reverse transcription kit  (Thermo Fisher)


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    Name:
    Arcturus Paradise PLUS Whole Transcript Reverse Transcription Kit WT RT
    Description:
    The ARCTURUS Paradise PLUS Whole Transcript RT WT RT Reagent System provides an efficient and reliable solution for purifying RNA from archived Formalin Fixed Paraffin Embedded FFPE tissue samples followed by reverse transcription of whole transcript for use in real time PCR FFPE samples introduce unique challenges for gene expression profiling and gene expression quantitation including chemical modification and fragmentation of RNA molecules Storage of FFPE samples adds to these challenges through increased RNA degradation over time The Paradise PLUS WT RT Reagent System was specifically developed and specially optimized to provide high quality RNA and robust reverse transcription RT from highly fragmented RNA obtained from archived FFPE samples over 20 years old Figure 2 Key product features • Simple workflow minimal sample handling reduces chance of contamination Figure 3 • Efficient RNA extraction and isolation saves time and minimizes sample loss• Optimized RT reactions generate cDNA representing the entire length of the transcript• Accurate transcript analysis results represent high medium and low abundance expressing genesOptimized RNA Extraction and Isolation from Archived FFPE TissuesFigure 1 shows a comparison between total RNA yields using the Paradise PLUS WT RT Reagent System and a similar commercially available protocol The average yield of the three replicate isolations illustrates the abundance of total RNA made available using the Paradise PLUS WT RT Reagent System The Paradise PLUS WT RT Reagent System produces abundant total RNA for reverse transcription and subsequent qPCR analysis up to three times more total RNA than obtained when a similar commercially available product was used for the same sample Superior Amplification Possible Over Wide Range of TargetsThe sensitivity of the Paradise PLUS WT RT Reagent System was compared to similar reagent products available on the market that generate cDNA for use in real time PCR reactions As Figure 4 illustrates improved Ct values were seen for all high medium and low expressing genes when the Paradise PLUS product was used Exon spanning primers at varying distances from the 3 end of the transcript 500 bp to 5 kb were used to show increased sensitivity and improved transcription success compared to other commercially available products The Paradise PLUS WT RT Reagent System successfully amplified all genes suggesting the reagents provided in the kit are optimized to work together saving you time and effort while producing high quality results across the entire transcript Integrated Systems for MicrogenomicsThe Paradise PLUS Reagent System is validated as part of Arcturus complete Systems for Microgenomics an integrated solution for utilizing small quantities of RNA for gene expression analysis and features the ARCTURUSXT Laser Capture Microdissection LCM Instrument to procure pure cell populations ARCTURUS Systems for Microgenomics permit accurate and sensitive microarray assays that reveal molecular otherwise obscured in whole tissue samples or cell mixtures For Research Use Only Not for use in diagnostics procedures
    Catalog Number:
    kit0305
    Price:
    None
    Applications:
    Laser Capture Microdissection|PCR & Real-Time PCR|Reverse Transcription|Gene Expression Analysis & Genotyping|LCM Kits & Reagents
    Category:
    Kits and Assays
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    Structured Review

    Thermo Fisher reverse transcription kit
    The ARCTURUS Paradise PLUS Whole Transcript RT WT RT Reagent System provides an efficient and reliable solution for purifying RNA from archived Formalin Fixed Paraffin Embedded FFPE tissue samples followed by reverse transcription of whole transcript for use in real time PCR FFPE samples introduce unique challenges for gene expression profiling and gene expression quantitation including chemical modification and fragmentation of RNA molecules Storage of FFPE samples adds to these challenges through increased RNA degradation over time The Paradise PLUS WT RT Reagent System was specifically developed and specially optimized to provide high quality RNA and robust reverse transcription RT from highly fragmented RNA obtained from archived FFPE samples over 20 years old Figure 2 Key product features • Simple workflow minimal sample handling reduces chance of contamination Figure 3 • Efficient RNA extraction and isolation saves time and minimizes sample loss• Optimized RT reactions generate cDNA representing the entire length of the transcript• Accurate transcript analysis results represent high medium and low abundance expressing genesOptimized RNA Extraction and Isolation from Archived FFPE TissuesFigure 1 shows a comparison between total RNA yields using the Paradise PLUS WT RT Reagent System and a similar commercially available protocol The average yield of the three replicate isolations illustrates the abundance of total RNA made available using the Paradise PLUS WT RT Reagent System The Paradise PLUS WT RT Reagent System produces abundant total RNA for reverse transcription and subsequent qPCR analysis up to three times more total RNA than obtained when a similar commercially available product was used for the same sample Superior Amplification Possible Over Wide Range of TargetsThe sensitivity of the Paradise PLUS WT RT Reagent System was compared to similar reagent products available on the market that generate cDNA for use in real time PCR reactions As Figure 4 illustrates improved Ct values were seen for all high medium and low expressing genes when the Paradise PLUS product was used Exon spanning primers at varying distances from the 3 end of the transcript 500 bp to 5 kb were used to show increased sensitivity and improved transcription success compared to other commercially available products The Paradise PLUS WT RT Reagent System successfully amplified all genes suggesting the reagents provided in the kit are optimized to work together saving you time and effort while producing high quality results across the entire transcript Integrated Systems for MicrogenomicsThe Paradise PLUS Reagent System is validated as part of Arcturus complete Systems for Microgenomics an integrated solution for utilizing small quantities of RNA for gene expression analysis and features the ARCTURUSXT Laser Capture Microdissection LCM Instrument to procure pure cell populations ARCTURUS Systems for Microgenomics permit accurate and sensitive microarray assays that reveal molecular otherwise obscured in whole tissue samples or cell mixtures For Research Use Only Not for use in diagnostics procedures
    https://www.bioz.com/result/reverse transcription kit/product/Thermo Fisher
    Average 99 stars, based on 241 article reviews
    Price from $9.99 to $1999.99
    reverse transcription kit - by Bioz Stars, 2021-01
    99/100 stars

