pcr amplification  (Roche)


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    Structured Review

    Roche pcr amplification
    Metagenomic analysis of the <t>16S</t> rRNA gene amplicons using MinION™ nanopore sequencing. a Workflow of 16S rRNA amplicon sequencing on the MinION™ platform. Sequencing libraries are generated by the four-primer <t>PCR-based</t> strategy, enabling simplified post-PCR adapter attachment. At the initial stage of PCR, the 16S rRNA gene is amplified with the inner primer pairs. The resulting PCR products are targeted for amplification with the outer primers to introduce the barcode and tag sequences at both ends, to which adapter molecules can be attached in a single-step reaction. b, c Taxonomic assignments of a mock community analyzed by MinION™ sequencing. The V1-V9 or V3-V4 region of the 16S rRNA gene was amplified from a pre-characterized mock community sample comprising ten bacterial species and sequenced on the MinION™ platform. Three thousand reads were randomly selected from the processed data set and aligned directly to the reference genome database of 5850 representative bacterial species. The pie charts represent taxonomic profiles at the (b) genus and (c) species levels. Slices corresponding to misclassified (assigned to bacteria not present in the mock community) or unclassified (not classified at the species level but placed in a higher taxonomic rank) reads are exploded. The relative abundance (%) of each taxon is shown.
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    Images

    1) Product Images from "Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION™ nanopore sequencing confers species-level resolution"

    Article Title: Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION™ nanopore sequencing confers species-level resolution

    Journal: bioRxiv

    doi: 10.1101/2020.05.06.078147

    Metagenomic analysis of the 16S rRNA gene amplicons using MinION™ nanopore sequencing. a Workflow of 16S rRNA amplicon sequencing on the MinION™ platform. Sequencing libraries are generated by the four-primer PCR-based strategy, enabling simplified post-PCR adapter attachment. At the initial stage of PCR, the 16S rRNA gene is amplified with the inner primer pairs. The resulting PCR products are targeted for amplification with the outer primers to introduce the barcode and tag sequences at both ends, to which adapter molecules can be attached in a single-step reaction. b, c Taxonomic assignments of a mock community analyzed by MinION™ sequencing. The V1-V9 or V3-V4 region of the 16S rRNA gene was amplified from a pre-characterized mock community sample comprising ten bacterial species and sequenced on the MinION™ platform. Three thousand reads were randomly selected from the processed data set and aligned directly to the reference genome database of 5850 representative bacterial species. The pie charts represent taxonomic profiles at the (b) genus and (c) species levels. Slices corresponding to misclassified (assigned to bacteria not present in the mock community) or unclassified (not classified at the species level but placed in a higher taxonomic rank) reads are exploded. The relative abundance (%) of each taxon is shown.
    Figure Legend Snippet: Metagenomic analysis of the 16S rRNA gene amplicons using MinION™ nanopore sequencing. a Workflow of 16S rRNA amplicon sequencing on the MinION™ platform. Sequencing libraries are generated by the four-primer PCR-based strategy, enabling simplified post-PCR adapter attachment. At the initial stage of PCR, the 16S rRNA gene is amplified with the inner primer pairs. The resulting PCR products are targeted for amplification with the outer primers to introduce the barcode and tag sequences at both ends, to which adapter molecules can be attached in a single-step reaction. b, c Taxonomic assignments of a mock community analyzed by MinION™ sequencing. The V1-V9 or V3-V4 region of the 16S rRNA gene was amplified from a pre-characterized mock community sample comprising ten bacterial species and sequenced on the MinION™ platform. Three thousand reads were randomly selected from the processed data set and aligned directly to the reference genome database of 5850 representative bacterial species. The pie charts represent taxonomic profiles at the (b) genus and (c) species levels. Slices corresponding to misclassified (assigned to bacteria not present in the mock community) or unclassified (not classified at the species level but placed in a higher taxonomic rank) reads are exploded. The relative abundance (%) of each taxon is shown.

    Techniques Used: Nanopore Sequencing, Amplification, Sequencing, Generated, Polymerase Chain Reaction, Introduce

    2) Product Images from "RNAi combining Sleeping Beauty transposon system inhibits ex vivo expression of foot-and-mouth disease virus VP1 in transgenic sheep cells"

    Article Title: RNAi combining Sleeping Beauty transposon system inhibits ex vivo expression of foot-and-mouth disease virus VP1 in transgenic sheep cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-09302-1

    Inhibitory effects of VP1 -shRNA on FMDV- VP1 . ( A ) Schematic map of the pLL3.7- VP1 -shRNA expression vector. ( B ) The VP1 gene amplification using overlapping PCR; 1: a-h, 2: a-g, 3: a-f, 4: a-e, 5: a-d, 6: a-c, 7: a-b, and M: marker. ( C ) Use of Dual-Glo luciferase to detect the inhibitory effect of targeted genes. 239FT cells were co-transfected with pll3.7-shRNA and psiCheck2 genes with different ratios, and the expression of Dual-Glo luciferase reporter genes was measured after 48 h. Data were expressed as the means ± S.E.M. (n = 3). Columns with different superscripts differ significantly, P
    Figure Legend Snippet: Inhibitory effects of VP1 -shRNA on FMDV- VP1 . ( A ) Schematic map of the pLL3.7- VP1 -shRNA expression vector. ( B ) The VP1 gene amplification using overlapping PCR; 1: a-h, 2: a-g, 3: a-f, 4: a-e, 5: a-d, 6: a-c, 7: a-b, and M: marker. ( C ) Use of Dual-Glo luciferase to detect the inhibitory effect of targeted genes. 239FT cells were co-transfected with pll3.7-shRNA and psiCheck2 genes with different ratios, and the expression of Dual-Glo luciferase reporter genes was measured after 48 h. Data were expressed as the means ± S.E.M. (n = 3). Columns with different superscripts differ significantly, P

    Techniques Used: shRNA, Expressing, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Marker, Luciferase, Transfection

