breakpoint within PDGFRA in SUPT13 T-ALL cells. *** P
    Figure Legend Snippet: Discovery of new cell line models for the known rearrangements, EGFRvIII and FIP1L1/PDGFRA . ( A ) Heatmap depicting genomic breakpoints within EGFR in the glioblastoma cell lines, CAS-1 and DKMG. ( B ) Identification of EGFRvIII in DKMG cells by paired-end RNA-seq. Paired-end reads supporting the rearrangement are depicted. ( C ) Verification of EGFRvIII expression by RT-PCR (top panel) and Western blotting (bottom panel) in DKMG. RT-PCR was done using primers flanking the exon 1/exon 8 junction of EGFRvIII , and Western blotting was done using an antibody specific to the EGFRvIII isoform. Control samples include U87 glioblastoma cells without EGFR rearrangement, U87-vIII cells engineered to express exogenous EGFRvIII , and A431 epidermoid carcinoma cells with EGFR amplification. ( D ) RBA identification of expression-level breakpoint within PDGFRA in SUPT13 T-ALL cells. *** P

    Techniques Used: RNA Sequencing Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Amplification

    Discovery of APIP/SLC1A2 in colon cancer. ( A ) Array CGH heatmap displaying genomic breakpoints disrupting SLC1A2 in the SNU-C1 colon cancer cell line and the SNU-16 gastric cancer cell line. SNU-16 is known to harbor CD44/SLC1A2 and its array CGH profile is depicted for comparison. Unsmoothed log 2 ratios are displayed. ( B ) Paired-end RNA seq uncovers APIP/SLC1A2 in SNU-C1. A subset of paired-end reads mapping to APIP/SLC1A2 as well as the gene fusion structure are displayed (left panel). The structure of the known gastric cancer gene fusion CD44/SLC1A2 is depicted for comparison (right panel). An internal start codon within exon 2 of SLC1A2 is predicted to initiate translation in both rearrangements. Inset : experimental validation of APIP/SLC1A2 by RT-PCR with primers flanking the gene fusion junction. ( C , D ) Gene expression profiling depicts high-level expression of APIP in normal colon ( C ) and overexpression of SLC1A2 in SNU-C1 ( D ). Mean-centered gene expression ratios are depicted by a log 2 pseudocolor scale and ranked in descending order from left to right.
    Figure Legend Snippet: Discovery of APIP/SLC1A2 in colon cancer. ( A ) Array CGH heatmap displaying genomic breakpoints disrupting SLC1A2 in the SNU-C1 colon cancer cell line and the SNU-16 gastric cancer cell line. SNU-16 is known to harbor CD44/SLC1A2 and its array CGH profile is depicted for comparison. Unsmoothed log 2 ratios are displayed. ( B ) Paired-end RNA seq uncovers APIP/SLC1A2 in SNU-C1. A subset of paired-end reads mapping to APIP/SLC1A2 as well as the gene fusion structure are displayed (left panel). The structure of the known gastric cancer gene fusion CD44/SLC1A2 is depicted for comparison (right panel). An internal start codon within exon 2 of SLC1A2 is predicted to initiate translation in both rearrangements. Inset : experimental validation of APIP/SLC1A2 by RT-PCR with primers flanking the gene fusion junction. ( C , D ) Gene expression profiling depicts high-level expression of APIP in normal colon ( C ) and overexpression of SLC1A2 in SNU-C1 ( D ). Mean-centered gene expression ratios are depicted by a log 2 pseudocolor scale and ranked in descending order from left to right.

    Techniques Used: RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Over Expression