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    Related Articles

    SYBR Green Assay:

    Article Title: MicroRNA-93 contributes to the suppression of lung inflammatory responses in LPS-induced acute lung injury in mice via the TLR4/MyD88/NF-κB signaling pathway
    Article Snippet: .. The reverse transcription of miR-93 was performed using the miScript II RT kit and the reverse transcription kit (Invitrogen; Thermo Fisher Scientific, Inc.), respectively. miR-93 expression was measured using Exiqon SYBR-Green Master Mix (Exiqon) on a Light Cycler instrument (Bio-Rad Laboratories, Inc.). ..

    Article Title: Characterization of the Entire Cystatin Gene Family in Barley and Their Target Cathepsin L-Like Cysteine-Proteases, Partners in the Hordein Mobilization during Seed Germination 1Characterization of the Entire Cystatin Gene Family in Barley and Their Target Cathepsin L-Like Cysteine-Proteases, Partners in the Hordein Mobilization during Seed Germination 1 [W]
    Article Snippet: .. Total RNA was extracted from frozen samples by the phenol/chloroform method, followed by precipitation with 3 m LiCl ( ) and digestion with DNase. cDNAs were synthesized from 1 μ g of RNA using the High Reverse Transcription kit (Applied Biosystems) following the manufacturer's instructions. qRT-PCR analyses were performed for duplicate samples in a 7300 Real-Time PCR System (Applied Biosystems) using the SYBR Green detection system. ..

    Synthesized:

    Article Title: Effect of intravenous immunoglobulin on the function of Treg cells derived from immunosuppressed mice with Pseudomonas aeruginosa pneumonia
    Article Snippet: .. Total cDNA was produced by reverse transcription via a Thermo Fisher reverse transcription kit (purchased from Thermo Fisher Scientific). qRT-PCR was carried out using a Thermo Fisher PCR kit with FOXP3 and glyceradehyde-3-phosphate-dehydrogenase (GAPDH) primers (synthesized by TaKaRa Biological Engineering Co., Ltd. Shanghai, China). ..

    Article Title: Characterization of the Entire Cystatin Gene Family in Barley and Their Target Cathepsin L-Like Cysteine-Proteases, Partners in the Hordein Mobilization during Seed Germination 1Characterization of the Entire Cystatin Gene Family in Barley and Their Target Cathepsin L-Like Cysteine-Proteases, Partners in the Hordein Mobilization during Seed Germination 1 [W]
    Article Snippet: .. Total RNA was extracted from frozen samples by the phenol/chloroform method, followed by precipitation with 3 m LiCl ( ) and digestion with DNase. cDNAs were synthesized from 1 μ g of RNA using the High Reverse Transcription kit (Applied Biosystems) following the manufacturer's instructions. qRT-PCR analyses were performed for duplicate samples in a 7300 Real-Time PCR System (Applied Biosystems) using the SYBR Green detection system. ..

