reverse transcription kit  (Thermo Fisher)


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    Name:
    RevertAid First Strand cDNA Synthesis Kit
    Description:
    Thermo Scientific RevertAid First Strand cDNA Synthesis Kit is a complete system for efficient synthesis of first strand cDNA from RNA templates The kit uses RevertAid Reverse Transcriptase RT a recombinant M MuLV RT which maintains activity at 42 50°C and is suitable for synthesis of cDNA up to 13 kb RiboLock RNase Inhibitor supplied with the kit effectively protects RNA templates from degradation The RevertAid First Strand cDNA Synthesis Kit is supplied with both oligo dT 18 and random hexamer primers The oligo dT 18 primer anneals selectively to the poly A tail of mRNA Random hexamer primers do not require the presence of the poly A tail therefore they can be used for transcription of the 5 end regions of mRNA or cDNA synthesis of RNA species lacking a poly A tail e g microRNAs Gene specific primers may also be used with the kit Highlights• Full length first strand cDNA up to 13 kb• Optimum reaction temperature 42°C• Complete kit all the components for the RT reaction are includedApplications• First strand cDNA synthesis for RT PCR and RT qPCR• Construction of full length cDNA libraries• Antisense RNA synthesis
    Catalog Number:
    k1621
    Price:
    None
    Applications:
    Cloning|cDNA Libraries & Library Construction
    Category:
    Kits and Assays
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    Structured Review

    Thermo Fisher reverse transcription kit
    Thermo Scientific RevertAid First Strand cDNA Synthesis Kit is a complete system for efficient synthesis of first strand cDNA from RNA templates The kit uses RevertAid Reverse Transcriptase RT a recombinant M MuLV RT which maintains activity at 42 50°C and is suitable for synthesis of cDNA up to 13 kb RiboLock RNase Inhibitor supplied with the kit effectively protects RNA templates from degradation The RevertAid First Strand cDNA Synthesis Kit is supplied with both oligo dT 18 and random hexamer primers The oligo dT 18 primer anneals selectively to the poly A tail of mRNA Random hexamer primers do not require the presence of the poly A tail therefore they can be used for transcription of the 5 end regions of mRNA or cDNA synthesis of RNA species lacking a poly A tail e g microRNAs Gene specific primers may also be used with the kit Highlights• Full length first strand cDNA up to 13 kb• Optimum reaction temperature 42°C• Complete kit all the components for the RT reaction are includedApplications• First strand cDNA synthesis for RT PCR and RT qPCR• Construction of full length cDNA libraries• Antisense RNA synthesis
    https://www.bioz.com/result/reverse transcription kit/product/Thermo Fisher
    Average 90 stars, based on 241 article reviews
    Price from $9.99 to $1999.99
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    90/100 stars

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    Staining:

    Article Title: Evolutionarily novel genes are expressed in transgenic fish tumors and their orthologs are involved in development of progressive traits in humans
    Article Snippet: .. Ethidium bromide staining of RNA in agarose gels visualizes two predominant bands of small and large ribosomal RNA, a bands of low molecular mass RNA. cDNA synthesis was performed from equal amounts of RNA using Revert Aid® First Strand cDNA Synthesis Kit (Thermo, USA) with random hexanucleotide, following the manufacturer guidelines. ..

    Spectrophotometry:

    Article Title: Anti-Inflammatory Effect of IL-37-Producing T-Cell Population in DSS-Induced Chronic Inflammatory Bowel Disease in Mice
    Article Snippet: .. The Nanodrop spectrophotometer (Thermo Scientific, Waltham, MA, USA) was used to measure absorbance. cDNAs were synthesized by using the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher, Waltham, MA, USA) according to manufacturer’s instructions. .. The following primer pairs were used: IFN-γ, sense: 5′-CGGCACAGTCATTGAAAGCC-3′ and anti-sense: 5′-TGCATCCTTTTTCGCCTTGC-3′; TNF-α, sense: 5′-ATGAGCACAGAAAGCATGATC-3′ and anti-sense: 5′-TACAGGCTTGTCACTCGAATT-3′; IL-1β, sense: 5′-CAGGATGAGGACATGAGCACC-3′ and anti-sense: 5′-CTCTGCAGACTCAAACTCCAC-3′; IL-37, sense: 5′-TCAGCTGAAGAAGGAGAAACTG-3′ and anti-sense 5′-TTATCTGTCACCCCAACAGG-3′; IL-10, sense: 5′-CTTGCACTACCAAAGCCACA-3′ and anti-sense: 5′-GTTAT GTCTTCCCGGCTGT-3′; GAPDH, sense: 5′-CTCTCTGCTCCTCCCTGT-3′ and anti-sense: 5′-GCAACAATCTCCACTTTG-3′. qPCR amplification reactions were prepared with the SYBR Green PCR Kit (Bio-Rad, Hercules, CA, USA) and performed using the CFX96 Real-Time System (Bio-Rad).

