combined random primers  (Thermo Fisher)


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    Name:
    Random Primers
    Description:
    Random Primers are oligodeoxyribonucleotides mostly hexamers used to prepare labeled DNA probes from templates for filter hybridization or in situ hybridization and to prime mRNAs with or without poly A for cDNA synthesis These primers are truly random and are suitable for DNA synthesis using Klenow fragments with DNA templates or for cDNA synthesis using reverse transcriptase with mRNA templates To avoid storage buffer interference Random Primers are supplied in a low salt concentration buffer Performance and Quality Testing Incorporation of a radioactively labeled nucleotide is verified using control DNA in a Random Primers labeling reaction
    Catalog Number:
    48190011
    Price:
    None
    Applications:
    PCR & Real-Time PCR|RT-PCR|Reverse Transcription|Two-Step RT-PCR
    Category:
    Oligos Primers Probes Nucleotides
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    Structured Review

    Thermo Fisher combined random primers
    Random Primers are oligodeoxyribonucleotides mostly hexamers used to prepare labeled DNA probes from templates for filter hybridization or in situ hybridization and to prime mRNAs with or without poly A for cDNA synthesis These primers are truly random and are suitable for DNA synthesis using Klenow fragments with DNA templates or for cDNA synthesis using reverse transcriptase with mRNA templates To avoid storage buffer interference Random Primers are supplied in a low salt concentration buffer Performance and Quality Testing Incorporation of a radioactively labeled nucleotide is verified using control DNA in a Random Primers labeling reaction
    https://www.bioz.com/result/combined random primers/product/Thermo Fisher
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    combined random primers - by Bioz Stars, 2020-07
    99/100 stars

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    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: MicroRNAs are Transported in Plasma and Delivered to Recipient Cells by High-Density Lipoproteins
    Article Snippet: .. For real-time PCR, total RNA was reverse transcribed using random primers (mRNA) or specific miRNA primers and TaqMan Reverse Transcription or MicroRNA Reverse Transcription Kits (Applied Biosystems), as per instructions. .. Real-time PCR (7300 Real-Time PCR System, Applied Biosystems) and TaqMan probes (Applied Biosystems) were used to quantify mRNA and miRNA levels.

    Article Title: Functional screening in human cardiac organoids reveals a metabolic mechanism for cardiomyocyte cell cycle arrest
    Article Snippet: .. RNA was reverse transcribed using SuperScript III (random primers), and qPCR was performed using SYBR Mastermix (Thermo Fisher Scientific) on a Applied Biosystems Step One Plus to determine cycle number (Ct). .. The 2−ΔΔCt method was used to determine gene expression changes using HRPT1 as a housekeeping gene.

    Article Title: A Nuclear Export Block Triggers the Decay of Newly Synthesized Polyadenylated RNA
    Article Snippet: .. RNAs were then reverse transcribed using a 100 μM dT18 oligonucleotide with 250 ng/μl random hexamers (Invitrogen, 48190-011) and Invitrogen SuperScript II (18064-014) kit according to the manufacturer’s recommendation. qPCR reactions were prepared using the Invitrogen Platinum SYBR Green qPCR Super-Mix-UDG (11733-046) kit and run on Aria MX Real Time PCR System from Agilent Technologies. ..

    Article Title: RPAD (RNase R Treatment, Polyadenylation, and Poly(A)+ RNA Depletion) Method to Isolate Highly Pure Circular RNA
    Article Snippet: .. Control RNA, RNase R-treated RNA, and RPAD-prepared RNA Random primers (Thermo Fisher Scientific) dNTP mix (Thermo Fisher Scientific) Ribolock RNase inhibitor (Thermo Fisher Scientific) Maxima Reverse Transcriptase (Thermo Fisher Scientific) Mir-X™ miRNA First Strand Synthesis Kit (Clonetech, Takara Bio) MicroAmp® Optical 96-Well Reaction Plate (Thermo Fisher Scientific) MicroAmp® Optical Adhesive Film (Thermo Fisher Scientific) MPS 1000 Mini Plate Spinner KAPA SYBR® FAST qPCR mix (Thermo Fisher Scientific) Thermomixture (Eppendorf) Veriti® 96-Well Thermal Cycler (Thermo Fisher Scientific) QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific) .. Prepare a 1 μM working primer mix of forward and reverse primers in nuclease-free water for using in the qPCR reaction.