    Isolation:

    Article Title: Treatment with adipose tissue-derived mesenchymal stem cells exerts anti-diabetic effects, improves long-term complications, and attenuates inflammation in type 2 diabetic rats
    Article Snippet: .. Quantitative real-time PCR Total RNA was isolated from rat adipose tissue, liver tissue, renal tissue, and lung tissue using Trizol reagent (Life technologies, Frederick, USA) and was reverse-transcribed with a reverse transcription kit (Thermo Scientific, CA, USA)) according to the manufacturer’s instructions. .. Real-time polymerase chain reaction (RT-PCR) was performed on ABI Prism thermal cycler model StepOnePlus (Applied Biosystems, CA, USA) using a SYBR Green PCR master mix (Applied Biosystems).

    Quantitative RT-PCR:

    Article Title: Effect of intravenous immunoglobulin on the function of Treg cells derived from immunosuppressed mice with Pseudomonas aeruginosa pneumonia
    Article Snippet: .. Total cDNA was produced by reverse transcription via a Thermo Fisher reverse transcription kit (purchased from Thermo Fisher Scientific). qRT-PCR was carried out using a Thermo Fisher PCR kit with FOXP3 and glyceradehyde-3-phosphate-dehydrogenase (GAPDH) primers (synthesized by TaKaRa Biological Engineering Co., Ltd. Shanghai, China). ..

    Article Title: Characterization of the Entire Cystatin Gene Family in Barley and Their Target Cathepsin L-Like Cysteine-Proteases, Partners in the Hordein Mobilization during Seed Germination 1Characterization of the Entire Cystatin Gene Family in Barley and Their Target Cathepsin L-Like Cysteine-Proteases, Partners in the Hordein Mobilization during Seed Germination 1 [W]
    Article Snippet: .. Total RNA was extracted from frozen samples by the phenol/chloroform method, followed by precipitation with 3 m LiCl ( ) and digestion with DNase. cDNAs were synthesized from 1 μ g of RNA using the High Reverse Transcription kit (Applied Biosystems) following the manufacturer's instructions. qRT-PCR analyses were performed for duplicate samples in a 7300 Real-Time PCR System (Applied Biosystems) using the SYBR Green detection system. ..

    Produced:

    Article Title: Effect of intravenous immunoglobulin on the function of Treg cells derived from immunosuppressed mice with Pseudomonas aeruginosa pneumonia
    Article Snippet: .. Total cDNA was produced by reverse transcription via a Thermo Fisher reverse transcription kit (purchased from Thermo Fisher Scientific). qRT-PCR was carried out using a Thermo Fisher PCR kit with FOXP3 and glyceradehyde-3-phosphate-dehydrogenase (GAPDH) primers (synthesized by TaKaRa Biological Engineering Co., Ltd. Shanghai, China). ..

    Concentration Assay:

    Article Title: Tryptase and Protease-Activated Receptor 2 Expression Levels in Irritable Bowel Syndrome
    Article Snippet: .. Total RNA was extracted, and reversed by using reverse transcription kit after measuring concentration of total RNA with Nano drop. ..

    Polymerase Chain Reaction:

    Article Title: Effect of intravenous immunoglobulin on the function of Treg cells derived from immunosuppressed mice with Pseudomonas aeruginosa pneumonia
    Article Snippet: .. Total cDNA was produced by reverse transcription via a Thermo Fisher reverse transcription kit (purchased from Thermo Fisher Scientific). qRT-PCR was carried out using a Thermo Fisher PCR kit with FOXP3 and glyceradehyde-3-phosphate-dehydrogenase (GAPDH) primers (synthesized by TaKaRa Biological Engineering Co., Ltd. Shanghai, China). ..

    Expressing:

    Article Title: MicroRNA-93 contributes to the suppression of lung inflammatory responses in LPS-induced acute lung injury in mice via the TLR4/MyD88/NF-κB signaling pathway
    Article Snippet: .. The reverse transcription of miR-93 was performed using the miScript II RT kit and the reverse transcription kit (Invitrogen; Thermo Fisher Scientific, Inc.), respectively. miR-93 expression was measured using Exiqon SYBR-Green Master Mix (Exiqon) on a Light Cycler instrument (Bio-Rad Laboratories, Inc.). ..

    Article Title: Rho/ROCK-dependent inhibition of 3T3-L1 adipogenesis by G-protein-deamidating dermonecrotic toxins: differential regulation of Notch1, Pref1/Dlk1, and ?-catenin signaling
    Article Snippet: .. The levels of mRNA expression were determined by RT-PCR using Reverse-iT RT-PCR kit (ABgene). ..

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Rho/ROCK-dependent inhibition of 3T3-L1 adipogenesis by G-protein-deamidating dermonecrotic toxins: differential regulation of Notch1, Pref1/Dlk1, and ?-catenin signaling
    Article Snippet: .. The levels of mRNA expression were determined by RT-PCR using Reverse-iT RT-PCR kit (ABgene). ..