    Synthesized:

    Article Title: Anti-Inflammatory Effect of IL-37-Producing T-Cell Population in DSS-Induced Chronic Inflammatory Bowel Disease in Mice
    Article Snippet: .. The Nanodrop spectrophotometer (Thermo Scientific, Waltham, MA, USA) was used to measure absorbance. cDNAs were synthesized by using the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher, Waltham, MA, USA) according to manufacturer’s instructions. .. The following primer pairs were used: IFN-γ, sense: 5′-CGGCACAGTCATTGAAAGCC-3′ and anti-sense: 5′-TGCATCCTTTTTCGCCTTGC-3′; TNF-α, sense: 5′-ATGAGCACAGAAAGCATGATC-3′ and anti-sense: 5′-TACAGGCTTGTCACTCGAATT-3′; IL-1β, sense: 5′-CAGGATGAGGACATGAGCACC-3′ and anti-sense: 5′-CTCTGCAGACTCAAACTCCAC-3′; IL-37, sense: 5′-TCAGCTGAAGAAGGAGAAACTG-3′ and anti-sense 5′-TTATCTGTCACCCCAACAGG-3′; IL-10, sense: 5′-CTTGCACTACCAAAGCCACA-3′ and anti-sense: 5′-GTTAT GTCTTCCCGGCTGT-3′; GAPDH, sense: 5′-CTCTCTGCTCCTCCCTGT-3′ and anti-sense: 5′-GCAACAATCTCCACTTTG-3′. qPCR amplification reactions were prepared with the SYBR Green PCR Kit (Bio-Rad, Hercules, CA, USA) and performed using the CFX96 Real-Time System (Bio-Rad).

    Article Title: Involvement of CD147 on multidrug resistance through the regulation of P-glycoprotein expression in K562/ADR leukemic cell line
    Article Snippet: .. Single-stranded cDNA was synthesized by reverse transcription from 1 μg of total RNA using the RevertAid strand cDNA Synthesis Kit (Thermo Scientific, USA). .. Then, 1 μg cDNA was amplified by PCR using a DreamTag Green PCR Master Mix (Thermo ScientificTM).

