magnetic mrna isolation kit  (New England Biolabs)


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    Name:
    Magnetic mRNA Isolation Kit
    Description:
    Magnetic mRNA Isolation Kit 25 isolations
    Catalog Number:
    s1550s
    Price:
    327
    Size:
    25 isolations
    Category:
    mRNA Purification Kits
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    New England Biolabs magnetic mrna isolation kit
    Magnetic mRNA Isolation Kit
    Magnetic mRNA Isolation Kit 25 isolations
    https://www.bioz.com/result/magnetic mrna isolation kit/product/New England Biolabs
    Average 99 stars, based on 156 article reviews
    Price from $9.99 to $1999.99
    magnetic mrna isolation kit - by Bioz Stars, 2020-09
    99/100 stars

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    1) Product Images from "Rbfox2 is Critical for Maintaining Alternative Polyadenylation and Mitochondrial Health in Myoblasts"

    Article Title: Rbfox2 is Critical for Maintaining Alternative Polyadenylation and Mitochondrial Health in Myoblasts

    Journal: bioRxiv

    doi: 10.1101/2020.05.13.093013

    RBFOX2-regulated tandem-APA modulates 3’UTR length and affects mRNA levels. (A) The number of downregulated and upregulated genes in RBFOX2 KD H9c2 cells. (B) Gene ontology analysis of downregulated or upregulated genes in RBFOX2 depleted H9c2 cells. (C) The number of genes undergoing tandem-APA generating shorter or longer 3’UTRs in RBFOX2 KD H9c2 cells. (D) The number of genes that undergo both APA and mRNA level changes in RBFOX2 KD H9c2 cells. (E) Hdlbp 3 ’ UTR shortening mediated via tandem-APA in RBFOX2 depleted H9c2 cells identified by PAC-seq (top panel) or by nanopore sequencing (bottom panel).
    Figure Legend Snippet: RBFOX2-regulated tandem-APA modulates 3’UTR length and affects mRNA levels. (A) The number of downregulated and upregulated genes in RBFOX2 KD H9c2 cells. (B) Gene ontology analysis of downregulated or upregulated genes in RBFOX2 depleted H9c2 cells. (C) The number of genes undergoing tandem-APA generating shorter or longer 3’UTRs in RBFOX2 KD H9c2 cells. (D) The number of genes that undergo both APA and mRNA level changes in RBFOX2 KD H9c2 cells. (E) Hdlbp 3 ’ UTR shortening mediated via tandem-APA in RBFOX2 depleted H9c2 cells identified by PAC-seq (top panel) or by nanopore sequencing (bottom panel).

    Techniques Used: Nanopore Sequencing

    RBFOX2 binding sites are enriched upstream of poly(A) sites in 3’UTRs that undergo APA changes. (A) Metagene analysis of RBFOX2 CLIP-binding distribution with respect to PASs in all detectable PASs, or in upregulated/downregulated PASs identified in RBFOX2 KD H9c2 cells. (B) Genome browser image of Rab7a 3’UTR displays tandem-APA changes determined by PAC-seq (left). Tandem-APA change in Rab7a in control vs RBFOX2 KD H9c2 cells determined by RT-qPCR (right). The ratio of “distal or long” to “common” mRNA expression levels of Rab7a by RT-qPCR in control cells was normalized to 1. Statistical significance was calculated using unpaired t-test to compare two different groups in three independent experiments (n=3). Data represent means ± SD. *p=0.0466. (C) RBFOX2 CLIP-seq reads mapped to the Rab7a 3’UTR. (D) FLAG western blot of FLAG-RBFOX2 pulled down with in-vitro transcribed and biotinylated Rab7a transcripts in induced or uninduced HEK293 cells. No RNA was used as a negative control for non-specific protein binding in induced or uninduced cells.
    Figure Legend Snippet: RBFOX2 binding sites are enriched upstream of poly(A) sites in 3’UTRs that undergo APA changes. (A) Metagene analysis of RBFOX2 CLIP-binding distribution with respect to PASs in all detectable PASs, or in upregulated/downregulated PASs identified in RBFOX2 KD H9c2 cells. (B) Genome browser image of Rab7a 3’UTR displays tandem-APA changes determined by PAC-seq (left). Tandem-APA change in Rab7a in control vs RBFOX2 KD H9c2 cells determined by RT-qPCR (right). The ratio of “distal or long” to “common” mRNA expression levels of Rab7a by RT-qPCR in control cells was normalized to 1. Statistical significance was calculated using unpaired t-test to compare two different groups in three independent experiments (n=3). Data represent means ± SD. *p=0.0466. (C) RBFOX2 CLIP-seq reads mapped to the Rab7a 3’UTR. (D) FLAG western blot of FLAG-RBFOX2 pulled down with in-vitro transcribed and biotinylated Rab7a transcripts in induced or uninduced HEK293 cells. No RNA was used as a negative control for non-specific protein binding in induced or uninduced cells.

    Techniques Used: Binding Assay, Cross-linking Immunoprecipitation, Quantitative RT-PCR, Expressing, Western Blot, In Vitro, Negative Control, Protein Binding

    2) Product Images from "Ongoing resolution of duplicate gene functions shapes the diversification of a metabolic network"

    Article Title: Ongoing resolution of duplicate gene functions shapes the diversification of a metabolic network

    Journal: eLife

    doi: 10.7554/eLife.19027

    The regulation of PGM1 by galactose was inferred as the ancestral state. ( A ) mRNA levels of S. uvarum PGM1 and PGM2 during mid-log phase in SC + 2% galactose, SC + 5% glycerol, and SC + 2% glucose. Note that PGM2 , which encodes the major isoform of phosphoglucomutase, has long been known to be transcriptionally induced by ~three–four fold in galactose, but it lacks a Gal4 binding site and does not appear to be a direct target in S. cerevisiae ( Oh and Hopper, 1990 ; Rubio-Texeira, 2005 ). These features are broadly shared with S. uvarum PGM2 , which is transcriptionally induced two-fold by galactose relative to glycerol but is not transcriptionally up-regulated in the gal80∆ gal80b∆ mutant; nor does it have a consensus Gal4 site. ( B ) Conservation of putative Gal4 binding sites upstream of PGM1 in S. uvarum , S. eubayanus , S. arboricola , and two outgroup species. The orange dot indicates the inferred loss of direct regulation of PGM1 by Gal4 based on the presence or absence of putative Gal4 binding sites (CGGN 11 CCG). The distances upstream from the start codon are shown at the right. The putative sites are shown as red boxes at the relative position of the upstream sequences of PGM1 . Note that, in Kazachstania nagansihii , the upstream intergenic region of PGM1 ortholog is 1958 bp, an unusually long intergenic region for yeasts, and contains a divergent promoter that also drives expression of the PMU1 ortholog. DOI: http://dx.doi.org/10.7554/eLife.19027.015
    Figure Legend Snippet: The regulation of PGM1 by galactose was inferred as the ancestral state. ( A ) mRNA levels of S. uvarum PGM1 and PGM2 during mid-log phase in SC + 2% galactose, SC + 5% glycerol, and SC + 2% glucose. Note that PGM2 , which encodes the major isoform of phosphoglucomutase, has long been known to be transcriptionally induced by ~three–four fold in galactose, but it lacks a Gal4 binding site and does not appear to be a direct target in S. cerevisiae ( Oh and Hopper, 1990 ; Rubio-Texeira, 2005 ). These features are broadly shared with S. uvarum PGM2 , which is transcriptionally induced two-fold by galactose relative to glycerol but is not transcriptionally up-regulated in the gal80∆ gal80b∆ mutant; nor does it have a consensus Gal4 site. ( B ) Conservation of putative Gal4 binding sites upstream of PGM1 in S. uvarum , S. eubayanus , S. arboricola , and two outgroup species. The orange dot indicates the inferred loss of direct regulation of PGM1 by Gal4 based on the presence or absence of putative Gal4 binding sites (CGGN 11 CCG). The distances upstream from the start codon are shown at the right. The putative sites are shown as red boxes at the relative position of the upstream sequences of PGM1 . Note that, in Kazachstania nagansihii , the upstream intergenic region of PGM1 ortholog is 1958 bp, an unusually long intergenic region for yeasts, and contains a divergent promoter that also drives expression of the PMU1 ortholog. DOI: http://dx.doi.org/10.7554/eLife.19027.015