    Identification of SB transposon-mediated transgenic sheep. ( A ) Schematic diagram of the inserted part of the pUC- VP1 -shRNA expression vector, which was cut from pLL3.7 and ligated into the PUC19-IR/DR vector. ( B ) Pronuclear microinjection. (a): non-centrifuged fertilized ovine egg and (b): centrifuged fertilized egg (12000 × g for 5 min). ( C ) Electrophoresis of PCR products and ( D ) Southern blot analysis to identify the transgenic sheep, respectively. a: U6- VP1 -shRNA group and linearized pLL3.7- VP1 -shRNA plasmid group. b: IR/DR-U6- VP1 -shRNA + SB100 × group. WT = wild type as a negative control; “p”: positive control; “1–8”: transgenic lambs. ( E ) Inhibitory effects of shRNA on VP1 gene expression in ear fibroblasts of transgenic versus wild type sheep as determined by luciferase reporter assay. Each test was repeated three times for each individual. Tg ( = transgenic sheep), N = 8; WT (wild type), N = 8. ( F ) A photo of the transgenic lamb. Data were expressed as the means ± S.E.M. Columns with different superscripts differed significantly, P
    Figure Legend Snippet: Identification of SB transposon-mediated transgenic sheep. ( A ) Schematic diagram of the inserted part of the pUC- VP1 -shRNA expression vector, which was cut from pLL3.7 and ligated into the PUC19-IR/DR vector. ( B ) Pronuclear microinjection. (a): non-centrifuged fertilized ovine egg and (b): centrifuged fertilized egg (12000 × g for 5 min). ( C ) Electrophoresis of PCR products and ( D ) Southern blot analysis to identify the transgenic sheep, respectively. a: U6- VP1 -shRNA group and linearized pLL3.7- VP1 -shRNA plasmid group. b: IR/DR-U6- VP1 -shRNA + SB100 × group. WT = wild type as a negative control; “p”: positive control; “1–8”: transgenic lambs. ( E ) Inhibitory effects of shRNA on VP1 gene expression in ear fibroblasts of transgenic versus wild type sheep as determined by luciferase reporter assay. Each test was repeated three times for each individual. Tg ( = transgenic sheep), N = 8; WT (wild type), N = 8. ( F ) A photo of the transgenic lamb. Data were expressed as the means ± S.E.M. Columns with different superscripts differed significantly, P

    Techniques Used: Transgenic Assay, shRNA, Expressing, Plasmid Preparation, Electrophoresis, Polymerase Chain Reaction, Southern Blot, Negative Control, Positive Control, Luciferase, Reporter Assay

    3) Product Images from "Clinical significance of DNA methylation mRNA levels of TET family members in colorectal cancer"

    Article Title: Clinical significance of DNA methylation mRNA levels of TET family members in colorectal cancer

    Journal: Journal of Cancer Research and Clinical Oncology

    doi: 10.1007/s00432-014-1901-2

    DNA methylation assessment of TET1 , TET2 and TET3 gene regulatory region by bisulfite sequencing in tissue samples from patients with CRC. Primary cancerous and histopathologically unchanged tissues from the same patients with CRC (P1–P5) were used for genomic DNA isolation followed by bisulfite conversion of cytosine to uracil. The TET1, TET2 and TET3 regions containing 47, 64 and 40 CpG dinucleotides, respectively, were then amplified by a pair of primers complementary to the bisulfite-DNA modified sequence. The PCR products were purified with subsequent cloning into a plasmid vector. Plasmid DNA isolated from five positive bacterial clones was used for commercial sequencing. The results of bisulfite sequencing were assessed and presented using BiQ analyzer software and BDPC web server. Black , gray and white boxes represent methylated, unmethylated or undetermined CpG dinucleotide, respectively
    Figure Legend Snippet: DNA methylation assessment of TET1 , TET2 and TET3 gene regulatory region by bisulfite sequencing in tissue samples from patients with CRC. Primary cancerous and histopathologically unchanged tissues from the same patients with CRC (P1–P5) were used for genomic DNA isolation followed by bisulfite conversion of cytosine to uracil. The TET1, TET2 and TET3 regions containing 47, 64 and 40 CpG dinucleotides, respectively, were then amplified by a pair of primers complementary to the bisulfite-DNA modified sequence. The PCR products were purified with subsequent cloning into a plasmid vector. Plasmid DNA isolated from five positive bacterial clones was used for commercial sequencing. The results of bisulfite sequencing were assessed and presented using BiQ analyzer software and BDPC web server. Black , gray and white boxes represent methylated, unmethylated or undetermined CpG dinucleotide, respectively

    Techniques Used: DNA Methylation Assay, Methylation Sequencing, DNA Extraction, Amplification, Modification, Sequencing, Polymerase Chain Reaction, Purification, Clone Assay, Plasmid Preparation, Isolation, Software, Methylation

    DNA methylation effect on TET1 mRNA levels in cancerous tissue. The primary cancerous tissues from 113 patients with CRC were used for RNA isolation. Total RNA was reverse-transcribed, and cDNAs were investigated by RQ-PCR relative quantification analysis. The TET1 mRNA levels were corrected by the geometric mean of PBGD and hMRPL19 cDNA levels. The amount of TET1 mRNA was expressed as the decimal logarithm of multiples of cDNA copies in the calibrator
    Figure Legend Snippet: DNA methylation effect on TET1 mRNA levels in cancerous tissue. The primary cancerous tissues from 113 patients with CRC were used for RNA isolation. Total RNA was reverse-transcribed, and cDNAs were investigated by RQ-PCR relative quantification analysis. The TET1 mRNA levels were corrected by the geometric mean of PBGD and hMRPL19 cDNA levels. The amount of TET1 mRNA was expressed as the decimal logarithm of multiples of cDNA copies in the calibrator