    Agarose Gel Electrophoresis:

    Article Title: Efficient gene targeting mediated by a lentiviral vector-associated meganuclease
    Article Snippet: .. Digested DNA was separated on 1% agarose gel, blotted to Hybond-N+ nylon membrane (GE Healthcare Europe GmBH, Saclay, France) and hybridized with a 32 P-radiolabelled probe prepared from a 883-bp XmnI–EcoRI fragment from the EIE sequence, using Random Primers DNA Labelling System (Invitrogen, Cergy Pontoise, France). .. After washing (2× SSC/0.1% SDS, room temperature; 0.2× SSC/0.1% SDS, room temperature; 0.1× SSC/0.1% SDS, 42°C and 65°C, 2× SSC, room temperature) membranes were placed 3 days at −80°C for autoradiography.

    Synthesized:

    Article Title: A Proximal Promoter Element Required for Positive Transcriptional Control by Guanosine Tetraphosphate and DksA Protein during the Stringent Response
    Article Snippet: .. First strand cDNAs were synthesized on 3.5 μg of total RNA using 150-ng random primers, 1× first strand buffer, 5 m m DTT, 0.5 m m each of dATP, dCTP, dGTP, and dTTP, and 200 units of SuperScriptTM III reverse transcriptase (Invitrogen). ..

    SYBR Green Assay:

    Article Title: A Nuclear Export Block Triggers the Decay of Newly Synthesized Polyadenylated RNA
    Article Snippet: .. RNAs were then reverse transcribed using a 100 μM dT18 oligonucleotide with 250 ng/μl random hexamers (Invitrogen, 48190-011) and Invitrogen SuperScript II (18064-014) kit according to the manufacturer’s recommendation. qPCR reactions were prepared using the Invitrogen Platinum SYBR Green qPCR Super-Mix-UDG (11733-046) kit and run on Aria MX Real Time PCR System from Agilent Technologies. ..

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Activation of an oncogenic microRNA cistron by provirus integration
    Article Snippet: .. Using random DNA hexamer primers and reverse transcriptase, we created cDNA by using the Invitrogen (Carlsbad, CA) SuperScript III First-Strand Synthesis System for RT-PCR. .. PCR analysis was performed by using the ABI PRISM 7700 (Applied Biosystems) machine.

    Sequencing:

    Article Title: Efficient gene targeting mediated by a lentiviral vector-associated meganuclease
    Article Snippet: .. Digested DNA was separated on 1% agarose gel, blotted to Hybond-N+ nylon membrane (GE Healthcare Europe GmBH, Saclay, France) and hybridized with a 32 P-radiolabelled probe prepared from a 883-bp XmnI–EcoRI fragment from the EIE sequence, using Random Primers DNA Labelling System (Invitrogen, Cergy Pontoise, France). .. After washing (2× SSC/0.1% SDS, room temperature; 0.2× SSC/0.1% SDS, room temperature; 0.1× SSC/0.1% SDS, 42°C and 65°C, 2× SSC, room temperature) membranes were placed 3 days at −80°C for autoradiography.