    Techniques Used: Binding Assay, Mutagenesis, Expressing

    3) Product Images from "High through-put identification of miR-145 targets in human articular chondrocytes"

    Article Title: High through-put identification of miR-145 targets in human articular chondrocytes

    Journal: bioRxiv

    doi: 10.1101/2020.02.03.931683

    Analysis of the effect of miR-145 over-expression on mRNA levels of selected genes in freshly isolated HACs. A) 16 randomly selected mRNAs from CD-IP and CD2DB1 from both CD-IP and DT-RNA. B) 3 genes only present in DT-RNA and COL2A1. Control (C) or miR-145 mimics were transfected in freshly isolated HACs and RNA was extracted 48h after transfection. Values are presented as relative to that obtained in cells transfected with control mimics for each patient and normalized to RPLP0. Data represents average ± S.E.M. from 7 different experiments, each performed with different donor cells. * P
    Figure Legend Snippet: Analysis of the effect of miR-145 over-expression on mRNA levels of selected genes in freshly isolated HACs. A) 16 randomly selected mRNAs from CD-IP and CD2DB1 from both CD-IP and DT-RNA. B) 3 genes only present in DT-RNA and COL2A1. Control (C) or miR-145 mimics were transfected in freshly isolated HACs and RNA was extracted 48h after transfection. Values are presented as relative to that obtained in cells transfected with control mimics for each patient and normalized to RPLP0. Data represents average ± S.E.M. from 7 different experiments, each performed with different donor cells. * P

    Techniques Used: Over Expression, Isolation, Transfection

    4) Product Images from "Aphid feeding induces the relaxation of epigenetic control and the associated regulation of the defense response in Arabidopsis"

    Article Title: Aphid feeding induces the relaxation of epigenetic control and the associated regulation of the defense response in Arabidopsis

    Journal: bioRxiv

    doi: 10.1101/2020.01.24.916783

    Changes induced at TE expression by aphid feeding. A. Volcano plot depicting TE mRNA-seq expression in the comparison aphid vs control RNA samples. Dots colored in red indicated genes with significant upregulation. B. Volcano plot depicting gene expression in the comparison aphid vs control PARE samples. Dots colored in red indicated genes with significant upregulation. C. Global sRNAs profiles of control and stressed samples. D. TE-derived sRNA profiles of control and stressed samples. E. Relative accumulation of 21,22 and 24 nt sRNAs in control (C) and aphid infested samples (Mper) for TEs of different sizes. Values shown are relative to control, where accumulation values for each sRNA category were set to 1. F. Venn diagram showing the overlap between the TE populations identified from each of the different RNA sequencing analyses. G. Venn diagram depicting the overlap of TEs upregulated two fold in the PARE sequencing data and TEs losing or gaining two fold 24 nt sRNAs. G. Screenshot of a genome browser showing the accumulation of PARE reads and 24 nt sRNAs in control and aphid samples for two of the TEs upregulated in the PARE libraries and showing a decrease of 24 nt sRNA accumulation.
    Figure Legend Snippet: Changes induced at TE expression by aphid feeding. A. Volcano plot depicting TE mRNA-seq expression in the comparison aphid vs control RNA samples. Dots colored in red indicated genes with significant upregulation. B. Volcano plot depicting gene expression in the comparison aphid vs control PARE samples. Dots colored in red indicated genes with significant upregulation. C. Global sRNAs profiles of control and stressed samples. D. TE-derived sRNA profiles of control and stressed samples. E. Relative accumulation of 21,22 and 24 nt sRNAs in control (C) and aphid infested samples (Mper) for TEs of different sizes. Values shown are relative to control, where accumulation values for each sRNA category were set to 1. F. Venn diagram showing the overlap between the TE populations identified from each of the different RNA sequencing analyses. G. Venn diagram depicting the overlap of TEs upregulated two fold in the PARE sequencing data and TEs losing or gaining two fold 24 nt sRNAs. G. Screenshot of a genome browser showing the accumulation of PARE reads and 24 nt sRNAs in control and aphid samples for two of the TEs upregulated in the PARE libraries and showing a decrease of 24 nt sRNA accumulation.

    Techniques Used: Expressing, Derivative Assay, RNA Sequencing Assay, Sequencing

    5) Product Images from "Kinesin-2 and kinesin-9 have atypical functions during ciliogenesis in the male gametophyte of Marsilea vestita"

    Article Title: Kinesin-2 and kinesin-9 have atypical functions during ciliogenesis in the male gametophyte of Marsilea vestita

    Journal: BMC Cell Biology

    doi: 10.1186/s12860-016-0107-7

    Characterization of the kinesin-2 and kinesin-9 families in Marsilea . a – b Maximum likelihood trees were constructed using the motor domain from well-established kinesin-2 and -9 sequences. The motor domain from kinesin-1 (Kif5) was used as an out-group for these analyses. a Kinesin-2 motors can be separated into three subfamilies; kinesin-2α ( red ), -2β ( orange ), and -2γ ( green ). There are also many kinesin-2 sequences that do not correspond to any well-supported subfamily. The kinesin-2 motor in Marsilea (MvKinesin-2) is most similar to the kinesin-2 in Physcomitrella and does not fall into a kinesin-2 subfamily. b Kinesin-9 can be separated into two well-supported subfamilies, kinesin-9A ( red ) and kinesin-9B ( blue ). Marsilea has one kinesin-9A (MvKinesin-9A) and one kinesin-9B (MvKinesin-9B). c An increase in MvKinesin-2, -9A, and -9B mRNA abundance was detected using RT-PCR against poly(A+)-RNA isolated at 1–2, 3–5, and 6–8 h of gametophyte development. The abundance of centrin mRNA does not change during development and was used as a control
    Figure Legend Snippet: Characterization of the kinesin-2 and kinesin-9 families in Marsilea . a – b Maximum likelihood trees were constructed using the motor domain from well-established kinesin-2 and -9 sequences. The motor domain from kinesin-1 (Kif5) was used as an out-group for these analyses. a Kinesin-2 motors can be separated into three subfamilies; kinesin-2α ( red ), -2β ( orange ), and -2γ ( green ). There are also many kinesin-2 sequences that do not correspond to any well-supported subfamily. The kinesin-2 motor in Marsilea (MvKinesin-2) is most similar to the kinesin-2 in Physcomitrella and does not fall into a kinesin-2 subfamily. b Kinesin-9 can be separated into two well-supported subfamilies, kinesin-9A ( red ) and kinesin-9B ( blue ). Marsilea has one kinesin-9A (MvKinesin-9A) and one kinesin-9B (MvKinesin-9B). c An increase in MvKinesin-2, -9A, and -9B mRNA abundance was detected using RT-PCR against poly(A+)-RNA isolated at 1–2, 3–5, and 6–8 h of gametophyte development. The abundance of centrin mRNA does not change during development and was used as a control

    Techniques Used: Construct, Reverse Transcription Polymerase Chain Reaction, Isolation