    Techniques Used: DNA Methylation Assay, Isolation, Polymerase Chain Reaction

    DNA methylation assessment of TET1 , TET2 and TET3 gene regulatory region by HRM analysis in tissue samples from patients with CRC. Primary cancerous and histopathologically unchanged tissues from the same patients with CRC were used for genomic DNA isolation followed by bisulfite conversion of cytosine to uracil. a – c Represent HRM profiles of standard and example of patient DNA PCR product for TET1, TET2 and TET3, respectively. Methylation percentage of DNA fragments within the CpG island was determined by real-time PCR amplification of bisulfite-treated standard and patient DNA, followed by comparison of their HRM profiles. DNA standards were prepared by mixing different ratios of methylated and non-methylated bisulfite-treated standard DNA. HRM methylation analysis was performed using Light Cycler ® 480 or LightCycler ® 96 Gene Scanning software, Roche Diagnostics GmbH (Mannheim, Germany). Each PCR amplification and HRM profile analysis was performed in triplicate
    Figure Legend Snippet: DNA methylation assessment of TET1 , TET2 and TET3 gene regulatory region by HRM analysis in tissue samples from patients with CRC. Primary cancerous and histopathologically unchanged tissues from the same patients with CRC were used for genomic DNA isolation followed by bisulfite conversion of cytosine to uracil. a – c Represent HRM profiles of standard and example of patient DNA PCR product for TET1, TET2 and TET3, respectively. Methylation percentage of DNA fragments within the CpG island was determined by real-time PCR amplification of bisulfite-treated standard and patient DNA, followed by comparison of their HRM profiles. DNA standards were prepared by mixing different ratios of methylated and non-methylated bisulfite-treated standard DNA. HRM methylation analysis was performed using Light Cycler ® 480 or LightCycler ® 96 Gene Scanning software, Roche Diagnostics GmbH (Mannheim, Germany). Each PCR amplification and HRM profile analysis was performed in triplicate

    Techniques Used: DNA Methylation Assay, DNA Extraction, Polymerase Chain Reaction, Methylation, Real-time Polymerase Chain Reaction, Amplification, Software

    4) Product Images from "A tripartite ssDNA mycovirus from a plant pathogenic fungus is infectious as cloned DNA and purified virions"

    Article Title: A tripartite ssDNA mycovirus from a plant pathogenic fungus is infectious as cloned DNA and purified virions

    Journal: Science Advances

    doi: 10.1126/sciadv.aay9634

    Characterization of FgGMTV1 infecting F. graminearum strain HB58 and composition of the isolated virus particles. ( A ) Colony morphology of virus-carrying field strain HB58 of F. graminearum after 3 days of culture on potato dextrose agar (PDA) in the dark. ( B ) Viral particles observed under transmission electron microscopy. Scale bar, 50 nm. ( C ) One percent agarose gel electrophoresis of DNA extracted from virus particles. bp, base pair. ( D ) Electrophoresis analysis of enzyme-treated nucleic acid samples on 1% agarose gel. The samples were treated with DNase I, exonuclease III, S1 nuclease, and exonuclease I, respectively. ( E ) SDS–polyacrylamide gel electrophoresis analysis of purified virus particles from strain HB58 showing three protein bands. ( F ) Southern blot analyses of nucleic acids extracted from viral particles. Genomic DNA extracted from strain HB58 was used as a positive control. The forms of the viral DNA are indicated as OC dsDNA (open circular dsDNA), SC dsDNA (supercoiled dsDNA), and circular ssDNA. Three DNA fragments (probe A, probe B, and probe C, specific for DNA-A, DNA-B, and DNA-C, respectively) were PCR-amplified, labeled with DIG, and used as DNA probes. ( G ) Genome organization of the three components of FgGMTV1. The single ORF of each DNA component is displayed as a thick arrow. The positions of the potential stem-loop structure, common region major (CR-M) and common region stem-loop (CR-SL) are indicated. Photo credit: Pengfei Li, Institute of Plant Protection, Chinese Academy of Agricultural Sciences.
    Figure Legend Snippet: Characterization of FgGMTV1 infecting F. graminearum strain HB58 and composition of the isolated virus particles. ( A ) Colony morphology of virus-carrying field strain HB58 of F. graminearum after 3 days of culture on potato dextrose agar (PDA) in the dark. ( B ) Viral particles observed under transmission electron microscopy. Scale bar, 50 nm. ( C ) One percent agarose gel electrophoresis of DNA extracted from virus particles. bp, base pair. ( D ) Electrophoresis analysis of enzyme-treated nucleic acid samples on 1% agarose gel. The samples were treated with DNase I, exonuclease III, S1 nuclease, and exonuclease I, respectively. ( E ) SDS–polyacrylamide gel electrophoresis analysis of purified virus particles from strain HB58 showing three protein bands. ( F ) Southern blot analyses of nucleic acids extracted from viral particles. Genomic DNA extracted from strain HB58 was used as a positive control. The forms of the viral DNA are indicated as OC dsDNA (open circular dsDNA), SC dsDNA (supercoiled dsDNA), and circular ssDNA. Three DNA fragments (probe A, probe B, and probe C, specific for DNA-A, DNA-B, and DNA-C, respectively) were PCR-amplified, labeled with DIG, and used as DNA probes. ( G ) Genome organization of the three components of FgGMTV1. The single ORF of each DNA component is displayed as a thick arrow. The positions of the potential stem-loop structure, common region major (CR-M) and common region stem-loop (CR-SL) are indicated. Photo credit: Pengfei Li, Institute of Plant Protection, Chinese Academy of Agricultural Sciences.

    Techniques Used: Isolation, Transmission Assay, Electron Microscopy, Agarose Gel Electrophoresis, Electrophoresis, Polyacrylamide Gel Electrophoresis, Purification, Southern Blot, Positive Control, Polymerase Chain Reaction, Amplification, Labeling

    5) Product Images from "HSPA12A is required for adipocyte differentiation and diet-induced obesity through a positive feedback regulation with PPARγ"

    Article Title: HSPA12A is required for adipocyte differentiation and diet-induced obesity through a positive feedback regulation with PPARγ

    Journal: Cell Death and Differentiation

    doi: 10.1038/s41418-019-0300-2

    Deficiency of HSPA12A suppressed PPARγ and its target genes linking to adipocyte differentiation in mice. Inguinal WAT were collected from mice that fed with HFD or chow diet for 14 weeks. The expression of the indicated mRNA and proteins was analyzed by Real-time PCR ( a ) and Immunoblotting ( b ), respectively. Data are mean ± SEM, ** P
    Figure Legend Snippet: Deficiency of HSPA12A suppressed PPARγ and its target genes linking to adipocyte differentiation in mice. Inguinal WAT were collected from mice that fed with HFD or chow diet for 14 weeks. The expression of the indicated mRNA and proteins was analyzed by Real-time PCR ( a ) and Immunoblotting ( b ), respectively. Data are mean ± SEM, ** P