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    Thermo Fisher random primers dna labeling system
    ( A ) Number of 8-oxodGs per million of dGs (8-oxodg/10 6 dG) measured by LC-MS/MS in untreated (NT), UV-irradiated (UV) and NAC-treated (NAC) MCF10A cells, as indicated. ( B ) Efficiency of polyclonal anti-8-oxodG from Millipore (Ab M), or monoclonal anti-8-oxodG from Trevigen (Ab T), and of anti-IgG antibodies in immuno-precipitation assays of 8-oxodG-containing synthetic <t>ssDNA</t> or G4 structures, as indicated. ( C ) Anti-8oxodG immuno-precipitation assay (% of input <t>DNA,</t> measured by qPCR; y axis) with equal amount (64 pg) of both synthetic oligonucleotides (8-oxodG-100mer and dG-100mer) added to 1 μg of NAC-treated genomic DNA. C1 and C2 indicate the same genomic negative control regions as in panel G. ( D ) Screenshot from the UCSC genome browser of 3.7 Mb from human chromosome 19 showing (top to bottom): OxiDIP-Seq signal profile, 8-oxodG peaks (dots), Input DNA, CG%, and RefSeq genes. ( E ) Screenshot from the UCSC genome browser of 2.1 Mb from human chromosome 19 showing OxiDIP-Seq signal profiles of two independent experiments (Exp #1 and #2) and Input DNA. ( F ) Scatter plot showing the correlation of the OxiDIP-Seq signals obtained in two independent experiments (Exp #1 and #2). Pearson's correlation coefficient (r), as indicated. Figure inset shows magnification of the high-density region. ( G ) OxiDIP-qPCR showing 8-oxodG enrichments (% of Input DNA) at eight different positive (#1–8) and two negative (C1, C2) regions, in untreated (black bar), UV- (gray), or NAC-treated (white) MCF10A cells. Screenshots from the UCSC genome browser show genomic position, 8-oxodG signal intensity, and qPCR probes (black box) of the selected regions. Data from two independent OxiDIP-qPCR assays are shown (±S.D.; P
    Random Primers Dna Labeling System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 128 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/random primers dna labeling system/product/Thermo Fisher
    Average 96 stars, based on 128 article reviews
    Price from $9.99 to $1999.99
    random primers dna labeling system - by Bioz Stars, 2020-07
    96/100 stars
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    91
    Thermo Fisher mrna species
    eQTL analysis of CD58 and microRNA-548ac based on three different data sets. Expression values of CD58 <t>mRNA</t> (labeled in green) and hsa-miR-548ac molecules (labeled in red) measured using microarrays ( A ), <t>RNA-sequencing</t> ( B ), and quantitative real-time PCR ( C ) were plotted for each genotype group. Genotypes 0, 1, and 2 denote the number of MS risk alleles carried, defined either by SNP rs1335532 ( A ) or SNP rs1414273 ( B and C ). The average expression level per group is indicated by a red line. Welch t -test p -values are shown above the brackets for all pairwise genotype comparisons. ( A ) HapMap cohort data (in log2 scale) demonstrated a significant relationship between the MS-associated SNP and CD58 transcript levels in independent populations (n = 82 JPT and n = 82 GIH displayed). ( B ) This could be confirmed by Geuvadis cohort data, presented here for LCLs collected from 282 individuals living in Europe. Interestingly, the eQTL effect is in the opposite direction for hsa-miR-548ac: Significantly higher levels of this miRNA were seen in individuals with increased genetic risk of MS. Numbers below the data points specify the proportion of samples (of n = 276 analyzed LCLs) with zero miRNA read counts. ( C ) Data of the regional MS cohort (n = 32) further substantiated the differences in miRNA levels among the genotypes. A non-significant positive correlation of CD58 mRNA and hsa-miR-548ac expression was found in both the RNA-sequencing data ( B ) and the real-time PCR data ( C ). ( D ) The table gives the F -test p -values calculated for the complete data of the HapMap and Geuvadis cohorts, establishing the cis -mRNA-/miR-eQTL when accounting for population structure in the analysis of covariance (ANCOVA). ANOVA = analysis of variance, eQTL = expression quantitative trait locus, FIN = Finnish in Finland, GBR = British in England and Scotland, GIH = Gujarati Indians in Houston, JPT = Japanese in Tokyo, LCL = lymphoblastoid cell line, MS = multiple sclerosis, PBMC = peripheral blood mononuclear cells, PCR = polymerase chain reaction, SLR = simple linear regression, SNP = single-nucleotide polymorphism, TSI = Toscani in Italia.
    Mrna Species, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mrna species/product/Thermo Fisher
    Average 91 stars, based on 12 article reviews
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    93
    Thermo Fisher c9orf72 antisense specific reverse primer
    AFF2 regulates expression of expanded G 4 C 2 repeats. a Schematic of the CRISPR-Cas9-mediated deletions created in exon 8 of AFF2 to generate AFF2 knockout (KO) <t>C9ORF72</t> iPSC lines. b PCR amplification of the genomic region containing exon 8, showing that all lines were homozygous for the deletion. c AFF2 mRNA levels in parental and AFF2 -KO iPSC lines. Neurons from CRISPR-Cas9-edited lines were analyzed for expression of AFF2 ( d ), C9ORF72 -V3 ( e ), C9ORF72 -V2 ( f ), and C9ORF72 <t>antisense</t> ( g ) mRNAs. h Schematic of the location of the A/G SNP (rs10757668) in exon 2 of C9ORF72 . i Pyrosequencing quantification of the relative expression levels of the allele containing expanded G 4 C 2 repeats (G-allele) in parental and AFF2 -KO iPSC lines. Each data point represents one differentiation ( n = 3 independent differentiations). Values are mean ± s.e.m. * p
    C9orf72 Antisense Specific Reverse Primer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/c9orf72 antisense specific reverse primer/product/Thermo Fisher
    Average 93 stars, based on 1 article reviews
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    ( A ) Number of 8-oxodGs per million of dGs (8-oxodg/10 6 dG) measured by LC-MS/MS in untreated (NT), UV-irradiated (UV) and NAC-treated (NAC) MCF10A cells, as indicated. ( B ) Efficiency of polyclonal anti-8-oxodG from Millipore (Ab M), or monoclonal anti-8-oxodG from Trevigen (Ab T), and of anti-IgG antibodies in immuno-precipitation assays of 8-oxodG-containing synthetic ssDNA or G4 structures, as indicated. ( C ) Anti-8oxodG immuno-precipitation assay (% of input DNA, measured by qPCR; y axis) with equal amount (64 pg) of both synthetic oligonucleotides (8-oxodG-100mer and dG-100mer) added to 1 μg of NAC-treated genomic DNA. C1 and C2 indicate the same genomic negative control regions as in panel G. ( D ) Screenshot from the UCSC genome browser of 3.7 Mb from human chromosome 19 showing (top to bottom): OxiDIP-Seq signal profile, 8-oxodG peaks (dots), Input DNA, CG%, and RefSeq genes. ( E ) Screenshot from the UCSC genome browser of 2.1 Mb from human chromosome 19 showing OxiDIP-Seq signal profiles of two independent experiments (Exp #1 and #2) and Input DNA. ( F ) Scatter plot showing the correlation of the OxiDIP-Seq signals obtained in two independent experiments (Exp #1 and #2). Pearson's correlation coefficient (r), as indicated. Figure inset shows magnification of the high-density region. ( G ) OxiDIP-qPCR showing 8-oxodG enrichments (% of Input DNA) at eight different positive (#1–8) and two negative (C1, C2) regions, in untreated (black bar), UV- (gray), or NAC-treated (white) MCF10A cells. Screenshots from the UCSC genome browser show genomic position, 8-oxodG signal intensity, and qPCR probes (black box) of the selected regions. Data from two independent OxiDIP-qPCR assays are shown (±S.D.; P