    Kinesin-2 and kinesin-9 are involved in spermatogenesis. a Unique 350–400 nt regions used for constructing dsRNA. b dsRNA constructed from MvKinesin-2, -9A, and -9B, respectively, using poly(A+)-RNA isolated at 8 h as a template. c RT-PCR for MvKinesin-2, MvKinesin-9A, MvKinesin-9B, and centrin respectively, in the absence (-) of any dsRNA at 8 h of development. d – f RT-PCR for MvKinesin-2, MvKinesin-9A, MvKinesin-9B, and centrin, respectively, in the presence (+) of ( d ) MvKinesin-2 dsRNA, ( e ) MvKinesin-9A dsRNA, or ( f ) MvKinesin-9B dsRNA. Each transcript cannot be detected after its knockdown. Centrin mRNA does not change after the addition of various kinesin dsRNAs. g Untreated, control microspores developed for 8 h, embedded in methacrylate, sectioned, and stained with TBO. Spermatogenous (SP) and jacket ( j ) cells can easily be distinguished from each other. Sectioned microspores treated with ( h – i ) MvKinesin-2, ( j ) MvKinesin-9A, and ( k ) MvKinesin-9B dsRNA and stained with TBO
    Figure Legend Snippet: Kinesin-2 and kinesin-9 are involved in spermatogenesis. a Unique 350–400 nt regions used for constructing dsRNA. b dsRNA constructed from MvKinesin-2, -9A, and -9B, respectively, using poly(A+)-RNA isolated at 8 h as a template. c RT-PCR for MvKinesin-2, MvKinesin-9A, MvKinesin-9B, and centrin respectively, in the absence (-) of any dsRNA at 8 h of development. d – f RT-PCR for MvKinesin-2, MvKinesin-9A, MvKinesin-9B, and centrin, respectively, in the presence (+) of ( d ) MvKinesin-2 dsRNA, ( e ) MvKinesin-9A dsRNA, or ( f ) MvKinesin-9B dsRNA. Each transcript cannot be detected after its knockdown. Centrin mRNA does not change after the addition of various kinesin dsRNAs. g Untreated, control microspores developed for 8 h, embedded in methacrylate, sectioned, and stained with TBO. Spermatogenous (SP) and jacket ( j ) cells can easily be distinguished from each other. Sectioned microspores treated with ( h – i ) MvKinesin-2, ( j ) MvKinesin-9A, and ( k ) MvKinesin-9B dsRNA and stained with TBO

    Techniques Used: Construct, Isolation, Reverse Transcription Polymerase Chain Reaction, Staining

    6) Product Images from "Transcript-specific characteristics determine the contribution of endo- and exonucleolytic decay pathways during the degradation of nonsense-mediated decay substrates"

    Article Title: Transcript-specific characteristics determine the contribution of endo- and exonucleolytic decay pathways during the degradation of nonsense-mediated decay substrates

    Journal: RNA

    doi: 10.1261/rna.059659.116

    Robustness of the NMD system activated via different decay-inducing features. ( A – C ) Northern blots of RNA samples extracted from HeLa cells transfected with the indicated siRNAs, plasmids, and reporter constructs. Cotransfected β-globin ( A , C ) or LacZ-H4 ( B ) served as control mRNA. Endocleavage products (3′ fragments) are indicated. To eliminate effects caused by altered transfection efficiency or availability of the general gene expression machinery, decreasing amounts of TPI-WT-0H ([ A,B ] 3, 2, and 0 µg; [ C ] 4, 2, and 0 µg) were cotransfected with increasing amounts of reporter ([ A,B ] 1, 2, and 4 µg; [ C ] constant 3 µg) or competition vector ([ C ] 0, 2, and 4 µg). The 0H constructs lack the Northern blot probe binding sites and are not detected by the 3′ probe. Mean values of reporter and 3′ fragment signal ± SD ( n = 3) were quantified and normalized to the control. The ratio of 3′ fragment to reporter mRNA levels is indicated below the bars. A representative Western blot is shown at the bottom , using tubulin as loading control ( C ).
    Figure Legend Snippet: Robustness of the NMD system activated via different decay-inducing features. ( A – C ) Northern blots of RNA samples extracted from HeLa cells transfected with the indicated siRNAs, plasmids, and reporter constructs. Cotransfected β-globin ( A , C ) or LacZ-H4 ( B ) served as control mRNA. Endocleavage products (3′ fragments) are indicated. To eliminate effects caused by altered transfection efficiency or availability of the general gene expression machinery, decreasing amounts of TPI-WT-0H ([ A,B ] 3, 2, and 0 µg; [ C ] 4, 2, and 0 µg) were cotransfected with increasing amounts of reporter ([ A,B ] 1, 2, and 4 µg; [ C ] constant 3 µg) or competition vector ([ C ] 0, 2, and 4 µg). The 0H constructs lack the Northern blot probe binding sites and are not detected by the 3′ probe. Mean values of reporter and 3′ fragment signal ± SD ( n = 3) were quantified and normalized to the control. The ratio of 3′ fragment to reporter mRNA levels is indicated below the bars. A representative Western blot is shown at the bottom , using tubulin as loading control ( C ).

    Techniques Used: Northern Blot, Transfection, Construct, Expressing, Plasmid Preparation, Binding Assay, Western Blot

    Deletions of C-terminal UPF1 phosphorylation sites impair NMD differently. ( A ) UPF1 protein architecture is depicted schematically. All structural and functional domains are indicated; the presence of potential phosphorylation sites (SQ/TQ) are shown in red and blue, respectively. ( B , C ) Northern blots of RNA samples extracted from HeLa cells transfected with the indicated siRNAs and reporter constructs. Cotransfected LacZ-H4 ( B ) or β-globin ( C ) served as control mRNA. Endocleavage products (3′ fragments) are indicated. Mean values of reporter and 3′ fragment signal ± SD ( n = 3) were quantified and normalized to the XRN1 control knockdown. Representative Western blots are shown at the bottom , using tubulin as loading control.
    Figure Legend Snippet: Deletions of C-terminal UPF1 phosphorylation sites impair NMD differently. ( A ) UPF1 protein architecture is depicted schematically. All structural and functional domains are indicated; the presence of potential phosphorylation sites (SQ/TQ) are shown in red and blue, respectively. ( B , C ) Northern blots of RNA samples extracted from HeLa cells transfected with the indicated siRNAs and reporter constructs. Cotransfected LacZ-H4 ( B ) or β-globin ( C ) served as control mRNA. Endocleavage products (3′ fragments) are indicated. Mean values of reporter and 3′ fragment signal ± SD ( n = 3) were quantified and normalized to the XRN1 control knockdown. Representative Western blots are shown at the bottom , using tubulin as loading control.

    Techniques Used: Functional Assay, Northern Blot, Transfection, Construct, Western Blot

    Functional analysis of potential endogenous NMD targets identified during high-throughput sequencing. ( A – D ) A PCR-based approach was used to quantify 3′ fragment levels upon knockdown of NMD effectors. HeLa cells were transiently transfected with the indicated siRNAs and poly(A) + RNA was extracted. 3′ Decay intermediates were ligated to an RNA linker, followed by reverse transcription with an oligo(dT) primer and gene-specific PCR. Overall transcript levels were determined with primer pairs located downstream from the estimated endocleavage site (second panel each, indicated as “gene int.”). PCR with TATA-Box binding protein (TBP) primers was used for cDNA level determination. For each class of NMD targets ( A , B , C , D, respectively), the same set of cDNA was used and therefore the same TBP profiles are shown for each target type. Degradome-sequencing reads were plotted against their position on the indicated mRNAs. Endocleavage events within the selected targets were visualized in an enlarged view spanning 150 nt (±75 nt). The position of the second nucleotide of the respective stop codon is set as zero. Mapped reads per nucleotide were plotted against the mRNA length for each knockdown condition (Luciferase [Luc, black], XRN1 [orange], XRN1/SMG6 [blue]). ( A ) PDRG1, SURF6, and MED10 transcripts with a long 3′ UTR. ( B ) IFRD1 and BAG1 transcripts with an uORF. ( C ) C11orf31, encoding for Selenoprotein H, containing a selenocysteine (Sec) codon. ( D ) TMEM222, incorporation of an alternative exon (indicated in purple), harboring a PTC as well as RPL10A, a transcript gaining a PTC probably due to alternative splice site usage within the third intron.
    Figure Legend Snippet: Functional analysis of potential endogenous NMD targets identified during high-throughput sequencing. ( A – D ) A PCR-based approach was used to quantify 3′ fragment levels upon knockdown of NMD effectors. HeLa cells were transiently transfected with the indicated siRNAs and poly(A) + RNA was extracted. 3′ Decay intermediates were ligated to an RNA linker, followed by reverse transcription with an oligo(dT) primer and gene-specific PCR. Overall transcript levels were determined with primer pairs located downstream from the estimated endocleavage site (second panel each, indicated as “gene int.”). PCR with TATA-Box binding protein (TBP) primers was used for cDNA level determination. For each class of NMD targets ( A , B , C , D, respectively), the same set of cDNA was used and therefore the same TBP profiles are shown for each target type. Degradome-sequencing reads were plotted against their position on the indicated mRNAs. Endocleavage events within the selected targets were visualized in an enlarged view spanning 150 nt (±75 nt). The position of the second nucleotide of the respective stop codon is set as zero. Mapped reads per nucleotide were plotted against the mRNA length for each knockdown condition (Luciferase [Luc, black], XRN1 [orange], XRN1/SMG6 [blue]). ( A ) PDRG1, SURF6, and MED10 transcripts with a long 3′ UTR. ( B ) IFRD1 and BAG1 transcripts with an uORF. ( C ) C11orf31, encoding for Selenoprotein H, containing a selenocysteine (Sec) codon. ( D ) TMEM222, incorporation of an alternative exon (indicated in purple), harboring a PTC as well as RPL10A, a transcript gaining a PTC probably due to alternative splice site usage within the third intron.