    Techniques Used: Mouse Assay, Expressing, Real-time Polymerase Chain Reaction

    HSPA12A regulated adipocyte differentiation and PPARγ expression in vitro. a , b HSPA12A deficiency suppressed adipocyte differentiation. Differentiation was induced in primary SVF isolated from WT and Hspa12a -/- mice. Lipid droplets were examined by ORO staining ( a ). Expression of mRNA was examined using real-time PCR ( b ). Data are mean ± SEM, ** P
    Figure Legend Snippet: HSPA12A regulated adipocyte differentiation and PPARγ expression in vitro. a , b HSPA12A deficiency suppressed adipocyte differentiation. Differentiation was induced in primary SVF isolated from WT and Hspa12a -/- mice. Lipid droplets were examined by ORO staining ( a ). Expression of mRNA was examined using real-time PCR ( b ). Data are mean ± SEM, ** P

    Techniques Used: Expressing, In Vitro, Isolation, Mouse Assay, Staining, Real-time Polymerase Chain Reaction

    6) Product Images from "Use of an Automated Multiple-Locus, Variable-Number Tandem Repeat-Based Method for Rapid and High-Throughput Genotyping of Staphylococcus aureus Isolates"

    Article Title: Use of an Automated Multiple-Locus, Variable-Number Tandem Repeat-Based Method for Rapid and High-Throughput Genotyping of Staphylococcus aureus Isolates

    Journal:

    doi: 10.1128/JCM.43.7.3346-3355.2005

    Effect of lysis duration on DNA yields and PCR amplification results. (A) Amounts of total purified DNA obtained after the rapid lysis procedure (strain MW2), as determined by the fluorescence area under the curve (BioAnalyzer). The electropherogram shown
    Figure Legend Snippet: Effect of lysis duration on DNA yields and PCR amplification results. (A) Amounts of total purified DNA obtained after the rapid lysis procedure (strain MW2), as determined by the fluorescence area under the curve (BioAnalyzer). The electropherogram shown

    Techniques Used: Lysis, Polymerase Chain Reaction, Amplification, Purification, Fluorescence

    7) Product Images from "Messenger RNAs of Yeast Virus-Like Elements Contain Non-templated 5′ Poly(A) Leaders, and Their Expression Is Independent of eIF4E and Pab1"

    Article Title: Messenger RNAs of Yeast Virus-Like Elements Contain Non-templated 5′ Poly(A) Leaders, and Their Expression Is Independent of eIF4E and Pab1

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2019.02366

    K2ORF5 mRNA is outcompeted by cellular RNA in binding to the yeast cap-binding protein eIF4E in vitro . Electrophoretic analysis of semiquantitative RT-PCR detecting control HGT1 mRNA (upper panel) and K2ORF5 mRNA (lower panel) in samples as follows: Lane 1, glutathione-Sepharose with the bound GST-eIF4E fusion protein in the presence of excess K. lactis IFO1267 total RNA (input); lane 2, same as in line 1 but the reaction was performed without reverse transcriptase (negative control); lane 3, supernatant from the first wash step (unbound mRNA); lanes 4, 5, and 6, supernatants after the second, third, and sixth wash steps, respectively (unbound mRNA); lane 7, mRNA remaining bound on GST-S.c-eIF4E Sepharose after the sixth wash step. M, GeneRuler 100-bp DNA Ladder Plus (Thermo Scientific). PCR was performed using cDNA, Taq DNA polymerase, and the gene-specific primers listed in Supplementary Table S1 . All washing steps were performed with 70 volumes of buffer I. The initial abundances of HGT1 and K2ORF5 mRNA in the K. lactis total RNA were comparable as determined by qRT-PCR ( Supplementary Figure S3 ).
    Figure Legend Snippet: K2ORF5 mRNA is outcompeted by cellular RNA in binding to the yeast cap-binding protein eIF4E in vitro . Electrophoretic analysis of semiquantitative RT-PCR detecting control HGT1 mRNA (upper panel) and K2ORF5 mRNA (lower panel) in samples as follows: Lane 1, glutathione-Sepharose with the bound GST-eIF4E fusion protein in the presence of excess K. lactis IFO1267 total RNA (input); lane 2, same as in line 1 but the reaction was performed without reverse transcriptase (negative control); lane 3, supernatant from the first wash step (unbound mRNA); lanes 4, 5, and 6, supernatants after the second, third, and sixth wash steps, respectively (unbound mRNA); lane 7, mRNA remaining bound on GST-S.c-eIF4E Sepharose after the sixth wash step. M, GeneRuler 100-bp DNA Ladder Plus (Thermo Scientific). PCR was performed using cDNA, Taq DNA polymerase, and the gene-specific primers listed in Supplementary Table S1 . All washing steps were performed with 70 volumes of buffer I. The initial abundances of HGT1 and K2ORF5 mRNA in the K. lactis total RNA were comparable as determined by qRT-PCR ( Supplementary Figure S3 ).

    Techniques Used: Binding Assay, In Vitro, Reverse Transcription Polymerase Chain Reaction, Negative Control, Polymerase Chain Reaction, Quantitative RT-PCR