    Journal: Nucleic Acids Research

    Article Title: Genome-wide mapping of 8-oxo-7,8-dihydro-2′-deoxyguanosine reveals accumulation of oxidatively-generated damage at DNA replication origins within transcribed long genes of mammalian cells

    doi: 10.1093/nar/gky1152

    Figure Lengend Snippet: ( A ) Number of 8-oxodGs per million of dGs (8-oxodg/10 6 dG) measured by LC-MS/MS in untreated (NT), UV-irradiated (UV) and NAC-treated (NAC) MCF10A cells, as indicated. ( B ) Efficiency of polyclonal anti-8-oxodG from Millipore (Ab M), or monoclonal anti-8-oxodG from Trevigen (Ab T), and of anti-IgG antibodies in immuno-precipitation assays of 8-oxodG-containing synthetic ssDNA or G4 structures, as indicated. ( C ) Anti-8oxodG immuno-precipitation assay (% of input DNA, measured by qPCR; y axis) with equal amount (64 pg) of both synthetic oligonucleotides (8-oxodG-100mer and dG-100mer) added to 1 μg of NAC-treated genomic DNA. C1 and C2 indicate the same genomic negative control regions as in panel G. ( D ) Screenshot from the UCSC genome browser of 3.7 Mb from human chromosome 19 showing (top to bottom): OxiDIP-Seq signal profile, 8-oxodG peaks (dots), Input DNA, CG%, and RefSeq genes. ( E ) Screenshot from the UCSC genome browser of 2.1 Mb from human chromosome 19 showing OxiDIP-Seq signal profiles of two independent experiments (Exp #1 and #2) and Input DNA. ( F ) Scatter plot showing the correlation of the OxiDIP-Seq signals obtained in two independent experiments (Exp #1 and #2). Pearson's correlation coefficient (r), as indicated. Figure inset shows magnification of the high-density region. ( G ) OxiDIP-qPCR showing 8-oxodG enrichments (% of Input DNA) at eight different positive (#1–8) and two negative (C1, C2) regions, in untreated (black bar), UV- (gray), or NAC-treated (white) MCF10A cells. Screenshots from the UCSC genome browser show genomic position, 8-oxodG signal intensity, and qPCR probes (black box) of the selected regions. Data from two independent OxiDIP-qPCR assays are shown (±S.D.; P

    Article Snippet: Conversion of ssDNA to dsDNA was obtained by Random Primers DNA Labeling System (Cat. No. 18187-013, ThermoFisher Scientific).