    Techniques Used: Functional Assay, Next-Generation Sequencing, Polymerase Chain Reaction, Transfection, Binding Assay, Sequencing, Luciferase, Size-exclusion Chromatography

    SMG7 abundance influences endocleavage efficiency for long 3′ UTR targets. ( A ) Domain structure of SMG7, showing the N-terminal 14-3-3-like domain (interacts with UPF1) and α-helical extensions as well as the C-terminal PC region (interacts with POP2). ( B ) Northern blot of RNA samples extracted from HeLa cells transfected with the indicated siRNAs and reporter constructs. Cotransfected β-globin served as control mRNA. Endocleavage products (3′ fragments) are indicated. Mean values of reporter and 3′ fragment signal ± SD ( n = 3) were quantified and normalized to the XRN1 control knockdown. A representative Western blot is shown at the bottom , using tubulin as loading control. ( C ) Schematic representation of the transfected triosephosphate isomerase (TPI) reporter with a PTC at amino acid position 160. Exons are depicted as white (untranslated) or black (translated) boxes, introns as two connecting black lines, and the Northern probe binding sites as light gray boxes. Vector derived 5′ UTR intron and SV40 poly(A) signal (pA) are shown. ( D ) Northern blot of RNA samples extracted from HeLa cells transfected with the indicated siRNAs, plasmids, and reporter constructs using LacZ-4H as control mRNA. Endocleavage products (3′ fragments) are indicated. Mean values of reporter and 3′ fragment signal ± SD ( n = 3) were quantified and normalized to the XRN1 control knockdown. A representative Western blot is shown at the bottom , using tubulin as loading control.
    Figure Legend Snippet: SMG7 abundance influences endocleavage efficiency for long 3′ UTR targets. ( A ) Domain structure of SMG7, showing the N-terminal 14-3-3-like domain (interacts with UPF1) and α-helical extensions as well as the C-terminal PC region (interacts with POP2). ( B ) Northern blot of RNA samples extracted from HeLa cells transfected with the indicated siRNAs and reporter constructs. Cotransfected β-globin served as control mRNA. Endocleavage products (3′ fragments) are indicated. Mean values of reporter and 3′ fragment signal ± SD ( n = 3) were quantified and normalized to the XRN1 control knockdown. A representative Western blot is shown at the bottom , using tubulin as loading control. ( C ) Schematic representation of the transfected triosephosphate isomerase (TPI) reporter with a PTC at amino acid position 160. Exons are depicted as white (untranslated) or black (translated) boxes, introns as two connecting black lines, and the Northern probe binding sites as light gray boxes. Vector derived 5′ UTR intron and SV40 poly(A) signal (pA) are shown. ( D ) Northern blot of RNA samples extracted from HeLa cells transfected with the indicated siRNAs, plasmids, and reporter constructs using LacZ-4H as control mRNA. Endocleavage products (3′ fragments) are indicated. Mean values of reporter and 3′ fragment signal ± SD ( n = 3) were quantified and normalized to the XRN1 control knockdown. A representative Western blot is shown at the bottom , using tubulin as loading control.

    Techniques Used: Northern Blot, Transfection, Construct, Western Blot, Binding Assay, Plasmid Preparation, Derivative Assay

    7) Product Images from "Human Survival Motor Neuron genes generate a vast repertoire of circular RNAs"

    Article Title: Human Survival Motor Neuron genes generate a vast repertoire of circular RNAs

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz034

    Linear SMN transcripts showing inclusion of novel exons 9, 10, 11 and 12. ( A ) An overview of the 3′ portion of the SMN pre-mRNA. Splicing events are indicated by dotted lines. Exons are depicted as colored shapes, and introns are shown as lines/broken lines. Exon sizes are indicated by numbers in black below exons and intron sizes are indicated by numbers in gray above introns. Names, assigned numbers and the locations of primers used to amplify linear SMN transcripts are presented. ‘Δ’ indicates SMN transcripts with skipped exon(s). ( B, C ) Detection of novel exons 9, 10, 11 and 12 without (−) and with (+) RNase R treatment. Primers used are shown at the top of each gel. After purification of total RNA, we isolated intact poly(A) + RNA using magnetic beads and generated cDNA employing oligo(dT) 12–18 primer. The identities of bands marked on the left of each gel were determined by cloning and sequencing. Abbreviations: NS, non-specific.
    Figure Legend Snippet: Linear SMN transcripts showing inclusion of novel exons 9, 10, 11 and 12. ( A ) An overview of the 3′ portion of the SMN pre-mRNA. Splicing events are indicated by dotted lines. Exons are depicted as colored shapes, and introns are shown as lines/broken lines. Exon sizes are indicated by numbers in black below exons and intron sizes are indicated by numbers in gray above introns. Names, assigned numbers and the locations of primers used to amplify linear SMN transcripts are presented. ‘Δ’ indicates SMN transcripts with skipped exon(s). ( B, C ) Detection of novel exons 9, 10, 11 and 12 without (−) and with (+) RNase R treatment. Primers used are shown at the top of each gel. After purification of total RNA, we isolated intact poly(A) + RNA using magnetic beads and generated cDNA employing oligo(dT) 12–18 primer. The identities of bands marked on the left of each gel were determined by cloning and sequencing. Abbreviations: NS, non-specific.

    Techniques Used: Purification, Isolation, Magnetic Beads, Generated, Clone Assay, Sequencing

    SMN circRNA production in human tissues. ( A ) MESDA depicting the splicing pattern of linear SMN pre-mRNA in different human tissues. Band identities are given on the right side of the gel. ‘Δ’ indicates SMN transcripts with skipped exon(s). ‘+6B’ indicates a full-length SMN transcript that also contains Alu-derived exon 6B. Tissue type is given at the top of each lane. Abbreviations: Br, brain; Sc, spinal cord; Ht, heart; Sk, skeletal muscle; Sm, smooth muscle; Lv, liver; Kd, kidney; Ln, lung; Ut, uterus; Ts, testis. ( B ) Ethidium bromide stained gels of divergent RT-PCR using human-tissue-derived RNA. Labeling is the same as in Figure 2 and in (A).
    Figure Legend Snippet: SMN circRNA production in human tissues. ( A ) MESDA depicting the splicing pattern of linear SMN pre-mRNA in different human tissues. Band identities are given on the right side of the gel. ‘Δ’ indicates SMN transcripts with skipped exon(s). ‘+6B’ indicates a full-length SMN transcript that also contains Alu-derived exon 6B. Tissue type is given at the top of each lane. Abbreviations: Br, brain; Sc, spinal cord; Ht, heart; Sk, skeletal muscle; Sm, smooth muscle; Lv, liver; Kd, kidney; Ln, lung; Ut, uterus; Ts, testis. ( B ) Ethidium bromide stained gels of divergent RT-PCR using human-tissue-derived RNA. Labeling is the same as in Figure 2 and in (A).