    Precise manipulation of pGKL VLEs in vivo revealed an essential role of pGKL promoters in mRNA capping and non-template-based 5′ polyadenylation. (A) A closer view of the native pGKL2 region subjected to homologous recombination with a PCR cassette depicted in Supplementary Figure S5 shows a tightly packed VLE genome. The 3′ end of the K2ORF3 coding region overlaps the K2ORF2 promoter, 5′ UTR, and the first four nucleotides of the K2ORF2 coding region. The pGKL2 VLEs displayed in (B,C) are in the reverse orientation of those in Figure 1 . Shades of gray indicate the degree of transcript capping, as shown in Figure 1 . (B) A PCR cassette containing an antibiotic resistance gene (G418) under the control of the ORF2 promoter from pGKL1 ( K1UCR2 ) and the ORF1 promoter from pGKL1 ( K1UCR1 ) ( Supplementary Figure S5 ) was inserted into the K2ORF2 promoter region by homologous recombination in vivo . The resulting VLE, pRKL2-1, contains two genes, aminoglycoside 3′-phosphotransferase (coding for G418 resistance) and K2ORF2 , that are artificially controlled by the pGKL1 promoters K1UCR2 and K1UCR1 , respectively. Shades of gray indicate the degree of transcript capping, as shown in Figure 1 . The 5′ RACE results of pRKL2-1-encoded mRNAs are summarized in the text and in Supplementary Table S7 . (C) Electrophoretic analysis of pGKL VLEs in K. lactis clones. M, lambda DNA/ Eco 130I ( Sty I) marker (Fermentas); lanes 1 and 4, native pGKL VLEs from K. lactis IFO1267 (pGKL1 [8874 bp] is labeled with an asterisk, and pGKL2 [13447 bp] is labeled with an arrow); lane 2, linear VLEs purified from K. lactis IFO1267 carrying both the recombinant (higher MW) and wild-type pGKL2 VLEs; lane 3, linear VLEs purified from K. lactis IFO1267 containing the recombinant pRKL2-1 VLE (14353 bp). The shorter wild-type pGKL2 was lost after cultivation for ≈60 generations in selective medium containing G418.
    Figure Legend Snippet: Precise manipulation of pGKL VLEs in vivo revealed an essential role of pGKL promoters in mRNA capping and non-template-based 5′ polyadenylation. (A) A closer view of the native pGKL2 region subjected to homologous recombination with a PCR cassette depicted in Supplementary Figure S5 shows a tightly packed VLE genome. The 3′ end of the K2ORF3 coding region overlaps the K2ORF2 promoter, 5′ UTR, and the first four nucleotides of the K2ORF2 coding region. The pGKL2 VLEs displayed in (B,C) are in the reverse orientation of those in Figure 1 . Shades of gray indicate the degree of transcript capping, as shown in Figure 1 . (B) A PCR cassette containing an antibiotic resistance gene (G418) under the control of the ORF2 promoter from pGKL1 ( K1UCR2 ) and the ORF1 promoter from pGKL1 ( K1UCR1 ) ( Supplementary Figure S5 ) was inserted into the K2ORF2 promoter region by homologous recombination in vivo . The resulting VLE, pRKL2-1, contains two genes, aminoglycoside 3′-phosphotransferase (coding for G418 resistance) and K2ORF2 , that are artificially controlled by the pGKL1 promoters K1UCR2 and K1UCR1 , respectively. Shades of gray indicate the degree of transcript capping, as shown in Figure 1 . The 5′ RACE results of pRKL2-1-encoded mRNAs are summarized in the text and in Supplementary Table S7 . (C) Electrophoretic analysis of pGKL VLEs in K. lactis clones. M, lambda DNA/ Eco 130I ( Sty I) marker (Fermentas); lanes 1 and 4, native pGKL VLEs from K. lactis IFO1267 (pGKL1 [8874 bp] is labeled with an asterisk, and pGKL2 [13447 bp] is labeled with an arrow); lane 2, linear VLEs purified from K. lactis IFO1267 carrying both the recombinant (higher MW) and wild-type pGKL2 VLEs; lane 3, linear VLEs purified from K. lactis IFO1267 containing the recombinant pRKL2-1 VLE (14353 bp). The shorter wild-type pGKL2 was lost after cultivation for ≈60 generations in selective medium containing G418.

    Techniques Used: In Vivo, Homologous Recombination, Polymerase Chain Reaction, Clone Assay, Lambda DNA Preparation, Marker, Labeling, Purification, Recombinant

    Differences in the lengths of the 5′ mRNA poly(A) leaders of individual pGKL ORFs. The box whisker plot represents the number of templated and non-templated consecutive adenosine nucleotides at the 5′ ends of pGKL mRNAs. The bottom and top of the box indicate the first and third quartiles, respectively. The whiskers indicate the 10th and 90th percentiles. The median is indicated as a solid black line. Outliers are not indicated. The open reading frames are ranked from left to right according to the prevalent lengths of the 5′ poly(A) leaders of their mRNAs. The value of 12 adenosine nucleotides represents an optimal length of the Pab1 binding site and is indicated as a solid gray line. The pGKL1 mRNAs belong to those with the longest 5′ poly(A) leaders. The data did not follow a normal distribution according to the Shapiro–Wilk test. The results were statistically analyzed using the non-parametric Kruskal–Wallis test, which supported rejection of the null hypothesis, p -value = 2.172940e-48. To discern gene pairs, the transcripts of which significantly differed in the lengths of their 5′ poly(A) leaders, we performed Dunn post hoc tests followed by adjustment of p -values according to the Benjamini–Hochberg FDR method. Adjusted p -values corresponding to all possible gene pairs are depicted in Supplementary Table S10 . In total, 458 sequences obtained by 5′ RACE-PCR were used for this analysis ( Supplementary Table S5A ).
    Figure Legend Snippet: Differences in the lengths of the 5′ mRNA poly(A) leaders of individual pGKL ORFs. The box whisker plot represents the number of templated and non-templated consecutive adenosine nucleotides at the 5′ ends of pGKL mRNAs. The bottom and top of the box indicate the first and third quartiles, respectively. The whiskers indicate the 10th and 90th percentiles. The median is indicated as a solid black line. Outliers are not indicated. The open reading frames are ranked from left to right according to the prevalent lengths of the 5′ poly(A) leaders of their mRNAs. The value of 12 adenosine nucleotides represents an optimal length of the Pab1 binding site and is indicated as a solid gray line. The pGKL1 mRNAs belong to those with the longest 5′ poly(A) leaders. The data did not follow a normal distribution according to the Shapiro–Wilk test. The results were statistically analyzed using the non-parametric Kruskal–Wallis test, which supported rejection of the null hypothesis, p -value = 2.172940e-48. To discern gene pairs, the transcripts of which significantly differed in the lengths of their 5′ poly(A) leaders, we performed Dunn post hoc tests followed by adjustment of p -values according to the Benjamini–Hochberg FDR method. Adjusted p -values corresponding to all possible gene pairs are depicted in Supplementary Table S10 . In total, 458 sequences obtained by 5′ RACE-PCR were used for this analysis ( Supplementary Table S5A ).