    Techniques: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Irradiation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control

    ( A ) Number of 8-oxodGs per million of dGs (8-oxodg/10 6 dG) measured by LC-MS/MS in untreated (NT), UV-irradiated (UV) and NAC-treated (NAC) MCF10A cells, as indicated. ( B ) Efficiency of polyclonal anti-8-oxodG from Millipore (Ab M), or monoclonal anti-8-oxodG from Trevigen (Ab T), and of anti-IgG antibodies in immuno-precipitation assays of 8-oxodG-containing synthetic ssDNA or G4 structures, as indicated. ( C ) Anti-8oxodG immuno-precipitation assay (% of input DNA, measured by qPCR; y axis) with equal amount (64 pg) of both synthetic oligonucleotides (8-oxodG-100mer and dG-100mer) added to 1 μg of NAC-treated genomic DNA. C1 and C2 indicate the same genomic negative control regions as in panel G. ( D ) Screenshot from the UCSC genome browser of 3.7 Mb from human chromosome 19 showing (top to bottom): OxiDIP-Seq signal profile, 8-oxodG peaks (dots), Input DNA, CG%, and RefSeq genes. ( E ) Screenshot from the UCSC genome browser of 2.1 Mb from human chromosome 19 showing OxiDIP-Seq signal profiles of two independent experiments (Exp #1 and #2) and Input DNA. ( F ) Scatter plot showing the correlation of the OxiDIP-Seq signals obtained in two independent experiments (Exp #1 and #2). Pearson's correlation coefficient (r), as indicated. Figure inset shows magnification of the high-density region. ( G ) OxiDIP-qPCR showing 8-oxodG enrichments (% of Input DNA) at eight different positive (#1–8) and two negative (C1, C2) regions, in untreated (black bar), UV- (gray), or NAC-treated (white) MCF10A cells. Screenshots from the UCSC genome browser show genomic position, 8-oxodG signal intensity, and qPCR probes (black box) of the selected regions. Data from two independent OxiDIP-qPCR assays are shown (±S.D.; P

    Journal: Nucleic Acids Research

    Article Title: Genome-wide mapping of 8-oxo-7,8-dihydro-2′-deoxyguanosine reveals accumulation of oxidatively-generated damage at DNA replication origins within transcribed long genes of mammalian cells

    doi: 10.1093/nar/gky1152

    Figure Lengend Snippet: ( A ) Number of 8-oxodGs per million of dGs (8-oxodg/10 6 dG) measured by LC-MS/MS in untreated (NT), UV-irradiated (UV) and NAC-treated (NAC) MCF10A cells, as indicated. ( B ) Efficiency of polyclonal anti-8-oxodG from Millipore (Ab M), or monoclonal anti-8-oxodG from Trevigen (Ab T), and of anti-IgG antibodies in immuno-precipitation assays of 8-oxodG-containing synthetic ssDNA or G4 structures, as indicated. ( C ) Anti-8oxodG immuno-precipitation assay (% of input DNA, measured by qPCR; y axis) with equal amount (64 pg) of both synthetic oligonucleotides (8-oxodG-100mer and dG-100mer) added to 1 μg of NAC-treated genomic DNA. C1 and C2 indicate the same genomic negative control regions as in panel G. ( D ) Screenshot from the UCSC genome browser of 3.7 Mb from human chromosome 19 showing (top to bottom): OxiDIP-Seq signal profile, 8-oxodG peaks (dots), Input DNA, CG%, and RefSeq genes. ( E ) Screenshot from the UCSC genome browser of 2.1 Mb from human chromosome 19 showing OxiDIP-Seq signal profiles of two independent experiments (Exp #1 and #2) and Input DNA. ( F ) Scatter plot showing the correlation of the OxiDIP-Seq signals obtained in two independent experiments (Exp #1 and #2). Pearson's correlation coefficient (r), as indicated. Figure inset shows magnification of the high-density region. ( G ) OxiDIP-qPCR showing 8-oxodG enrichments (% of Input DNA) at eight different positive (#1–8) and two negative (C1, C2) regions, in untreated (black bar), UV- (gray), or NAC-treated (white) MCF10A cells. Screenshots from the UCSC genome browser show genomic position, 8-oxodG signal intensity, and qPCR probes (black box) of the selected regions. Data from two independent OxiDIP-qPCR assays are shown (±S.D.; P

    Article Snippet: Conversion of ssDNA to dsDNA was obtained by Random Primers DNA Labeling System (Cat. No. 18187-013, ThermoFisher Scientific).