    Techniques Used: Derivative Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Labeling

    8) Product Images from "Lysine-specific demethylase 1 promotes brown adipose tissue thermogenesis via repressing glucocorticoid activation"

    Article Title: Lysine-specific demethylase 1 promotes brown adipose tissue thermogenesis via repressing glucocorticoid activation

    Journal: Genes & Development

    doi: 10.1101/gad.285312.116

    Simultaneous deletion of HSD11B1 substantially rescues the metabolic phenotype of the AdLKO mice. ( A ) mRNA levels of HSD11B1 in the control and LSD1-deficient BAT. (AdLHKO) Heterozygous LSD1 knockout. Mean ± SEM. n = 5. (***) P
    Figure Legend Snippet: Simultaneous deletion of HSD11B1 substantially rescues the metabolic phenotype of the AdLKO mice. ( A ) mRNA levels of HSD11B1 in the control and LSD1-deficient BAT. (AdLHKO) Heterozygous LSD1 knockout. Mean ± SEM. n = 5. (***) P

    Techniques Used: Mouse Assay, Knock-Out

    9) Product Images from "Lysine-specific demethylase 1 promotes brown adipose tissue thermogenesis via repressing glucocorticoid activation"

    Article Title: Lysine-specific demethylase 1 promotes brown adipose tissue thermogenesis via repressing glucocorticoid activation

    Journal: Genes & Development

    doi: 10.1101/gad.285312.116

    Simultaneous deletion of HSD11B1 substantially rescues the metabolic phenotype of the AdLKO mice. ( A ) mRNA levels of HSD11B1 in the control and LSD1-deficient BAT. (AdLHKO) Heterozygous LSD1 knockout. Mean ± SEM. n = 5. (***) P
    Figure Legend Snippet: Simultaneous deletion of HSD11B1 substantially rescues the metabolic phenotype of the AdLKO mice. ( A ) mRNA levels of HSD11B1 in the control and LSD1-deficient BAT. (AdLHKO) Heterozygous LSD1 knockout. Mean ± SEM. n = 5. (***) P

    Techniques Used: Mouse Assay, Knock-Out

    10) Product Images from "Reciprocal Regulation of Aquaporin-2 Abundance and Degradation by Protein Kinase A and p38-MAP Kinase"

    Article Title: Reciprocal Regulation of Aquaporin-2 Abundance and Degradation by Protein Kinase A and p38-MAP Kinase

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2009111190

    FSK increases the AQP2 protein abundance in IMCD cells independent from transcriptional regulation. (A) IMCD cells were left untreated or treated with FSK (10 μM, 15 to 240 minutes). The AQP2 mRNA level was quantified by real-time PCR. Shown are
    Figure Legend Snippet: FSK increases the AQP2 protein abundance in IMCD cells independent from transcriptional regulation. (A) IMCD cells were left untreated or treated with FSK (10 μM, 15 to 240 minutes). The AQP2 mRNA level was quantified by real-time PCR. Shown are

    Techniques Used: Real-time Polymerase Chain Reaction

    11) Product Images from "m6A RNA methylation regulates the UV-induced DNA damage response"

    Article Title: m6A RNA methylation regulates the UV-induced DNA damage response

    Journal: Nature

    doi: 10.1038/nature21671

    FTO, but not ALKBH5, modulates m 6 A RNA levels and duration at damage sites a , WT or FTO KO U2OS cells were subjected to UVA laser microirradiation, incubated at 37°C for 4 min, then stained for FTO and γH2A.X as indicated. Middle panel shows a representative γH2A.X-positive cell with FTO colocalizing at the damage site (arrow). The percentage of cells showing colocalization of signals is indicated on the right. b , Western blot of FTO in WT or FTO KO U2OS cells. Actin is shown as a loading control. c , U2OS cells expressing control (Control KD) or one of two independent shRNAs targeting ALKBH5 (ALKBH5 KD1, KD2) were subject to qPCR analysis of ALKBH5 mRNA levels and normalized to GAPDH. d , WT or FTO KO U2OS cells were microirradiated by UVA laser, incubated at 37°C for 4 min, and costained for m 6 A and γH2A.X. Two different exposures for m 6 A are shown. The percentage of γH2A.X-positive cells showing colocalizing m 6 A signal is indicated on the right. e , WT, FTO KO, and two ALKBH5 KD U2OS cell lines were irradiated with 50 J UVC, incubated at 37°C for 2 min, and costained for m 6 A and γH2A.X. Relative m 6 A intensity is indicated on the right. All images are representative of at least 50 cells in triplicate. Scale bar, 20 μm.
    Figure Legend Snippet: FTO, but not ALKBH5, modulates m 6 A RNA levels and duration at damage sites a , WT or FTO KO U2OS cells were subjected to UVA laser microirradiation, incubated at 37°C for 4 min, then stained for FTO and γH2A.X as indicated. Middle panel shows a representative γH2A.X-positive cell with FTO colocalizing at the damage site (arrow). The percentage of cells showing colocalization of signals is indicated on the right. b , Western blot of FTO in WT or FTO KO U2OS cells. Actin is shown as a loading control. c , U2OS cells expressing control (Control KD) or one of two independent shRNAs targeting ALKBH5 (ALKBH5 KD1, KD2) were subject to qPCR analysis of ALKBH5 mRNA levels and normalized to GAPDH. d , WT or FTO KO U2OS cells were microirradiated by UVA laser, incubated at 37°C for 4 min, and costained for m 6 A and γH2A.X. Two different exposures for m 6 A are shown. The percentage of γH2A.X-positive cells showing colocalizing m 6 A signal is indicated on the right. e , WT, FTO KO, and two ALKBH5 KD U2OS cell lines were irradiated with 50 J UVC, incubated at 37°C for 2 min, and costained for m 6 A and γH2A.X. Relative m 6 A intensity is indicated on the right. All images are representative of at least 50 cells in triplicate. Scale bar, 20 μm.

    Techniques Used: Incubation, Staining, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Irradiation

    12) Product Images from "The Mediator co-activator complex regulates Ty1 retromobility by controlling the balance between Ty1i and Ty1 promoters"

    Article Title: The Mediator co-activator complex regulates Ty1 retromobility by controlling the balance between Ty1i and Ty1 promoters

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007232

    Ty1i situated downstream of a repressed Ty1 element is repressed by Mediator tail module triad subunits. (A) Schematic of the GAL1 :Ty1 Δ POL cassette in pGTy1 Δ POL showing forward primer locations for detection of Ty1 RNA (blue) versus Ty1i RNA (red). Note that because Ty1i is contained within Ty1, the Ty1i primer reports both Ty1i and Ty1 transcripts. Both amplifications utilized the same reverse primer (purple), that crosses the deletion junction and contains sequences unique to the pGTy1 Δ POL element. A reverse primer specific for ACT1 mRNA was also used to synthesize cDNA used as a template for the PCR amplification. No PCR product was detected using the Δ POL reverse primer when RNA from yeast lacking pGTy1 Δ POL was used as template. (B) Quantitation of the products of Reverse Transciption-PCR reactions using polyA + RNA isolated from wild-type yeast bearing plasmid pGTy1 Δ POL, and grown in glucose-containing broth, and from a genomic DNA control. Aliquots were taken from reactions at the indicated number of cycles and analyzed by agarose gel electrophoresis ( S6 Fig ). Levels of RT-PCR amplification products using Ty1, Ty1i, and ACT1 primers are indicated. (C) Northern blot probed with a single-stranded riboprobe that hybridizes to Ty1, Ty1i, Ty1 Δ POL and Ty1i Δ POL RNA. Note that the full-length Ty1 Δ POL transcript cannot be distinguished from the rRNA band. Image is representative of three biological replicates. (D) Quantification for Ty1, Ty1i, and Ty1i Δ POL RNA from three biological replicate northern analyses. Graph bar colors correspond to WT strain and the congenic spt3Δ strain as a negative control (grey bars), Mediator head subunit gene deletion strain (red bars), middle subunit gene deletion strains (blue bars) and tail subunit gene deletion strains (yellow bars). Error bars represent s.d.
    Figure Legend Snippet: Ty1i situated downstream of a repressed Ty1 element is repressed by Mediator tail module triad subunits. (A) Schematic of the GAL1 :Ty1 Δ POL cassette in pGTy1 Δ POL showing forward primer locations for detection of Ty1 RNA (blue) versus Ty1i RNA (red). Note that because Ty1i is contained within Ty1, the Ty1i primer reports both Ty1i and Ty1 transcripts. Both amplifications utilized the same reverse primer (purple), that crosses the deletion junction and contains sequences unique to the pGTy1 Δ POL element. A reverse primer specific for ACT1 mRNA was also used to synthesize cDNA used as a template for the PCR amplification. No PCR product was detected using the Δ POL reverse primer when RNA from yeast lacking pGTy1 Δ POL was used as template. (B) Quantitation of the products of Reverse Transciption-PCR reactions using polyA + RNA isolated from wild-type yeast bearing plasmid pGTy1 Δ POL, and grown in glucose-containing broth, and from a genomic DNA control. Aliquots were taken from reactions at the indicated number of cycles and analyzed by agarose gel electrophoresis ( S6 Fig ). Levels of RT-PCR amplification products using Ty1, Ty1i, and ACT1 primers are indicated. (C) Northern blot probed with a single-stranded riboprobe that hybridizes to Ty1, Ty1i, Ty1 Δ POL and Ty1i Δ POL RNA. Note that the full-length Ty1 Δ POL transcript cannot be distinguished from the rRNA band. Image is representative of three biological replicates. (D) Quantification for Ty1, Ty1i, and Ty1i Δ POL RNA from three biological replicate northern analyses. Graph bar colors correspond to WT strain and the congenic spt3Δ strain as a negative control (grey bars), Mediator head subunit gene deletion strain (red bars), middle subunit gene deletion strains (blue bars) and tail subunit gene deletion strains (yellow bars). Error bars represent s.d.