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  • 99
    Thermo Fisher taqman microrna reverse transcription kit
    Quantitative real-time PCR analyses comparing miRNA expression patterns in tissues using different extraction methods. Fold abundance of miRNAs quantitated using <t>TaqMan</t> <t>MicroRNA</t> Assays via qRT-PCR from brain ( a ), liver ( b ) and lung ( c ) tissues. miRNA expression was normalised using sno202 as a reference gene. Values indicate the average normalised miRNA expression in different tissues on a Log2 scale with SEM error bars. * p
    Taqman Microrna Reverse Transcription Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3219 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taqman microrna reverse transcription kit/product/Thermo Fisher
    Average 99 stars, based on 3219 article reviews
    Price from $9.99 to $1999.99
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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
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    99
    Thermo Fisher high capacity cdna reverse transcription kit
    HFD-fed iRhom2 KO mice have increased thermogenesis and browning of the white adipose tissue. A-C Thermal images (A), and BAT (B) or body (C) temperature of iRhom2 KO and WT mice fed with SD and HFD for 19 weeks. One experiment with 8 mice per group. D RT-PCR analysis of UCP1, PGC1α, Cidea, PRDM16, and Cox8b expression in SD-fed iRhom2 KO and HFD-fed WT and iRhom2 KO mice BAT samples compared to WT SD-fed control samples. Two experiments with 3–4 replicates in each. E, G Representative photographs of eWAT (E) and sWAT (G) UCP1 immunohistochemistry derived from iRhom2 KO and WT mice fed with HFD for 20 weeks. Scale bar = 100 μm. F, H Graphics showing the UCP1 percentage of area stained in the eWAT (F) and sWAT (H) of the mice described above. One experiment with 8 mice per group (with 2 photographs analyzed per mouse). I RT-PCR analysis of UCP1 in brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT or iRhom2 KO mice. Three independent experiments. J RT-PCR analysis of UCP1 in immortalized WT brown preadipocytes transduced with empty vector or iRhom2-HA, differentiated in vitro and stimulated with norepinephrine for 6 h. Two independent experiments. K HA expression in differentiated immortalized WT brown preadipocytes transduced with retrovirus containing iRhom2 <t>cDNA</t> fused to C-terminal HA tag (iR2-HA). As a negative control, we used the same cells transduced with retrovirus containing the empty vector (EV), and as a loading control we measured p97 protein level. Two independent experiments. L Mitochondrial oxygen consumption rate (OCR) of brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT and iRhom2 KO mice and stimulated or not with norepinephrine. The results were normalized to the protein content. Two experiments with one or two independent samples per genotype, respectively. M Mitochondrial proton leak of the cells described in L normalized to the protein content. Error bars represent SEM; * represents p
    High Capacity Cdna Reverse Transcription Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10196 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/high capacity cdna reverse transcription kit/product/Thermo Fisher
    Average 99 stars, based on 10196 article reviews
    Price from $9.99 to $1999.99
    high capacity cdna reverse transcription kit - by Bioz Stars, 2020-08
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    Quantitative real-time PCR analyses comparing miRNA expression patterns in tissues using different extraction methods. Fold abundance of miRNAs quantitated using TaqMan MicroRNA Assays via qRT-PCR from brain ( a ), liver ( b ) and lung ( c ) tissues. miRNA expression was normalised using sno202 as a reference gene. Values indicate the average normalised miRNA expression in different tissues on a Log2 scale with SEM error bars. * p

    Journal: BMC Biotechnology

    Article Title: Total RNA extraction from tissues for microRNA and target gene expression analysis: not all kits are created equal

    doi: 10.1186/s12896-018-0421-6

    Figure Lengend Snippet: Quantitative real-time PCR analyses comparing miRNA expression patterns in tissues using different extraction methods. Fold abundance of miRNAs quantitated using TaqMan MicroRNA Assays via qRT-PCR from brain ( a ), liver ( b ) and lung ( c ) tissues. miRNA expression was normalised using sno202 as a reference gene. Values indicate the average normalised miRNA expression in different tissues on a Log2 scale with SEM error bars. * p

    Article Snippet: Reverse transcription and quantitative real time PCR (qRT-PCR) For miRNA detection, 10 ng of the RNA samples were reverse transcribed in technical duplicates using a TaqMan microRNA reverse transcription kit (Thermo Fisher Scientific) with Taqman microRNA primers specific for the miRNAs of interest, following manufacturer’s instructions (see Additional file : Table S1).

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

    Quantitative real-time PCR analyses of 6 miRNAs comparing different RNA extraction methods. RNA was extracted from murine brain ( a ), liver ( b ) and lung ( c ) tissues using the five indicated methods and endogenous miRNA expression quantitated using TaqMan MicroRNA Assays via qRT-PCR. miRNA expression was normalised using sno202 as a reference gene. Values indicate the average normalised miRNA expression (technical duplicates from triplicate biological isolations) in different tissues on a Log2 scale with SEM error bars. * p

    Journal: BMC Biotechnology

    Article Title: Total RNA extraction from tissues for microRNA and target gene expression analysis: not all kits are created equal

    doi: 10.1186/s12896-018-0421-6

    Figure Lengend Snippet: Quantitative real-time PCR analyses of 6 miRNAs comparing different RNA extraction methods. RNA was extracted from murine brain ( a ), liver ( b ) and lung ( c ) tissues using the five indicated methods and endogenous miRNA expression quantitated using TaqMan MicroRNA Assays via qRT-PCR. miRNA expression was normalised using sno202 as a reference gene. Values indicate the average normalised miRNA expression (technical duplicates from triplicate biological isolations) in different tissues on a Log2 scale with SEM error bars. * p

    Article Snippet: Reverse transcription and quantitative real time PCR (qRT-PCR) For miRNA detection, 10 ng of the RNA samples were reverse transcribed in technical duplicates using a TaqMan microRNA reverse transcription kit (Thermo Fisher Scientific) with Taqman microRNA primers specific for the miRNAs of interest, following manufacturer’s instructions (see Additional file : Table S1).