    Techniques: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Irradiation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control

    eQTL analysis of CD58 and microRNA-548ac based on three different data sets. Expression values of CD58 mRNA (labeled in green) and hsa-miR-548ac molecules (labeled in red) measured using microarrays ( A ), RNA-sequencing ( B ), and quantitative real-time PCR ( C ) were plotted for each genotype group. Genotypes 0, 1, and 2 denote the number of MS risk alleles carried, defined either by SNP rs1335532 ( A ) or SNP rs1414273 ( B and C ). The average expression level per group is indicated by a red line. Welch t -test p -values are shown above the brackets for all pairwise genotype comparisons. ( A ) HapMap cohort data (in log2 scale) demonstrated a significant relationship between the MS-associated SNP and CD58 transcript levels in independent populations (n = 82 JPT and n = 82 GIH displayed). ( B ) This could be confirmed by Geuvadis cohort data, presented here for LCLs collected from 282 individuals living in Europe. Interestingly, the eQTL effect is in the opposite direction for hsa-miR-548ac: Significantly higher levels of this miRNA were seen in individuals with increased genetic risk of MS. Numbers below the data points specify the proportion of samples (of n = 276 analyzed LCLs) with zero miRNA read counts. ( C ) Data of the regional MS cohort (n = 32) further substantiated the differences in miRNA levels among the genotypes. A non-significant positive correlation of CD58 mRNA and hsa-miR-548ac expression was found in both the RNA-sequencing data ( B ) and the real-time PCR data ( C ). ( D ) The table gives the F -test p -values calculated for the complete data of the HapMap and Geuvadis cohorts, establishing the cis -mRNA-/miR-eQTL when accounting for population structure in the analysis of covariance (ANCOVA). ANOVA = analysis of variance, eQTL = expression quantitative trait locus, FIN = Finnish in Finland, GBR = British in England and Scotland, GIH = Gujarati Indians in Houston, JPT = Japanese in Tokyo, LCL = lymphoblastoid cell line, MS = multiple sclerosis, PBMC = peripheral blood mononuclear cells, PCR = polymerase chain reaction, SLR = simple linear regression, SNP = single-nucleotide polymorphism, TSI = Toscani in Italia.

    Journal: PLoS Genetics

    Article Title: A genetic variant associated with multiple sclerosis inversely affects the expression of CD58 and microRNA-548ac from the same gene

    doi: 10.1371/journal.pgen.1007961

    Figure Lengend Snippet: eQTL analysis of CD58 and microRNA-548ac based on three different data sets. Expression values of CD58 mRNA (labeled in green) and hsa-miR-548ac molecules (labeled in red) measured using microarrays ( A ), RNA-sequencing ( B ), and quantitative real-time PCR ( C ) were plotted for each genotype group. Genotypes 0, 1, and 2 denote the number of MS risk alleles carried, defined either by SNP rs1335532 ( A ) or SNP rs1414273 ( B and C ). The average expression level per group is indicated by a red line. Welch t -test p -values are shown above the brackets for all pairwise genotype comparisons. ( A ) HapMap cohort data (in log2 scale) demonstrated a significant relationship between the MS-associated SNP and CD58 transcript levels in independent populations (n = 82 JPT and n = 82 GIH displayed). ( B ) This could be confirmed by Geuvadis cohort data, presented here for LCLs collected from 282 individuals living in Europe. Interestingly, the eQTL effect is in the opposite direction for hsa-miR-548ac: Significantly higher levels of this miRNA were seen in individuals with increased genetic risk of MS. Numbers below the data points specify the proportion of samples (of n = 276 analyzed LCLs) with zero miRNA read counts. ( C ) Data of the regional MS cohort (n = 32) further substantiated the differences in miRNA levels among the genotypes. A non-significant positive correlation of CD58 mRNA and hsa-miR-548ac expression was found in both the RNA-sequencing data ( B ) and the real-time PCR data ( C ). ( D ) The table gives the F -test p -values calculated for the complete data of the HapMap and Geuvadis cohorts, establishing the cis -mRNA-/miR-eQTL when accounting for population structure in the analysis of covariance (ANCOVA). ANOVA = analysis of variance, eQTL = expression quantitative trait locus, FIN = Finnish in Finland, GBR = British in England and Scotland, GIH = Gujarati Indians in Houston, JPT = Japanese in Tokyo, LCL = lymphoblastoid cell line, MS = multiple sclerosis, PBMC = peripheral blood mononuclear cells, PCR = polymerase chain reaction, SLR = simple linear regression, SNP = single-nucleotide polymorphism, TSI = Toscani in Italia.