    Discovery and characterization of EWSR1/CREM in melanoma. ( A ) Array CGH heatmap displaying intragenic EWSR1 breakpoints identified in the SH-4 and CHL-1 melanoma cell lines. ( B ) Paired-end RNA-seq identification of EWSR1/CREM in CHL-1. Paired-end reads supporting the rearrangement are depicted along with the predicted gene fusion structure. CREM contributes a basic leucine zipper motif (ZIP), while EWSR1 contributes the EWS Activation Domain (EAD). ( C ) RT-PCR verification of EWSR1/CREM in CHL-1. ( D ) Quantitative RT-PCR using primers flanking the gene fusion junction verifies EWSR1/CREM knockdown following transfection of an siRNA pool targeting the 3′ end of CREM . ( E , F , G ) Transfection of CHL-1 with CREM -targeting siRNA pool results in ( E ) decreased cell proliferation, ( F ) decreased invasion, and ( G ) a higher fraction of senescent cells, compared to non-targeting control (NTC). ** P
    Figure Legend Snippet: Discovery and characterization of EWSR1/CREM in melanoma. ( A ) Array CGH heatmap displaying intragenic EWSR1 breakpoints identified in the SH-4 and CHL-1 melanoma cell lines. ( B ) Paired-end RNA-seq identification of EWSR1/CREM in CHL-1. Paired-end reads supporting the rearrangement are depicted along with the predicted gene fusion structure. CREM contributes a basic leucine zipper motif (ZIP), while EWSR1 contributes the EWS Activation Domain (EAD). ( C ) RT-PCR verification of EWSR1/CREM in CHL-1. ( D ) Quantitative RT-PCR using primers flanking the gene fusion junction verifies EWSR1/CREM knockdown following transfection of an siRNA pool targeting the 3′ end of CREM . ( E , F , G ) Transfection of CHL-1 with CREM -targeting siRNA pool results in ( E ) decreased cell proliferation, ( F ) decreased invasion, and ( G ) a higher fraction of senescent cells, compared to non-targeting control (NTC). ** P

    Techniques Used: RNA Sequencing Assay, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Transfection

    Identification and characterization of novel RAF1 gene fusions in pancreatic cancer and anaplastic astrocytoma. ( A ) Array CGH heatmaps displaying intragenic RAF1 genomic breakpoints identified in the PL5 pancreatic cancer cell line ( left panel ) and the D-538MG anaplastic astrocytoma cell line ( right panel ). Unsmoothed log 2 ratios are displayed. ( B ) Identification of ATG7/RAF1 (left) and BCL6/RAF1 (right) in PL5 and D-538MG cells, respectively, by paired-end RNA-seq. A subset of the paired-end reads supporting each gene fusion is displayed. Both gene fusions are in-frame and the RAF1 serine threonine kinase domain (STK) is retained in both fusions. ( C ) Experimental validation of gene fusions by RT-PCR, using primers flanking the respective gene fusion junction. ( D ) Western blotting verifies knockdown of ATG7/RAF1 in PL5 following transfection of a RAF1 -targeting siRNA pool. ATG7/RAF1 protein levels were monitored using an anti- RAF1 antibody, with anti- GAPDH providing a loading control. ( E ) Decreased cell proliferation and ( F ) invasion rates of PL5 following transfection of a RAF1 -targeting siRNA pool, compared to transfection of a non-targeting control (NTC) siRNA pool. ** P
    Figure Legend Snippet: Identification and characterization of novel RAF1 gene fusions in pancreatic cancer and anaplastic astrocytoma. ( A ) Array CGH heatmaps displaying intragenic RAF1 genomic breakpoints identified in the PL5 pancreatic cancer cell line ( left panel ) and the D-538MG anaplastic astrocytoma cell line ( right panel ). Unsmoothed log 2 ratios are displayed. ( B ) Identification of ATG7/RAF1 (left) and BCL6/RAF1 (right) in PL5 and D-538MG cells, respectively, by paired-end RNA-seq. A subset of the paired-end reads supporting each gene fusion is displayed. Both gene fusions are in-frame and the RAF1 serine threonine kinase domain (STK) is retained in both fusions. ( C ) Experimental validation of gene fusions by RT-PCR, using primers flanking the respective gene fusion junction. ( D ) Western blotting verifies knockdown of ATG7/RAF1 in PL5 following transfection of a RAF1 -targeting siRNA pool. ATG7/RAF1 protein levels were monitored using an anti- RAF1 antibody, with anti- GAPDH providing a loading control. ( E ) Decreased cell proliferation and ( F ) invasion rates of PL5 following transfection of a RAF1 -targeting siRNA pool, compared to transfection of a non-targeting control (NTC) siRNA pool. ** P