    Techniques: Real-time Polymerase Chain Reaction, RNA Extraction, Expressing, Quantitative RT-PCR

    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

    HFD-fed iRhom2 KO mice have increased thermogenesis and browning of the white adipose tissue. A-C Thermal images (A), and BAT (B) or body (C) temperature of iRhom2 KO and WT mice fed with SD and HFD for 19 weeks. One experiment with 8 mice per group. D RT-PCR analysis of UCP1, PGC1α, Cidea, PRDM16, and Cox8b expression in SD-fed iRhom2 KO and HFD-fed WT and iRhom2 KO mice BAT samples compared to WT SD-fed control samples. Two experiments with 3–4 replicates in each. E, G Representative photographs of eWAT (E) and sWAT (G) UCP1 immunohistochemistry derived from iRhom2 KO and WT mice fed with HFD for 20 weeks. Scale bar = 100 μm. F, H Graphics showing the UCP1 percentage of area stained in the eWAT (F) and sWAT (H) of the mice described above. One experiment with 8 mice per group (with 2 photographs analyzed per mouse). I RT-PCR analysis of UCP1 in brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT or iRhom2 KO mice. Three independent experiments. J RT-PCR analysis of UCP1 in immortalized WT brown preadipocytes transduced with empty vector or iRhom2-HA, differentiated in vitro and stimulated with norepinephrine for 6 h. Two independent experiments. K HA expression in differentiated immortalized WT brown preadipocytes transduced with retrovirus containing iRhom2 cDNA fused to C-terminal HA tag (iR2-HA). As a negative control, we used the same cells transduced with retrovirus containing the empty vector (EV), and as a loading control we measured p97 protein level. Two independent experiments. L Mitochondrial oxygen consumption rate (OCR) of brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT and iRhom2 KO mice and stimulated or not with norepinephrine. The results were normalized to the protein content. Two experiments with one or two independent samples per genotype, respectively. M Mitochondrial proton leak of the cells described in L normalized to the protein content. Error bars represent SEM; * represents p

    Journal: Molecular Metabolism

    Article Title: Deletion of iRhom2 protects against diet-induced obesity by increasing thermogenesis

    doi: 10.1016/j.molmet.2019.10.006

    Figure Lengend Snippet: HFD-fed iRhom2 KO mice have increased thermogenesis and browning of the white adipose tissue. A-C Thermal images (A), and BAT (B) or body (C) temperature of iRhom2 KO and WT mice fed with SD and HFD for 19 weeks. One experiment with 8 mice per group. D RT-PCR analysis of UCP1, PGC1α, Cidea, PRDM16, and Cox8b expression in SD-fed iRhom2 KO and HFD-fed WT and iRhom2 KO mice BAT samples compared to WT SD-fed control samples. Two experiments with 3–4 replicates in each. E, G Representative photographs of eWAT (E) and sWAT (G) UCP1 immunohistochemistry derived from iRhom2 KO and WT mice fed with HFD for 20 weeks. Scale bar = 100 μm. F, H Graphics showing the UCP1 percentage of area stained in the eWAT (F) and sWAT (H) of the mice described above. One experiment with 8 mice per group (with 2 photographs analyzed per mouse). I RT-PCR analysis of UCP1 in brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT or iRhom2 KO mice. Three independent experiments. J RT-PCR analysis of UCP1 in immortalized WT brown preadipocytes transduced with empty vector or iRhom2-HA, differentiated in vitro and stimulated with norepinephrine for 6 h. Two independent experiments. K HA expression in differentiated immortalized WT brown preadipocytes transduced with retrovirus containing iRhom2 cDNA fused to C-terminal HA tag (iR2-HA). As a negative control, we used the same cells transduced with retrovirus containing the empty vector (EV), and as a loading control we measured p97 protein level. Two independent experiments. L Mitochondrial oxygen consumption rate (OCR) of brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT and iRhom2 KO mice and stimulated or not with norepinephrine. The results were normalized to the protein content. Two experiments with one or two independent samples per genotype, respectively. M Mitochondrial proton leak of the cells described in L normalized to the protein content. Error bars represent SEM; * represents p

    Article Snippet: First-strand cDNA was synthesized from total RNA using the SuperScript® III First-Strand Synthesis SuperMix or the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific).