    Article Snippet: From each sample, 400 ng of total RNA was reverse transcribed with random primers for mRNA species (High-Capacity cDNA Reverse Transcription Kit), and 10 ng of total RNA was reverse transcribed with specific primers provided with each TaqMan miRNA assay to convert mature miRNAs to cDNA (TaqMan MicroRNA Reverse Transcription Kit, Thermo Fisher Scientific).

    Techniques: Expressing, Labeling, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    Biogenesis of microRNA-548ac and genetic variants in the CD58 gene locus. ( A ) Diagram of the processing of an mRNA and an intronic miRNA from the same transcript (adapted from [ 49 ]). Both RNA splicing by the spliceosome and miRNA stem-loop cropping by the Drosha-DGCR8 complex occur cotranscriptionally. Drosha may cleave the miRNA-harboring intron before splicing commitment of the flanking exons. The resulting precursor miRNA is subsequently processed into a mature miRNA, which is loaded into the RNA-induced silencing complex (RISC). ( B ) Annotated secondary structure of hsa-mir-548ac. Highlighted in gray is the 22 nt long sequence of the mature miRNA isoform as assigned by Jima et al . (miRBase accession MIMAT0018938) [ 43 ]. The red circle marks the only common single-nucleotide polymorphism (SNP) within the stem-loop region. The G allele is overrepresented in MS patients. ( C ) Genetic variants in pairwise linkage disequilibrium (LD) with SNP rs1414273. This plot was generated using the LDproxy module of the web-based analysis tool LDlink [ 97 ]. Shown are r 2 LD values of proximal SNPs based on all subpopulations of the 1000 Genomes project (orange dots), recombination rate as estimated from HapMap data (gray line), and the position and exon-intron structure of nearby genes on chromosome 1 (chr1, GRCh37 assembly). The MS-associated SNP rs1335532 is in strong LD with SNP rs1414273 (blue) (correlated forward strand alleles: A = C, G = T). The entire block of LD spans about 50 kb but does not include the promoter region of CD58, which is encoded on the minus strand in the reference genome. ( D ) Worldwide distribution of SNP rs1335532 alleles. Global allele frequencies were visualized as two-color pie charts with the HGDP Selection Browser [ 98 ]. The disease susceptibility variant (A, blue) is the major allele in European populations and the minor allele in East Asian and Southern African populations. cM/Mb = centimorgan per megabase, HGDP = Human Genome Diversity Panel, MS = multiple sclerosis.

    Journal: PLoS Genetics

    Article Title: A genetic variant associated with multiple sclerosis inversely affects the expression of CD58 and microRNA-548ac from the same gene