    Techniques Used: Polymerase Chain Reaction, Amplification, Quantitation Assay, Isolation, Plasmid Preparation, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Northern Blot, Negative Control

    13) Product Images from "Aphid feeding induces the relaxation of epigenetic control and the associated regulation of the defense response in Arabidopsis"

    Article Title: Aphid feeding induces the relaxation of epigenetic control and the associated regulation of the defense response in Arabidopsis

    Journal: bioRxiv

    doi: 10.1101/2020.01.24.916783

    Changes induced at TE expression by aphid feeding. A. Volcano plot depicting TE mRNA-seq expression in the comparison aphid vs control RNA samples. Dots colored in red indicated genes with significant upregulation. B. Volcano plot depicting gene expression in the comparison aphid vs control PARE samples. Dots colored in red indicated genes with significant upregulation. C. Global sRNAs profiles of control and stressed samples. D. TE-derived sRNA profiles of control and stressed samples. E. Relative accumulation of 21,22 and 24 nt sRNAs in control (C) and aphid infested samples (Mper) for TEs of different sizes. Values shown are relative to control, where accumulation values for each sRNA category were set to 1. F. Venn diagram showing the overlap between the TE populations identified from each of the different RNA sequencing analyses. G. Venn diagram depicting the overlap of TEs upregulated two fold in the PARE sequencing data and TEs losing or gaining two fold 24 nt sRNAs. G. Screenshot of a genome browser showing the accumulation of PARE reads and 24 nt sRNAs in control and aphid samples for two of the TEs upregulated in the PARE libraries and showing a decrease of 24 nt sRNA accumulation.
    Figure Legend Snippet: Changes induced at TE expression by aphid feeding. A. Volcano plot depicting TE mRNA-seq expression in the comparison aphid vs control RNA samples. Dots colored in red indicated genes with significant upregulation. B. Volcano plot depicting gene expression in the comparison aphid vs control PARE samples. Dots colored in red indicated genes with significant upregulation. C. Global sRNAs profiles of control and stressed samples. D. TE-derived sRNA profiles of control and stressed samples. E. Relative accumulation of 21,22 and 24 nt sRNAs in control (C) and aphid infested samples (Mper) for TEs of different sizes. Values shown are relative to control, where accumulation values for each sRNA category were set to 1. F. Venn diagram showing the overlap between the TE populations identified from each of the different RNA sequencing analyses. G. Venn diagram depicting the overlap of TEs upregulated two fold in the PARE sequencing data and TEs losing or gaining two fold 24 nt sRNAs. G. Screenshot of a genome browser showing the accumulation of PARE reads and 24 nt sRNAs in control and aphid samples for two of the TEs upregulated in the PARE libraries and showing a decrease of 24 nt sRNA accumulation.

    Techniques Used: Expressing, Derivative Assay, RNA Sequencing Assay, Sequencing

    14) Product Images from "Pro-angiogenic Ginsenosides F1 and Rh1 Inhibit Vascular Leakage by Modulating NR4A1"

    Article Title: Pro-angiogenic Ginsenosides F1 and Rh1 Inhibit Vascular Leakage by Modulating NR4A1

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-41115-2

    F1 and Rh1 regulate NR4A1 expression and transcriptional activity. ( A ) Effect of F1 and Rh1 on VEGF-induced NR4A1 mRNA expression in HUVECs. The mRNA expression of NR4A1 after treatment with F1 or Rh1 in VEGF-stimulated cells was analyzed by Real-time RT PCR. β-actin was used as a reference gene in the analysis. Data are presented as mean ± SD (n = 3). ( B ) Effect of F1 and Rh1 on VEGF-induced NR4A1 protein expression in HUVECs. The protein expressions of NR4A1 after treatment with F1 or Rh1 in VEGF-stimulated cells were assessed with by Western blot analysis. Data are presented as mean ± SD (n = 3). Full-length blots are presented in Supplementary Fig. 9 ( C ) Effect of F1 and Rh1 on NR4A1 transcriptional activity. NurRE-Luc reporter plasmid-transfected HEK293T cells were treated with the indicated condition for 1 hr (0.1 μM of Csn-B and 10 μM of F1 or Rh1). NR4A1 transcriptional activities were analyzed using the dual luciferase reporter gene assay. Data are presented as mean ± SD (n = 3). ( D ) Effect of F1 and Rh1 on Csn-B-induced NR4A1 mRNA expression in HUVECs. NR4A1 mRNA expressions after co-treatment of F1 or Rh1 with Csn-B were analyzed by Real-time RT PCR. β-actin was used as a reference gene in the analysis. Data are presented as mean ± SD (n = 3). ( E ) Effect of F1 and Rh1 on the VEGF-induced junctional disruption in HUVECs. The cells were treated with F1 or Rh1 (10 μM, 1 hr) after VEGF stimulation (2.5 nM, 1 hr). VE-cadherin (green) was stained with immunofluorescence and the nucleus (blue) was stained by DAPI. Scale bars, 10 μm. ( F ) Effect of F1 and Rh1 on the Csn-B-induced junctional disruption in HUVECs. The cells were co-treated with 1 μM of Csn-B and 10 μM of F1 or Rh1 for an hour. VE cadherin (green) was stained by immunofluorescence and the nucleus (blue) was stained by DAPI. Scale bars, 10 μm. ( G ) Schematic representation of the inhibitory effect of F1 and Rh1 on VEGF-induced vascular leakage in ECs. Statistical significance was calculated based on three independent experiments (* P
    Figure Legend Snippet: F1 and Rh1 regulate NR4A1 expression and transcriptional activity. ( A ) Effect of F1 and Rh1 on VEGF-induced NR4A1 mRNA expression in HUVECs. The mRNA expression of NR4A1 after treatment with F1 or Rh1 in VEGF-stimulated cells was analyzed by Real-time RT PCR. β-actin was used as a reference gene in the analysis. Data are presented as mean ± SD (n = 3). ( B ) Effect of F1 and Rh1 on VEGF-induced NR4A1 protein expression in HUVECs. The protein expressions of NR4A1 after treatment with F1 or Rh1 in VEGF-stimulated cells were assessed with by Western blot analysis. Data are presented as mean ± SD (n = 3). Full-length blots are presented in Supplementary Fig. 9 ( C ) Effect of F1 and Rh1 on NR4A1 transcriptional activity. NurRE-Luc reporter plasmid-transfected HEK293T cells were treated with the indicated condition for 1 hr (0.1 μM of Csn-B and 10 μM of F1 or Rh1). NR4A1 transcriptional activities were analyzed using the dual luciferase reporter gene assay. Data are presented as mean ± SD (n = 3). ( D ) Effect of F1 and Rh1 on Csn-B-induced NR4A1 mRNA expression in HUVECs. NR4A1 mRNA expressions after co-treatment of F1 or Rh1 with Csn-B were analyzed by Real-time RT PCR. β-actin was used as a reference gene in the analysis. Data are presented as mean ± SD (n = 3). ( E ) Effect of F1 and Rh1 on the VEGF-induced junctional disruption in HUVECs. The cells were treated with F1 or Rh1 (10 μM, 1 hr) after VEGF stimulation (2.5 nM, 1 hr). VE-cadherin (green) was stained with immunofluorescence and the nucleus (blue) was stained by DAPI. Scale bars, 10 μm. ( F ) Effect of F1 and Rh1 on the Csn-B-induced junctional disruption in HUVECs. The cells were co-treated with 1 μM of Csn-B and 10 μM of F1 or Rh1 for an hour. VE cadherin (green) was stained by immunofluorescence and the nucleus (blue) was stained by DAPI. Scale bars, 10 μm. ( G ) Schematic representation of the inhibitory effect of F1 and Rh1 on VEGF-induced vascular leakage in ECs. Statistical significance was calculated based on three independent experiments (* P