    Techniques Used: Whisker Assay, Binding Assay, Polymerase Chain Reaction

    8) Product Images from "Excessive folic acid supplementation in pregnant mice impairs insulin secretion and induces the expression of genes associated with fatty liver in their offspring"

    Article Title: Excessive folic acid supplementation in pregnant mice impairs insulin secretion and induces the expression of genes associated with fatty liver in their offspring

    Journal: Heliyon

    doi: 10.1016/j.heliyon.2020.e03597

    mRNA expression of genes involved in carbohydrate metabolism. Analysis of the mRNA expression of genes involved in carbohydrate metabolism was performed in offspring liver using real-time RT-PCR. A) Pparγ1 , B) Pparγ2 , C) Acaca , D) Acacb , E) C idec , F) Acox1 , G) Fasn , H) Gpd1 , I) Pck1 , J) Pck2 , K) Ggat1 , and L) G gat2 . Control group (CN; white bars); high-folic acid group (HFA; black bars), The fold difference between the HFA and CN groups are shown at the top of the graphs (∗, P
    Figure Legend Snippet: mRNA expression of genes involved in carbohydrate metabolism. Analysis of the mRNA expression of genes involved in carbohydrate metabolism was performed in offspring liver using real-time RT-PCR. A) Pparγ1 , B) Pparγ2 , C) Acaca , D) Acacb , E) C idec , F) Acox1 , G) Fasn , H) Gpd1 , I) Pck1 , J) Pck2 , K) Ggat1 , and L) G gat2 . Control group (CN; white bars); high-folic acid group (HFA; black bars), The fold difference between the HFA and CN groups are shown at the top of the graphs (∗, P

    Techniques Used: Expressing, Quantitative RT-PCR

    9) Product Images from "A highly pathogenic porcine reproductive and respiratory syndrome virus generated from an infectious cDNA clone retains the in vivo virulence and transmissibility properties of the parental virus"

    Article Title: A highly pathogenic porcine reproductive and respiratory syndrome virus generated from an infectious cDNA clone retains the in vivo virulence and transmissibility properties of the parental virus

    Journal: Virology

    doi: 10.1016/j.virol.2004.04.046

    The cloned virus retains in vivo markers of virulence and is genetically stable. Three groups of pigs (four pigs per group) were sham-inoculated or inoculated with either vParental or vFL12 at TCID 50 /ml of 10 5.2 and 10 4.8 , respectively. (A) Temperatures were recorded daily from 2 days before inoculation to 16 dpi. Average temperatures from four different animals in each group are shown. (B) Serum samples were collected at 4, 7, 14, and 22 dpi and virus titers were determined and expressed as TCID 50 /ml. Values represent average titers from four animals. (C) Serum antibody titers were determined with the Idexx ELISA kit. Values represent average titers from four animals. Dashed line at 0.4 S/P ratio designates threshold value above which titers are considered positive for anti-PRRSV antibodies. Error bars in panels A, B, and C represent standard deviation. (D) Serum from 14 dpi, recovered from inoculated animals, was used to distinguish between vFL12 and vParental by examining for the presence of the Bsr GI genetic tag (left panel). Viral RNA extracted from sera was amplified by RT-PCR and the products were digested with Bsr GI. Similar analysis (lane 7) was performed with virus in serum from a sentinel pig at 8 days postcohabitation with pigs infected with vFL12 (right panel). Gel image is inverted. Error bars represent standard deviation.
    Figure Legend Snippet: The cloned virus retains in vivo markers of virulence and is genetically stable. Three groups of pigs (four pigs per group) were sham-inoculated or inoculated with either vParental or vFL12 at TCID 50 /ml of 10 5.2 and 10 4.8 , respectively. (A) Temperatures were recorded daily from 2 days before inoculation to 16 dpi. Average temperatures from four different animals in each group are shown. (B) Serum samples were collected at 4, 7, 14, and 22 dpi and virus titers were determined and expressed as TCID 50 /ml. Values represent average titers from four animals. (C) Serum antibody titers were determined with the Idexx ELISA kit. Values represent average titers from four animals. Dashed line at 0.4 S/P ratio designates threshold value above which titers are considered positive for anti-PRRSV antibodies. Error bars in panels A, B, and C represent standard deviation. (D) Serum from 14 dpi, recovered from inoculated animals, was used to distinguish between vFL12 and vParental by examining for the presence of the Bsr GI genetic tag (left panel). Viral RNA extracted from sera was amplified by RT-PCR and the products were digested with Bsr GI. Similar analysis (lane 7) was performed with virus in serum from a sentinel pig at 8 days postcohabitation with pigs infected with vFL12 (right panel). Gel image is inverted. Error bars represent standard deviation.

    Techniques Used: Clone Assay, In Vivo, Enzyme-linked Immunosorbent Assay, Standard Deviation, Amplification, Reverse Transcription Polymerase Chain Reaction, Infection

    10) Product Images from "Stolbur Phytoplasma Genome Survey Achieved Using a Suppression Subtractive Hybridization Approach with High Specificity †"

    Article Title: Stolbur Phytoplasma Genome Survey Achieved Using a Suppression Subtractive Hybridization Approach with High Specificity †

    Journal:

    doi: 10.1128/AEM.72.5.3274-3283.2006

    PCR amplification of SSH orphan sequences. PCRs were performed on healthy periwinkle DNA (lane 1) and stolbur phytoplasma-infected periwinkle DNA (lane 2) for HincII-SSH libraries and RsaI-SSH libraries. Clone names are indicated according to libraries.
    Figure Legend Snippet: PCR amplification of SSH orphan sequences. PCRs were performed on healthy periwinkle DNA (lane 1) and stolbur phytoplasma-infected periwinkle DNA (lane 2) for HincII-SSH libraries and RsaI-SSH libraries. Clone names are indicated according to libraries.