    doi: 10.1371/journal.pgen.1007961

    Figure Lengend Snippet: Biogenesis of microRNA-548ac and genetic variants in the CD58 gene locus. ( A ) Diagram of the processing of an mRNA and an intronic miRNA from the same transcript (adapted from [ 49 ]). Both RNA splicing by the spliceosome and miRNA stem-loop cropping by the Drosha-DGCR8 complex occur cotranscriptionally. Drosha may cleave the miRNA-harboring intron before splicing commitment of the flanking exons. The resulting precursor miRNA is subsequently processed into a mature miRNA, which is loaded into the RNA-induced silencing complex (RISC). ( B ) Annotated secondary structure of hsa-mir-548ac. Highlighted in gray is the 22 nt long sequence of the mature miRNA isoform as assigned by Jima et al . (miRBase accession MIMAT0018938) [ 43 ]. The red circle marks the only common single-nucleotide polymorphism (SNP) within the stem-loop region. The G allele is overrepresented in MS patients. ( C ) Genetic variants in pairwise linkage disequilibrium (LD) with SNP rs1414273. This plot was generated using the LDproxy module of the web-based analysis tool LDlink [ 97 ]. Shown are r 2 LD values of proximal SNPs based on all subpopulations of the 1000 Genomes project (orange dots), recombination rate as estimated from HapMap data (gray line), and the position and exon-intron structure of nearby genes on chromosome 1 (chr1, GRCh37 assembly). The MS-associated SNP rs1335532 is in strong LD with SNP rs1414273 (blue) (correlated forward strand alleles: A = C, G = T). The entire block of LD spans about 50 kb but does not include the promoter region of CD58, which is encoded on the minus strand in the reference genome. ( D ) Worldwide distribution of SNP rs1335532 alleles. Global allele frequencies were visualized as two-color pie charts with the HGDP Selection Browser [ 98 ]. The disease susceptibility variant (A, blue) is the major allele in European populations and the minor allele in East Asian and Southern African populations. cM/Mb = centimorgan per megabase, HGDP = Human Genome Diversity Panel, MS = multiple sclerosis.

    Article Snippet: From each sample, 400 ng of total RNA was reverse transcribed with random primers for mRNA species (High-Capacity cDNA Reverse Transcription Kit), and 10 ng of total RNA was reverse transcribed with specific primers provided with each TaqMan miRNA assay to convert mature miRNAs to cDNA (TaqMan MicroRNA Reverse Transcription Kit, Thermo Fisher Scientific).

    Techniques: Sequencing, Generated, Blocking Assay, Selection, Variant Assay

    AFF2 regulates expression of expanded G 4 C 2 repeats. a Schematic of the CRISPR-Cas9-mediated deletions created in exon 8 of AFF2 to generate AFF2 knockout (KO) C9ORF72 iPSC lines. b PCR amplification of the genomic region containing exon 8, showing that all lines were homozygous for the deletion. c AFF2 mRNA levels in parental and AFF2 -KO iPSC lines. Neurons from CRISPR-Cas9-edited lines were analyzed for expression of AFF2 ( d ), C9ORF72 -V3 ( e ), C9ORF72 -V2 ( f ), and C9ORF72 antisense ( g ) mRNAs. h Schematic of the location of the A/G SNP (rs10757668) in exon 2 of C9ORF72 . i Pyrosequencing quantification of the relative expression levels of the allele containing expanded G 4 C 2 repeats (G-allele) in parental and AFF2 -KO iPSC lines. Each data point represents one differentiation ( n = 3 independent differentiations). Values are mean ± s.e.m. * p

    Journal: Nature Communications

    Article Title: Transcription elongation factor AFF2/FMR2 regulates expression of expanded GGGGCC repeat-containing C9ORF72 allele in ALS/FTD

    doi: 10.1038/s41467-019-13477-8

    Figure Lengend Snippet: AFF2 regulates expression of expanded G 4 C 2 repeats. a Schematic of the CRISPR-Cas9-mediated deletions created in exon 8 of AFF2 to generate AFF2 knockout (KO) C9ORF72 iPSC lines. b PCR amplification of the genomic region containing exon 8, showing that all lines were homozygous for the deletion. c AFF2 mRNA levels in parental and AFF2 -KO iPSC lines. Neurons from CRISPR-Cas9-edited lines were analyzed for expression of AFF2 ( d ), C9ORF72 -V3 ( e ), C9ORF72 -V2 ( f ), and C9ORF72 antisense ( g ) mRNAs. h Schematic of the location of the A/G SNP (rs10757668) in exon 2 of C9ORF72 . i Pyrosequencing quantification of the relative expression levels of the allele containing expanded G 4 C 2 repeats (G-allele) in parental and AFF2 -KO iPSC lines. Each data point represents one differentiation ( n = 3 independent differentiations). Values are mean ± s.e.m. * p

    Article Snippet: RNA (500–900 ng) was reverse transcribed into cDNA with random hexamers or C9ORF72 antisense-specific reverse primer and MultiScribe reverse transcriptase (Thermo Fisher Scientific, Cat. No. N8080234) according to the manufacturer’s instructions. qPCR was done with an Applied Biosystems Quant Studio 3 system and SYBR Select Master Mix (Thermo Fisher Scientific, Cat. No. 4472908).

    Techniques: Expressing, CRISPR, Knock-Out, Polymerase Chain Reaction, Amplification