    Techniques Used: Expressing, Activity Assay, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Transfection, Luciferase, Reporter Gene Assay, Staining, Immunofluorescence

    15) Product Images from "High-Throughput Identification of MiR-145 Targets in Human Articular Chondrocytes"

    Article Title: High-Throughput Identification of MiR-145 Targets in Human Articular Chondrocytes

    Journal: Life

    doi: 10.3390/life10050058

    Analysis of the effect of miR-145 overexpression on mRNA levels of selected genes in freshly isolated HACs. ( A ) Sixteen randomly selected mRNAs from Di-IP and CD2DB1 from both Di-IP and DownT-RNA. ( B ) Three genes only present in DownT-RNA and COL2A1. Control (C) or miR-145 mimics were transfected in freshly isolated HACs and RNA was extracted 48 h after transfection. Values are presented as relative to that obtained in cells transfected with control mimics for each patient and normalized to RPLP0. Data represents average ± SEM from seven different experiments, each performed with different donor cells (19 yo. female; 8 yo. male; 28 yo. male; 45 yo. male; 16 yo. male; 13 yo. female; 31 yo. female). * p
    Figure Legend Snippet: Analysis of the effect of miR-145 overexpression on mRNA levels of selected genes in freshly isolated HACs. ( A ) Sixteen randomly selected mRNAs from Di-IP and CD2DB1 from both Di-IP and DownT-RNA. ( B ) Three genes only present in DownT-RNA and COL2A1. Control (C) or miR-145 mimics were transfected in freshly isolated HACs and RNA was extracted 48 h after transfection. Values are presented as relative to that obtained in cells transfected with control mimics for each patient and normalized to RPLP0. Data represents average ± SEM from seven different experiments, each performed with different donor cells (19 yo. female; 8 yo. male; 28 yo. male; 45 yo. male; 16 yo. male; 13 yo. female; 31 yo. female). * p

    Techniques Used: Over Expression, Isolation, Transfection

    16) Product Images from "Cardiomyocyte-specific Srsf3 deletion reveals a mitochondrial regulatory role"

    Article Title: Cardiomyocyte-specific Srsf3 deletion reveals a mitochondrial regulatory role

    Journal: bioRxiv

    doi: 10.1101/2020.07.03.186999

    RNA-seq reveals the oxidative phosphorylation cellular process as a target of Srsf3 in the heart. A) Volcano plot of the mRNA expression level (blue and red dots are significant, whereas grey dots are not) from Srsf3 Fl/Fl-βMHC-Cre mice compared to Srsf3 Fl/Fl mice (5-day-old mice; n=3 for each group). B) Venn diagram comparison of the heart transcriptomes from the cardiomyocyte-specific Srsf3 cKO at early age (5-day-old Srsf3 Fl/Fl-βMHC-Cre mice) and adulthood (5 days after the initial tamoxifen injection of approximately 3-month-old Srsf3 Fl/Fl-αMHC-MerCreMer mice, as recently reported ( Ortiz-Sanchez et al ., 2019 )). C, D) Metascape analysis performed on all mRNAs with a significant fold change in expression in Srsf3 Fl/Fl-βMHC-Cre mice. Each individual mRNA from the oxidative phosphorylation cluster identified is represented as a heat map. E) Scatterplot of all splicing events detected. Only the blue dots were retained (significant change in PSI higher than 10% compared to control mice). F) Number of each splicing event type ( i . e ., for the Srsf3 cKO mice compared to control mice). G) Venn diagram of all significant splicing events in a 5-day-old mouse heart compared to an adult mouse heart sample from E. Lara-Pezzi’s study ( Ortiz-Sanchez et al ., 2019 ). H) mRNA expression analysis by RT-qPCR of different mRNAs modulated by the absence of Srsf3 in cardiomyocytes implicated in oxidative phosphorylation. I) End-point PCR analysis of different spliced transcripts observed in the heart sample at an early age with the Srsf3 deletion model compared to the control.
    Figure Legend Snippet: RNA-seq reveals the oxidative phosphorylation cellular process as a target of Srsf3 in the heart. A) Volcano plot of the mRNA expression level (blue and red dots are significant, whereas grey dots are not) from Srsf3 Fl/Fl-βMHC-Cre mice compared to Srsf3 Fl/Fl mice (5-day-old mice; n=3 for each group). B) Venn diagram comparison of the heart transcriptomes from the cardiomyocyte-specific Srsf3 cKO at early age (5-day-old Srsf3 Fl/Fl-βMHC-Cre mice) and adulthood (5 days after the initial tamoxifen injection of approximately 3-month-old Srsf3 Fl/Fl-αMHC-MerCreMer mice, as recently reported ( Ortiz-Sanchez et al ., 2019 )). C, D) Metascape analysis performed on all mRNAs with a significant fold change in expression in Srsf3 Fl/Fl-βMHC-Cre mice. Each individual mRNA from the oxidative phosphorylation cluster identified is represented as a heat map. E) Scatterplot of all splicing events detected. Only the blue dots were retained (significant change in PSI higher than 10% compared to control mice). F) Number of each splicing event type ( i . e ., for the Srsf3 cKO mice compared to control mice). G) Venn diagram of all significant splicing events in a 5-day-old mouse heart compared to an adult mouse heart sample from E. Lara-Pezzi’s study ( Ortiz-Sanchez et al ., 2019 ). H) mRNA expression analysis by RT-qPCR of different mRNAs modulated by the absence of Srsf3 in cardiomyocytes implicated in oxidative phosphorylation. I) End-point PCR analysis of different spliced transcripts observed in the heart sample at an early age with the Srsf3 deletion model compared to the control.

    Techniques Used: RNA Sequencing Assay, Expressing, Mouse Assay, Injection, Quantitative RT-PCR, Polymerase Chain Reaction

    17) Product Images from "Aphid feeding induces the relaxation of epigenetic control and the associated regulation of the defense response in Arabidopsis"

    Article Title: Aphid feeding induces the relaxation of epigenetic control and the associated regulation of the defense response in Arabidopsis