    Techniques Used: RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection

    39) Product Images from "Characterization of West Nile Viruses Isolated from Captive American Flamingoes (Phoenicopterus ruber) in Medellin, Colombia"

    Article Title: Characterization of West Nile Viruses Isolated from Captive American Flamingoes (Phoenicopterus ruber) in Medellin, Colombia

    Journal: The American Journal of Tropical Medicine and Hygiene

    doi: 10.4269/ajtmh.2012.11-0655

    Flavivirus detection by reverse transcription-polymerase chain reaction (RT-PCR).
    Figure Legend Snippet: Flavivirus detection by reverse transcription-polymerase chain reaction (RT-PCR).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    40) Product Images from "Oral and Gastric Helicobacter Pylori: Effects and Associations"

    Article Title: Oral and Gastric Helicobacter Pylori: Effects and Associations

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0126923

    H . pylori - specific PCR for DNA extracted from oral cavity. In this figure, one can see the amplification of VacA in the three cases previously identified as oral cavity positives for H. pylori (Hp positive sample #1 to #3), in comparison with other three cases that are oral cavity H. pylori negatives (Hp negative sample #1 to #3). As a positive control (Hp positive control) PCR for DNA extracted from H . pylori (strain 7354) diluted in saliva was used. Blank—PCR negative control.
    Figure Legend Snippet: H . pylori - specific PCR for DNA extracted from oral cavity. In this figure, one can see the amplification of VacA in the three cases previously identified as oral cavity positives for H. pylori (Hp positive sample #1 to #3), in comparison with other three cases that are oral cavity H. pylori negatives (Hp negative sample #1 to #3). As a positive control (Hp positive control) PCR for DNA extracted from H . pylori (strain 7354) diluted in saliva was used. Blank—PCR negative control.

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

    Highly sensitive PCR for detection of H . pylori . In order to evaluate the sensitivity of VacA -specific PCR, 50ng of DNA from H . pylori was successively diluted (1 to 1:100000) in saliva from two random cases (#3 and #10) that were shown previously to be negative for the presence of H . pylori . The PCR allowed the amplification of the expected product for all different dilutions.
    Figure Legend Snippet: Highly sensitive PCR for detection of H . pylori . In order to evaluate the sensitivity of VacA -specific PCR, 50ng of DNA from H . pylori was successively diluted (1 to 1:100000) in saliva from two random cases (#3 and #10) that were shown previously to be negative for the presence of H . pylori . The PCR allowed the amplification of the expected product for all different dilutions.

    Techniques Used: Polymerase Chain Reaction, Amplification

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    Article Snippet: .. PCR amplification was carried out in a total volume of 25 μL containing 150 ng template DNA, 0.2 μM of each primer, and 1.5 μL of PCR Master Mix (Type–It Microsatellite Kit, Qiagen). .. PCR was performed in a GeneAmp PCR System 9700 thermal cycler (Applied BioSystem) as follows: an initial denaturation step at 95 °C for 5 minutes, denaturation at 90 °C for 30 seconds, annealing at 60 °C for 90 seconds, and extension at 72 °C for 30 seconds.

    Article Title: Temperature increase altered Daphnia community structure in artificially heated lakes: a potential scenario for a warmer future
    Article Snippet: .. COI gene sequences were amplified from 1 μL DNA template in 5 μL of PCR reaction mix, containing 2.5 μL Type-it Microsatellite Kit (Qiagen GmbH, Hilden, Germany), 0.25 μmol/L of each primer, and 2 μL of water. .. The PCR product was visualised by electrophoresis on 1% agarose gel, purified using Exonuclease I and Alkaline Phosphatase (Thermo Scientific, San Jose, USA) for 15 min at 37 °C and 15 min at 80 °C, and Sanger sequenced using the BigDye Terminator v3.1 kit and an ABI Prism 3130XL Analyzer (Applied Biosystems, Foster City, CA, USA).

    Article Title: Temperature increase altered Daphnia community structure in artificially heated lakes: a potential scenario for a warmer future
    Article Snippet: .. PCR reactions were conducted in a 5 μL mix that contained 2.5 μL Type-it Microsatellite Kit mix (Qiagen GmbH, Hilden, Germany), 0.2 μmol/L of each primer, 1 μL of DNA template, and enough water to make 5 μL. .. PCR products were diluted with 70 μL of water, and electrophoresis was conducted with an ABI Prism 3130XL Analyzer.