    Real-time Polymerase Chain Reaction:

    Article Title: Treatment with adipose tissue-derived mesenchymal stem cells exerts anti-diabetic effects, improves long-term complications, and attenuates inflammation in type 2 diabetic rats
    Article Snippet: .. Quantitative real-time PCR Total RNA was isolated from rat adipose tissue, liver tissue, renal tissue, and lung tissue using Trizol reagent (Life technologies, Frederick, USA) and was reverse-transcribed with a reverse transcription kit (Thermo Scientific, CA, USA)) according to the manufacturer’s instructions. .. Real-time polymerase chain reaction (RT-PCR) was performed on ABI Prism thermal cycler model StepOnePlus (Applied Biosystems, CA, USA) using a SYBR Green PCR master mix (Applied Biosystems).

    Article Title: Characterization of the Entire Cystatin Gene Family in Barley and Their Target Cathepsin L-Like Cysteine-Proteases, Partners in the Hordein Mobilization during Seed Germination 1Characterization of the Entire Cystatin Gene Family in Barley and Their Target Cathepsin L-Like Cysteine-Proteases, Partners in the Hordein Mobilization during Seed Germination 1 [W]
    Article Snippet: .. Total RNA was extracted from frozen samples by the phenol/chloroform method, followed by precipitation with 3 m LiCl ( ) and digestion with DNase. cDNAs were synthesized from 1 μ g of RNA using the High Reverse Transcription kit (Applied Biosystems) following the manufacturer's instructions. qRT-PCR analyses were performed for duplicate samples in a 7300 Real-Time PCR System (Applied Biosystems) using the SYBR Green detection system. ..

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  • 99
    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: 99/100, based on 11209 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mrna species/product/Thermo Fisher
    Average 99 stars, based on 11209 article reviews
    Price from $9.99 to $1999.99
    mrna species - by Bioz Stars, 2021-01
    99/100 stars
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    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

    BRD4 expression is sufficient to transform immortalized ovarian surface epithelial cells. (A) Analysis of BRD4 copy-number from 1042 cell lines (CCLE). (B) BRD4 long and short-isoform expression in BRD4-transduced IOSE cells, and several BRD4-non-amplified (OV0419F, OV110F, OV2022F and OV0452F) and BRD4-amplified (HOXF062 and OV0857F) patient derived xenograft models. BRD4 copy-number (CN) in different PDX models were determined using TaqMan ® Copy-number Assay by qPCR. (C) Colony formation potential of IOSE cells expressing BRD4 -/+ 500 nM JQ1 was assessed using a soft-agar assay. (D) Proliferation of BRD4 expressing IOSE cells was examined by measuring cell confluence over time via live cell imaging. (E) Cell cycle analysis of long and short BRD4-isoform expressing IOSE cells.

    Journal: PLoS ONE

    Article Title: BRD4 amplification facilitates an oncogenic gene expression program in high-grade serous ovarian cancer and confers sensitivity to BET inhibitors

    doi: 10.1371/journal.pone.0200826

    Figure Lengend Snippet: BRD4 expression is sufficient to transform immortalized ovarian surface epithelial cells. (A) Analysis of BRD4 copy-number from 1042 cell lines (CCLE). (B) BRD4 long and short-isoform expression in BRD4-transduced IOSE cells, and several BRD4-non-amplified (OV0419F, OV110F, OV2022F and OV0452F) and BRD4-amplified (HOXF062 and OV0857F) patient derived xenograft models. BRD4 copy-number (CN) in different PDX models were determined using TaqMan ® Copy-number Assay by qPCR. (C) Colony formation potential of IOSE cells expressing BRD4 -/+ 500 nM JQ1 was assessed using a soft-agar assay. (D) Proliferation of BRD4 expressing IOSE cells was examined by measuring cell confluence over time via live cell imaging. (E) Cell cycle analysis of long and short BRD4-isoform expressing IOSE cells.

    Article Snippet: 2 μg RNA was converted to cDNA with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems #4368814). qPCR reaction was set up with TaqMan® Gene Expression Master Mix (Thermo Fisher Scientific #4369016) and carried out on an Applied Biosystems 7500 HT instrument.

    Techniques: Expressing, Amplification, Derivative Assay, TaqMan Copy Number Assay, Real-time Polymerase Chain Reaction, Soft Agar Assay, Live Cell Imaging, Cell Cycle Assay

    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

    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