    Techniques: Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunohistochemistry, Derivative Assay, Staining, In Vitro, Transduction, Plasmid Preparation, Negative Control

    Interaction with K562 CML cells and its paracrine factors modified cell surface antigen expression in MSC. a Cell surface expression of CD13, CD73, CD90, CD95 and CD105 in control-MSC (CON-MSC) and CML-MSC was analyzed by flow cytometry. Mean (geometric) fluorescent intensity (MFI) was calculated for each marker against its isotype control. b , c Control-MSC were cultured in conditioned media derived from K562 cells for one week and their cell surface gene expression in control-MSC (CON) and conditioned media treated MSC (MSC + CM) was analyzed by flow cytometry. MFI of analyzed markers was normalized to control-MSC. c Representative flow cytometry histogram showing cell surface antigen expression levels in CON and MSC + CM conditions. Grey line represents the isotype control, blue and red line represents the stained cells. d , e Control-MSC were co-cultured without (CON) or with K562 cells (MSC + K) for one week and their cell surface gene expression profile was determined by flow cytometry. MFI was calculated for each antigen and normalized to control-MSC. e Representative flow cytometry histograms showing the cell surface antigen expression in CON (blue line), MSC + K (red line) and isotype control (grey line). f , g RNA was extracted from control-MSC (CON) and K562 co-cultured MSC (MSC + K) and reverse transcribed into cDNA. mRNA expression levels of ( f ) CD90, ( g ) CAT and MnSOD was analyzed by real-time PCR. (h) Mitochondrial ROS levels in CON and MSC + K were analyzed by staining with mitosox red and MFI of mitosox was normalized to unstained cells. Values are mean ± SE, *p

    Journal: Journal of Cell Communication and Signaling

    Article Title: K562 chronic myeloid leukemia cells modify osteogenic differentiation and gene expression of bone marrow stromal cells

    doi: 10.1007/s12079-017-0412-8

    Figure Lengend Snippet: Interaction with K562 CML cells and its paracrine factors modified cell surface antigen expression in MSC. a Cell surface expression of CD13, CD73, CD90, CD95 and CD105 in control-MSC (CON-MSC) and CML-MSC was analyzed by flow cytometry. Mean (geometric) fluorescent intensity (MFI) was calculated for each marker against its isotype control. b , c Control-MSC were cultured in conditioned media derived from K562 cells for one week and their cell surface gene expression in control-MSC (CON) and conditioned media treated MSC (MSC + CM) was analyzed by flow cytometry. MFI of analyzed markers was normalized to control-MSC. c Representative flow cytometry histogram showing cell surface antigen expression levels in CON and MSC + CM conditions. Grey line represents the isotype control, blue and red line represents the stained cells. d , e Control-MSC were co-cultured without (CON) or with K562 cells (MSC + K) for one week and their cell surface gene expression profile was determined by flow cytometry. MFI was calculated for each antigen and normalized to control-MSC. e Representative flow cytometry histograms showing the cell surface antigen expression in CON (blue line), MSC + K (red line) and isotype control (grey line). f , g RNA was extracted from control-MSC (CON) and K562 co-cultured MSC (MSC + K) and reverse transcribed into cDNA. mRNA expression levels of ( f ) CD90, ( g ) CAT and MnSOD was analyzed by real-time PCR. (h) Mitochondrial ROS levels in CON and MSC + K were analyzed by staining with mitosox red and MFI of mitosox was normalized to unstained cells. Values are mean ± SE, *p

    Article Snippet: The resulting RNA was reverse transcribed using high capacity cDNA synthesis kit and oligodT primers at 37°C for 120 min. Real-time PCR was performed using Power SyBr Green reagents in an ABI 7500 system (Thermo Fisher Scientific).

    Techniques: Modification, Expressing, Flow Cytometry, Cytometry, Marker, Cell Culture, Derivative Assay, Staining, Real-time Polymerase Chain Reaction