    Article Title: From cytogenetics to cytogenomics: whole-genome sequencing as a first-line test comprehensively captures the diverse spectrum of disease-causing genetic variation underlying intellectual disability
    Article Snippet: .. Primers were pooled and amplified using Type-it Microsatellite PCR Kit according to the manufacturer’s instructions (QIAGEN, Hilden, Germany). .. PCR products were analyzed using 3500xL Genetic Analyzer and GeneMapper v5 according to the manufacturer’s protocol (Applied Biosystems).

    Concentration Assay:

    Article Title: A genetically distinct hybrid zone occurs for two globally invasive mosquito fish species with striking phenotypic resemblance), A genetically distinct hybrid zone occurs for two globally invasive mosquito fish species with striking phenotypic resemblance
    Article Snippet: .. 25 μl reactions were run (5 μl DNA at ~20 ng/μl, 5 μl RNase free water, 0.625 μl each primer (0.1 μmol/L concentration), 12.5 μl of mastermix from Qiagen (Type‐It Microsatellite Kit, Valencia, CA, USA) for 5:00 min at 95°C, 35 cycles for 0:30 s at 95°C, 1:30 min at 64°C, 0:30 s at 72°C, 10:00 min at 68°C. .. Polymerase chain reaction products were processed on an ABI 3130xl (Applied Biosystems, Grand Island, NY, USA) genetic analyzer.

    Multiplex Assay:

    Article Title: A microsatellite based multiplex PCR method for the detection of chromosomal instability in gastric cancer
    Article Snippet: .. Amplification of 30 markers was performed in 5 multiplex PCRs using the Type-it Microsatellite PCR kit (Qiagen, Hilden, Germany) and the respective forward primers were labelled with the dyes FAM, HEX or ATTO550 (Eurofins Genomics, Ebersberg, Germany). .. 20 ng DNA was added to each PCR reaction in a final volume of 25 µl.

    Article Title: Genes with Restricted Introgression in a Field Cricket (Gryllus firmus/Gryllus pennsylvanicus) Hybrid Zone Are Concentrated on the X Chromosome and a Single Autosome
    Article Snippet: .. We used Qiagen Type-it microsatellite kit following manufacturer recommendations to run three multiplex PCRs. .. Each reaction contained five loci labeled with four different fluorescent dyes (Fam, Vic, Pet, and Ned).

    Amplification:

    Article Title: A microsatellite based multiplex PCR method for the detection of chromosomal instability in gastric cancer
    Article Snippet: .. Amplification of 30 markers was performed in 5 multiplex PCRs using the Type-it Microsatellite PCR kit (Qiagen, Hilden, Germany) and the respective forward primers were labelled with the dyes FAM, HEX or ATTO550 (Eurofins Genomics, Ebersberg, Germany). .. 20 ng DNA was added to each PCR reaction in a final volume of 25 µl.

    Article Title: Electrophoretic Techniques for the Detection of Human Microsatellite D19S884
    Article Snippet: .. PCR amplification was carried out in a total volume of 25 μL containing 150 ng template DNA, 0.2 μM of each primer, and 1.5 μL of PCR Master Mix (Type–It Microsatellite Kit, Qiagen). .. PCR was performed in a GeneAmp PCR System 9700 thermal cycler (Applied BioSystem) as follows: an initial denaturation step at 95 °C for 5 minutes, denaturation at 90 °C for 30 seconds, annealing at 60 °C for 90 seconds, and extension at 72 °C for 30 seconds.

    Article Title: Temperature increase altered Daphnia community structure in artificially heated lakes: a potential scenario for a warmer future
    Article Snippet: .. COI gene sequences were amplified from 1 μL DNA template in 5 μL of PCR reaction mix, containing 2.5 μL Type-it Microsatellite Kit (Qiagen GmbH, Hilden, Germany), 0.25 μmol/L of each primer, and 2 μL of water. .. The PCR product was visualised by electrophoresis on 1% agarose gel, purified using Exonuclease I and Alkaline Phosphatase (Thermo Scientific, San Jose, USA) for 15 min at 37 °C and 15 min at 80 °C, and Sanger sequenced using the BigDye Terminator v3.1 kit and an ABI Prism 3130XL Analyzer (Applied Biosystems, Foster City, CA, USA).