    Techniques Used: Polymerase Chain Reaction, Amplification, Infection

    11) Product Images from "Inhibition of Epstein-Barr Virus Infection by Lactoferrin"

    Article Title: Inhibition of Epstein-Barr Virus Infection by Lactoferrin

    Journal: Journal of Innate Immunity

    doi: 10.1159/000336178

    LF inhibits EBV entry. Primary B cells were incubated with hLF (50 µg/ml) or BSA (50 µg/ml) as control at 4°C for 1 h and hLF was washed away. After that, the cells were infected with EBV (MOI 50) at 4°C for 3 h and were then washed with PBS to remove unbound virus. a Total cellular and bound viral DNA was extracted, and the EBV copy number per cell (i.e. EBV titers) was determined using the Q-PCR assay of the EBV Bam HI-W fragment. The EBV titer of the control treatment group (first lane) was adjusted as 1. b Total cellular RNA of the treated primary B cells was extracted, and mRNA expression levels of EBV gene EBER were measured by Q-PCR assay. The expression level of the control group was adjusted as 1. Two-way ANOVA was used to evaluate differences between the three groups. ** p
    Figure Legend Snippet: LF inhibits EBV entry. Primary B cells were incubated with hLF (50 µg/ml) or BSA (50 µg/ml) as control at 4°C for 1 h and hLF was washed away. After that, the cells were infected with EBV (MOI 50) at 4°C for 3 h and were then washed with PBS to remove unbound virus. a Total cellular and bound viral DNA was extracted, and the EBV copy number per cell (i.e. EBV titers) was determined using the Q-PCR assay of the EBV Bam HI-W fragment. The EBV titer of the control treatment group (first lane) was adjusted as 1. b Total cellular RNA of the treated primary B cells was extracted, and mRNA expression levels of EBV gene EBER were measured by Q-PCR assay. The expression level of the control group was adjusted as 1. Two-way ANOVA was used to evaluate differences between the three groups. ** p

    Techniques Used: Incubation, Infection, Polymerase Chain Reaction, Expressing

    LF does not inhibit EBV replication. Lymphoblastoid cells ( a ) and P3HR1 cells ( b ) were treated with either hLF (10 or 100 µg/ml) or BSA (as control) for 3, 6, 15 or 21 days. hLF was presented throughout the reproduction process. The cells were harvested, and viral DNA copies (EBV titers) were determined by Q-PCR assay of the EBV Bam HI-W fragment. The EBV titer of the control treatment group (first lane) was adjusted as 1 in both cell types.
    Figure Legend Snippet: LF does not inhibit EBV replication. Lymphoblastoid cells ( a ) and P3HR1 cells ( b ) were treated with either hLF (10 or 100 µg/ml) or BSA (as control) for 3, 6, 15 or 21 days. hLF was presented throughout the reproduction process. The cells were harvested, and viral DNA copies (EBV titers) were determined by Q-PCR assay of the EBV Bam HI-W fragment. The EBV titer of the control treatment group (first lane) was adjusted as 1 in both cell types.

    Techniques Used: Polymerase Chain Reaction

    12) Product Images from "Essential Role of Chitinase in the Development of the Filarial Nematode Acanthocheilonema viteae ▿"

    Article Title: Essential Role of Chitinase in the Development of the Filarial Nematode Acanthocheilonema viteae ▿

    Journal:

    doi: 10.1128/IAI.00701-07

    Quantification of chitinase transcripts in different Acanthocheilonema viteae stages by real-time PCR. The ordinate depicts the log 10 values of relative chitinase expression, calculated by the 2 −ΔΔ C T method. The values represent
    Figure Legend Snippet: Quantification of chitinase transcripts in different Acanthocheilonema viteae stages by real-time PCR. The ordinate depicts the log 10 values of relative chitinase expression, calculated by the 2 −ΔΔ C T method. The values represent

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    13) Product Images from "The African buffalo parasite Theileria. sp. (buffalo) can infect and immortalize cattle leukocytes and encodes divergent orthologues of Theileria parva antigen genes"

    Article Title: The African buffalo parasite Theileria. sp. (buffalo) can infect and immortalize cattle leukocytes and encodes divergent orthologues of Theileria parva antigen genes

    Journal: International Journal for Parasitology: Parasites and Wildlife

    doi: 10.1016/j.ijppaw.2015.08.006

    PCR amplification of genes encoding Theileria parva antigens from Marula schizont-infected leukocyte cultures. Panel A, p104 primers; Panel B PIM, primers; Panel C p67 primers. The order of the schizont-infected lymphocyte samples is (1) N6; (2). N13; (3). N18; (4). N20; (5). N33; (6). N36; (7). N38; (8). N43; (9). N50; (10). N55; (11). N69; (12). N76; (13). N77, (14). N79; (15). N86, (16). N88; (17). N99; (18). N100; (19). N102; (20). N103; (21). N106; (22). N107.
    Figure Legend Snippet: PCR amplification of genes encoding Theileria parva antigens from Marula schizont-infected leukocyte cultures. Panel A, p104 primers; Panel B PIM, primers; Panel C p67 primers. The order of the schizont-infected lymphocyte samples is (1) N6; (2). N13; (3). N18; (4). N20; (5). N33; (6). N36; (7). N38; (8). N43; (9). N50; (10). N55; (11). N69; (12). N76; (13). N77, (14). N79; (15). N86, (16). N88; (17). N99; (18). N100; (19). N102; (20). N103; (21). N106; (22). N107.

    Techniques Used: Polymerase Chain Reaction, Amplification, Infection

    14) Product Images from "Characterization of calcineurin A and B genes in the abalone, Haliotis diversicolor, and their immune response role during bacterial infection"

    Article Title: Characterization of calcineurin A and B genes in the abalone, Haliotis diversicolor, and their immune response role during bacterial infection

    Journal: PeerJ

    doi: 10.7717/peerj.8868

    HcCNA and HcCNB gene expression patterns in response to V. parahaemolyticus challenge. The expression patterns of HcCNA and HcCNB were detected by quantitative real-time PCR in the mantle, gill, hepatopancreas, hemocytes, and foot (A, B, C, D, and E). The data was shown expression levels relative to the baseline. β - actin was used as an internal reference gene. Data denoted with difference uppercase and lowercase letters indicated significant difference at p
    Figure Legend Snippet: HcCNA and HcCNB gene expression patterns in response to V. parahaemolyticus challenge. The expression patterns of HcCNA and HcCNB were detected by quantitative real-time PCR in the mantle, gill, hepatopancreas, hemocytes, and foot (A, B, C, D, and E). The data was shown expression levels relative to the baseline. β - actin was used as an internal reference gene. Data denoted with difference uppercase and lowercase letters indicated significant difference at p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Tissue distributions of HcCNA . (A) and HcCNB (B) of H. diversicolor analyzed by real-time PCR. The relative expression was calculated by the Livak method (2 ΔΔ Cq ) using β - actin as a reference gene. Data was presented as mean relative expression of triplicate real-time reactions. Hem, hemocyte; Gil, gill; Hyp, hypobrachial gland; Man, mantle; Epi, epipodium; Gon, gonad; Sto, stomach; Foo, foot; Hep, hepatopancreas; Kid, kidney. Lowercase letters indicated significant differences at p
    Figure Legend Snippet: Tissue distributions of HcCNA . (A) and HcCNB (B) of H. diversicolor analyzed by real-time PCR. The relative expression was calculated by the Livak method (2 ΔΔ Cq ) using β - actin as a reference gene. Data was presented as mean relative expression of triplicate real-time reactions. Hem, hemocyte; Gil, gill; Hyp, hypobrachial gland; Man, mantle; Epi, epipodium; Gon, gonad; Sto, stomach; Foo, foot; Hep, hepatopancreas; Kid, kidney. Lowercase letters indicated significant differences at p