    Article Title: Large‐scale genetic panmixia in the blue shark (Prionace glauca): A single worldwide population, or a genetic lag‐time effect of the “grey zone” of differentiation?. Large‐scale genetic panmixia in the blue shark (Prionace glauca): A single worldwide population, or a genetic lag‐time effect of the “grey zone” of differentiation?
    Article Snippet: .. Except for TB01, loci were multiplexed (amplified and genotyped together) by sets of three loci using the “Type‐it microsatellite” kit by QIAGEN (QIAGEN GmbH, Hilden, Germany) in 25 μl reaction volumes containing 12.5 μl of Master Mix, 5.5 μl of pure water and 0.25 μl of each primer (at 10 μM). ..

    Article Title: From cytogenetics to cytogenomics: whole-genome sequencing as a first-line test comprehensively captures the diverse spectrum of disease-causing genetic variation underlying intellectual disability
    Article Snippet: .. Primers were pooled and amplified using Type-it Microsatellite PCR Kit according to the manufacturer’s instructions (QIAGEN, Hilden, Germany). .. PCR products were analyzed using 3500xL Genetic Analyzer and GeneMapper v5 according to the manufacturer’s protocol (Applied Biosystems).

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  • 88
    Qiagen 1x q solution
    Increasing the assay specificity of the  JAK2  V617F mutation-specific PCR.  Samples tested: 100% mutant control DNA (MUT 100%) analysed in triplicate, 100% wild-type control DNA (WT 100%) analysed in 10 replicates, non-template control (NTC) analysed in triplicate.  A . Mutant allele-specific PCR. The reactions contained two oligonucleotides: the mutant allele-specific forward primer and the reverse primer. The graph shows a significant number of false-positive amplifications. We observed a Cq value difference of 20 cycles between the MUT 100% and the first false-positive amplification.  B . Mutant allele-specific PCR with the introduction of the wild-type specific 3′ dideoxy blocker. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele specific blocker and the reverse primer. The graph still shows the presence of a number of false positives. We observed a Cq value difference of 23 cycles between the MUT 100% and the first false-positive amplification.  C . Mutant allele-specific PCR with the introduction of 1X Q-Solution. The reactions contained two oligonucleotides: the mutant allele-specific forward and the reverse primers. The graph shows a significant reduction of false-positive amplifications to a single false positive. We observed a Cq value difference of 16 cycles between the MUT 100% and the first false-positive amplification.  D . Mutant allele-specific PCR with the introduction of both the 3′ dideoxy blocker and 1X Q-Solution. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele-specific blocker and the reverse primer. One false-positive amplification was observed, at a very late Cq value. We observed a Cq value of 23 cycles difference between the MUT 100% and the first false-positive amplification.
    1x Q Solution, supplied by Qiagen, used in various techniques. Bioz Stars score: 88/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/1x q solution/product/Qiagen
    Average 88 stars, based on 31 article reviews
    Price from $9.99 to $1999.99
    1x q solution - by Bioz Stars, 2020-09
    88/100 stars
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    89
    Qiagen q solution buffer
    Increasing the assay specificity of the  JAK2  V617F mutation-specific PCR.  Samples tested: 100% mutant control DNA (MUT 100%) analysed in triplicate, 100% wild-type control DNA (WT 100%) analysed in 10 replicates, non-template control (NTC) analysed in triplicate.  A . Mutant allele-specific PCR. The reactions contained two oligonucleotides: the mutant allele-specific forward primer and the reverse primer. The graph shows a significant number of false-positive amplifications. We observed a Cq value difference of 20 cycles between the MUT 100% and the first false-positive amplification.  B . Mutant allele-specific PCR with the introduction of the wild-type specific 3′ dideoxy blocker. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele specific blocker and the reverse primer. The graph still shows the presence of a number of false positives. We observed a Cq value difference of 23 cycles between the MUT 100% and the first false-positive amplification.  C . Mutant allele-specific PCR with the introduction of 1X Q-Solution. The reactions contained two oligonucleotides: the mutant allele-specific forward and the reverse primers. The graph shows a significant reduction of false-positive amplifications to a single false positive. We observed a Cq value difference of 16 cycles between the MUT 100% and the first false-positive amplification.  D . Mutant allele-specific PCR with the introduction of both the 3′ dideoxy blocker and 1X Q-Solution. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele-specific blocker and the reverse primer. One false-positive amplification was observed, at a very late Cq value. We observed a Cq value of 23 cycles difference between the MUT 100% and the first false-positive amplification.
    Q Solution Buffer, supplied by Qiagen, used in various techniques. Bioz Stars score: 89/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/q solution buffer/product/Qiagen
    Average 89 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    q solution buffer - by Bioz Stars, 2020-09
    89/100 stars
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    85
    Qiagen q solution 5x
    Increasing the assay specificity of the  JAK2  V617F mutation-specific PCR.  Samples tested: 100% mutant control DNA (MUT 100%) analysed in triplicate, 100% wild-type control DNA (WT 100%) analysed in 10 replicates, non-template control (NTC) analysed in triplicate.  A . Mutant allele-specific PCR. The reactions contained two oligonucleotides: the mutant allele-specific forward primer and the reverse primer. The graph shows a significant number of false-positive amplifications. We observed a Cq value difference of 20 cycles between the MUT 100% and the first false-positive amplification.  B . Mutant allele-specific PCR with the introduction of the wild-type specific 3′ dideoxy blocker. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele specific blocker and the reverse primer. The graph still shows the presence of a number of false positives. We observed a Cq value difference of 23 cycles between the MUT 100% and the first false-positive amplification.  C . Mutant allele-specific PCR with the introduction of 1X Q-Solution. The reactions contained two oligonucleotides: the mutant allele-specific forward and the reverse primers. The graph shows a significant reduction of false-positive amplifications to a single false positive. We observed a Cq value difference of 16 cycles between the MUT 100% and the first false-positive amplification.  D . Mutant allele-specific PCR with the introduction of both the 3′ dideoxy blocker and 1X Q-Solution. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele-specific blocker and the reverse primer. One false-positive amplification was observed, at a very late Cq value. We observed a Cq value of 23 cycles difference between the MUT 100% and the first false-positive amplification.
    Q Solution 5x, supplied by Qiagen, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/q solution 5x/product/Qiagen
    Average 85 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    q solution 5x - by Bioz Stars, 2020-09
    85/100 stars
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    86
    Qiagen q solution pcr
    Increasing the assay specificity of the  JAK2  V617F mutation-specific PCR.  Samples tested: 100% mutant control DNA (MUT 100%) analysed in triplicate, 100% wild-type control DNA (WT 100%) analysed in 10 replicates, non-template control (NTC) analysed in triplicate.  A . Mutant allele-specific PCR. The reactions contained two oligonucleotides: the mutant allele-specific forward primer and the reverse primer. The graph shows a significant number of false-positive amplifications. We observed a Cq value difference of 20 cycles between the MUT 100% and the first false-positive amplification.  B . Mutant allele-specific PCR with the introduction of the wild-type specific 3′ dideoxy blocker. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele specific blocker and the reverse primer. The graph still shows the presence of a number of false positives. We observed a Cq value difference of 23 cycles between the MUT 100% and the first false-positive amplification.  C . Mutant allele-specific PCR with the introduction of 1X Q-Solution. The reactions contained two oligonucleotides: the mutant allele-specific forward and the reverse primers. The graph shows a significant reduction of false-positive amplifications to a single false positive. We observed a Cq value difference of 16 cycles between the MUT 100% and the first false-positive amplification.  D . Mutant allele-specific PCR with the introduction of both the 3′ dideoxy blocker and 1X Q-Solution. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele-specific blocker and the reverse primer. One false-positive amplification was observed, at a very late Cq value. We observed a Cq value of 23 cycles difference between the MUT 100% and the first false-positive amplification.
    Q Solution Pcr, supplied by Qiagen, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/q solution pcr/product/Qiagen
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    q solution pcr - by Bioz Stars, 2020-09
    86/100 stars
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    Increasing the assay specificity of the  JAK2  V617F mutation-specific PCR.  Samples tested: 100% mutant control DNA (MUT 100%) analysed in triplicate, 100% wild-type control DNA (WT 100%) analysed in 10 replicates, non-template control (NTC) analysed in triplicate.  A . Mutant allele-specific PCR. The reactions contained two oligonucleotides: the mutant allele-specific forward primer and the reverse primer. The graph shows a significant number of false-positive amplifications. We observed a Cq value difference of 20 cycles between the MUT 100% and the first false-positive amplification.  B . Mutant allele-specific PCR with the introduction of the wild-type specific 3′ dideoxy blocker. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele specific blocker and the reverse primer. The graph still shows the presence of a number of false positives. We observed a Cq value difference of 23 cycles between the MUT 100% and the first false-positive amplification.  C . Mutant allele-specific PCR with the introduction of 1X Q-Solution. The reactions contained two oligonucleotides: the mutant allele-specific forward and the reverse primers. The graph shows a significant reduction of false-positive amplifications to a single false positive. We observed a Cq value difference of 16 cycles between the MUT 100% and the first false-positive amplification.  D . Mutant allele-specific PCR with the introduction of both the 3′ dideoxy blocker and 1X Q-Solution. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele-specific blocker and the reverse primer. One false-positive amplification was observed, at a very late Cq value. We observed a Cq value of 23 cycles difference between the MUT 100% and the first false-positive amplification.