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    15) Product Images from "Haptoglobin polymorphisms in Latin American populations"

    Article Title: Haptoglobin polymorphisms in Latin American populations

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-70755-y

    Real-time PCR and HRM analysis of HP promoter polymorphisms. Typical results of amplicon for rs5471 and amplicon for rs5472 on Ghanaians (n = 95) are shown. Normalized and temperature-shifted melting curves of amplicons for rs5471 ( A ) and rs5472 ( C ) and normalized and temperature-shifted difference plots for rs5471 ( B ) and rs5472 ( D ).
    Figure Legend Snippet: Real-time PCR and HRM analysis of HP promoter polymorphisms. Typical results of amplicon for rs5471 and amplicon for rs5472 on Ghanaians (n = 95) are shown. Normalized and temperature-shifted melting curves of amplicons for rs5471 ( A ) and rs5472 ( C ) and normalized and temperature-shifted difference plots for rs5471 ( B ) and rs5472 ( D ).

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification

    DNA sequences of amplicons for real-time PCR and HRM analyses. DNA sequences of amplicons for rs5471 ( A ) and rs5472 ( B ). The primer pairs of each amplicon are indicated by arrows. Positions and dimorphic bases of rs5471 and rs5472 are also indicated.
    Figure Legend Snippet: DNA sequences of amplicons for real-time PCR and HRM analyses. DNA sequences of amplicons for rs5471 ( A ) and rs5472 ( B ). The primer pairs of each amplicon are indicated by arrows. Positions and dimorphic bases of rs5471 and rs5472 are also indicated.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification

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    Irradiation:

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    Amplification:

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    In Vitro:

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    Isolation:

    Article Title: HSPA12A is required for adipocyte differentiation and diet-induced obesity through a positive feedback regulation with PPARγ
    Article Snippet: .. Quantitative real-time PCR After total mRNA isolation and cDNA synthesis, PCR amplification was performed with SYBR Green PCR Master Mix (Roche). .. Quantitative real-time PCR were performed as described [ ].

    Construct:

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    SYBR Green Assay:

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    shRNA:

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    Labeling:

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    Expressing:

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    DNA Labeling:

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    Polymerase Chain Reaction:

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    Article Title: Excessive folic acid supplementation in pregnant mice impairs insulin secretion and induces the expression of genes associated with fatty liver in their offspring
    Article Snippet: .. To quantify the mRNA expression of selected genes, PCR amplification was performed in a Light Cycler Instrument (Roche Applied Science, Upper Bavaria, Germany). .. Real-time RT-PCR amplifications were carried out in a total volume of 10 μL, containing 400 nM of each gene-specific primer (Rikaken, Aichi, Japan), cDNA, and Light Cycler 480 Probes Master (Roche Applied Science).

    Article Title: Messenger RNAs of Yeast Virus-Like Elements Contain Non-templated 5′ Poly(A) Leaders, and Their Expression Is Independent of eIF4E and Pab1
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    Article Title: Clinical significance of DNA methylation mRNA levels of TET family members in colorectal cancer
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    Article Title: HSPA12A is required for adipocyte differentiation and diet-induced obesity through a positive feedback regulation with PPARγ
    Article Snippet: .. Quantitative real-time PCR After total mRNA isolation and cDNA synthesis, PCR amplification was performed with SYBR Green PCR Master Mix (Roche). .. Quantitative real-time PCR were performed as described [ ].

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    Article Snippet: .. The DNA was cross-linked to the membrane by ultraviolet (UV) irradiation for 10 min. Three DNA probes [359 base pair (bp) for probe A, 335 bp for probe B, and 326 bp for probe C, specific for DNA-A, DNA-B, and DNA-C, respectively] were produced by PCR amplification using specific primer pairs (table S6) and labeled with digoxigenin (DIG) using the DIG DNA Labeling and Detection Kit (Roche Diagnostics, Mannheim, Germany). .. After prehybridization and hybridization were complete, the blots were washed in 1× SSC (0.15 M sodium chloride and 0.015 M sodium citrate) and 0.1% SDS at 65°C for 30 min. Hybridization signals were detected on a Tanon 5200 imaging system (Tanon Science and Technology Co. Ltd.) using the DIG detection kit (Roche Diagnostics, Mannheim, Germany).

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    Produced:

    Article Title: A tripartite ssDNA mycovirus from a plant pathogenic fungus is infectious as cloned DNA and purified virions
    Article Snippet: .. The DNA was cross-linked to the membrane by ultraviolet (UV) irradiation for 10 min. Three DNA probes [359 base pair (bp) for probe A, 335 bp for probe B, and 326 bp for probe C, specific for DNA-A, DNA-B, and DNA-C, respectively] were produced by PCR amplification using specific primer pairs (table S6) and labeled with digoxigenin (DIG) using the DIG DNA Labeling and Detection Kit (Roche Diagnostics, Mannheim, Germany). .. After prehybridization and hybridization were complete, the blots were washed in 1× SSC (0.15 M sodium chloride and 0.015 M sodium citrate) and 0.1% SDS at 65°C for 30 min. Hybridization signals were detected on a Tanon 5200 imaging system (Tanon Science and Technology Co. Ltd.) using the DIG detection kit (Roche Diagnostics, Mannheim, Germany).

    Real-time Polymerase Chain Reaction:

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    Binding Assay:

    Article Title: Messenger RNAs of Yeast Virus-Like Elements Contain Non-templated 5′ Poly(A) Leaders, and Their Expression Is Independent of eIF4E and Pab1
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