    Journal: BMC Cancer

    Article Title: Quantitative threefold allele-specific PCR (QuanTAS-PCR) for highly sensitive JAK2 V617F mutant allele detection

    doi: 10.1186/1471-2407-13-206

    Figure Lengend Snippet: Increasing the assay specificity of the JAK2 V617F mutation-specific PCR. Samples tested: 100% mutant control DNA (MUT 100%) analysed in triplicate, 100% wild-type control DNA (WT 100%) analysed in 10 replicates, non-template control (NTC) analysed in triplicate. A . Mutant allele-specific PCR. The reactions contained two oligonucleotides: the mutant allele-specific forward primer and the reverse primer. The graph shows a significant number of false-positive amplifications. We observed a Cq value difference of 20 cycles between the MUT 100% and the first false-positive amplification. B . Mutant allele-specific PCR with the introduction of the wild-type specific 3′ dideoxy blocker. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele specific blocker and the reverse primer. The graph still shows the presence of a number of false positives. We observed a Cq value difference of 23 cycles between the MUT 100% and the first false-positive amplification. C . Mutant allele-specific PCR with the introduction of 1X Q-Solution. The reactions contained two oligonucleotides: the mutant allele-specific forward and the reverse primers. The graph shows a significant reduction of false-positive amplifications to a single false positive. We observed a Cq value difference of 16 cycles between the MUT 100% and the first false-positive amplification. D . Mutant allele-specific PCR with the introduction of both the 3′ dideoxy blocker and 1X Q-Solution. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele-specific blocker and the reverse primer. One false-positive amplification was observed, at a very late Cq value. We observed a Cq value of 23 cycles difference between the MUT 100% and the first false-positive amplification.

    Article Snippet: The JAK2 mutation-specific PCR reactions were performed in a 10 μl final volume comprising 1X PCR Buffer containing 1.5 mmol/L of MgCl2 , 0.5 mmol/L of extra MgCl2 (for a final concentration of 2 mmol/L of MgCl2 ), 200 μmol/L of each deoxynucleotide triphosphate, 200 nmol/L of the JAK2_Ex14_WT_Blocker_F oligo, 400 nmol/L of the JAK2_Ex14_Mut_F primer, 200 nmol/L of the JAK2_Ex14_R primer, 5 μmol/L of SYTO9, 1X Q-Solution (Qiagen), 0.25 units of HotStar Taq DNA polymerase and 2 μl of template (at a concentration of 16.5 ng/μl), for a total of about 10,000 JAK2 copies per reaction.

    Techniques: Mutagenesis, Polymerase Chain Reaction, Amplification