    Journal: bioRxiv

    doi: 10.1101/2020.01.24.916783

    Changes induced at TE expression by aphid feeding. A. Volcano plot depicting TE mRNA-seq expression in the comparison aphid vs control RNA samples. Dots colored in red indicated genes with significant upregulation. B. Volcano plot depicting gene expression in the comparison aphid vs control PARE samples. Dots colored in red indicated genes with significant upregulation. C. Global sRNAs profiles of control and stressed samples. D. TE-derived sRNA profiles of control and stressed samples. E. Relative accumulation of 21,22 and 24 nt sRNAs in control (C) and aphid infested samples (Mper) for TEs of different sizes. Values shown are relative to control, where accumulation values for each sRNA category were set to 1. F. Venn diagram showing the overlap between the TE populations identified from each of the different RNA sequencing analyses. G. Venn diagram depicting the overlap of TEs upregulated two fold in the PARE sequencing data and TEs losing or gaining two fold 24 nt sRNAs. G. Screenshot of a genome browser showing the accumulation of PARE reads and 24 nt sRNAs in control and aphid samples for two of the TEs upregulated in the PARE libraries and showing a decrease of 24 nt sRNA accumulation.
    Figure Legend Snippet: Changes induced at TE expression by aphid feeding. A. Volcano plot depicting TE mRNA-seq expression in the comparison aphid vs control RNA samples. Dots colored in red indicated genes with significant upregulation. B. Volcano plot depicting gene expression in the comparison aphid vs control PARE samples. Dots colored in red indicated genes with significant upregulation. C. Global sRNAs profiles of control and stressed samples. D. TE-derived sRNA profiles of control and stressed samples. E. Relative accumulation of 21,22 and 24 nt sRNAs in control (C) and aphid infested samples (Mper) for TEs of different sizes. Values shown are relative to control, where accumulation values for each sRNA category were set to 1. F. Venn diagram showing the overlap between the TE populations identified from each of the different RNA sequencing analyses. G. Venn diagram depicting the overlap of TEs upregulated two fold in the PARE sequencing data and TEs losing or gaining two fold 24 nt sRNAs. G. Screenshot of a genome browser showing the accumulation of PARE reads and 24 nt sRNAs in control and aphid samples for two of the TEs upregulated in the PARE libraries and showing a decrease of 24 nt sRNA accumulation.

    Techniques Used: Expressing, Derivative Assay, RNA Sequencing Assay, Sequencing

    18) Product Images from "Ongoing resolution of duplicate gene functions shapes the diversification of a metabolic network"

    Article Title: Ongoing resolution of duplicate gene functions shapes the diversification of a metabolic network

    Journal: eLife

    doi: 10.7554/eLife.19027

    The regulation of PGM1 by galactose was inferred as the ancestral state. ( A ) mRNA levels of S. uvarum PGM1 and PGM2 during mid-log phase in SC + 2% galactose, SC + 5% glycerol, and SC + 2% glucose. Note that PGM2 , which encodes the major isoform of phosphoglucomutase, has long been known to be transcriptionally induced by ~three–four fold in galactose, but it lacks a Gal4 binding site and does not appear to be a direct target in S. cerevisiae ( Oh and Hopper, 1990 ; Rubio-Texeira, 2005 ). These features are broadly shared with S. uvarum PGM2 , which is transcriptionally induced two-fold by galactose relative to glycerol but is not transcriptionally up-regulated in the gal80∆ gal80b∆ mutant; nor does it have a consensus Gal4 site. ( B ) Conservation of putative Gal4 binding sites upstream of PGM1 in S. uvarum , S. eubayanus , S. arboricola , and two outgroup species. The orange dot indicates the inferred loss of direct regulation of PGM1 by Gal4 based on the presence or absence of putative Gal4 binding sites (CGGN 11 CCG). The distances upstream from the start codon are shown at the right. The putative sites are shown as red boxes at the relative position of the upstream sequences of PGM1 . Note that, in Kazachstania nagansihii , the upstream intergenic region of PGM1 ortholog is 1958 bp, an unusually long intergenic region for yeasts, and contains a divergent promoter that also drives expression of the PMU1 ortholog. DOI: http://dx.doi.org/10.7554/eLife.19027.015
    Figure Legend Snippet: The regulation of PGM1 by galactose was inferred as the ancestral state. ( A ) mRNA levels of S. uvarum PGM1 and PGM2 during mid-log phase in SC + 2% galactose, SC + 5% glycerol, and SC + 2% glucose. Note that PGM2 , which encodes the major isoform of phosphoglucomutase, has long been known to be transcriptionally induced by ~three–four fold in galactose, but it lacks a Gal4 binding site and does not appear to be a direct target in S. cerevisiae ( Oh and Hopper, 1990 ; Rubio-Texeira, 2005 ). These features are broadly shared with S. uvarum PGM2 , which is transcriptionally induced two-fold by galactose relative to glycerol but is not transcriptionally up-regulated in the gal80∆ gal80b∆ mutant; nor does it have a consensus Gal4 site. ( B ) Conservation of putative Gal4 binding sites upstream of PGM1 in S. uvarum , S. eubayanus , S. arboricola , and two outgroup species. The orange dot indicates the inferred loss of direct regulation of PGM1 by Gal4 based on the presence or absence of putative Gal4 binding sites (CGGN 11 CCG). The distances upstream from the start codon are shown at the right. The putative sites are shown as red boxes at the relative position of the upstream sequences of PGM1 . Note that, in Kazachstania nagansihii , the upstream intergenic region of PGM1 ortholog is 1958 bp, an unusually long intergenic region for yeasts, and contains a divergent promoter that also drives expression of the PMU1 ortholog. DOI: http://dx.doi.org/10.7554/eLife.19027.015

    Techniques Used: Binding Assay, Mutagenesis, Expressing

    19) Product Images from "The Mediator co-activator complex regulates Ty1 retromobility by controlling the balance between Ty1i and Ty1 promoters"

    Article Title: The Mediator co-activator complex regulates Ty1 retromobility by controlling the balance between Ty1i and Ty1 promoters

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007232

    Ty1i situated downstream of a repressed Ty1 element is repressed by Mediator tail module triad subunits. (A) Schematic of the GAL1 :Ty1 Δ POL cassette in pGTy1 Δ POL showing forward primer locations for detection of Ty1 RNA (blue) versus Ty1i RNA (red). Note that because Ty1i is contained within Ty1, the Ty1i primer reports both Ty1i and Ty1 transcripts. Both amplifications utilized the same reverse primer (purple), that crosses the deletion junction and contains sequences unique to the pGTy1 Δ POL element. A reverse primer specific for ACT1 mRNA was also used to synthesize cDNA used as a template for the PCR amplification. No PCR product was detected using the Δ POL reverse primer when RNA from yeast lacking pGTy1 Δ POL was used as template. (B) Quantitation of the products of Reverse Transciption-PCR reactions using polyA + RNA isolated from wild-type yeast bearing plasmid pGTy1 Δ ). Levels of RT-PCR amplification products using Ty1, Ty1i, and ACT1 primers are indicated. (C) Northern blot probed with a single-stranded riboprobe that hybridizes to Ty1, Ty1i, Ty1 Δ POL and Ty1i Δ POL RNA. Note that the full-length Ty1 Δ POL transcript cannot be distinguished from the rRNA band. Image is representative of three biological replicates. (D) Quantification for Ty1, Ty1i, and Ty1i Δ POL RNA from three biological replicate northern analyses. Graph bar colors correspond to WT strain and the congenic spt3Δ strain as a negative control (grey bars), Mediator head subunit gene deletion strain (red bars), middle subunit gene deletion strains (blue bars) and tail subunit gene deletion strains (yellow bars). Error bars represent s.d.
    Figure Legend Snippet: Ty1i situated downstream of a repressed Ty1 element is repressed by Mediator tail module triad subunits. (A) Schematic of the GAL1 :Ty1 Δ POL cassette in pGTy1 Δ POL showing forward primer locations for detection of Ty1 RNA (blue) versus Ty1i RNA (red). Note that because Ty1i is contained within Ty1, the Ty1i primer reports both Ty1i and Ty1 transcripts. Both amplifications utilized the same reverse primer (purple), that crosses the deletion junction and contains sequences unique to the pGTy1 Δ POL element. A reverse primer specific for ACT1 mRNA was also used to synthesize cDNA used as a template for the PCR amplification. No PCR product was detected using the Δ POL reverse primer when RNA from yeast lacking pGTy1 Δ POL was used as template. (B) Quantitation of the products of Reverse Transciption-PCR reactions using polyA + RNA isolated from wild-type yeast bearing plasmid pGTy1 Δ ). Levels of RT-PCR amplification products using Ty1, Ty1i, and ACT1 primers are indicated. (C) Northern blot probed with a single-stranded riboprobe that hybridizes to Ty1, Ty1i, Ty1 Δ POL and Ty1i Δ POL RNA. Note that the full-length Ty1 Δ POL transcript cannot be distinguished from the rRNA band. Image is representative of three biological replicates. (D) Quantification for Ty1, Ty1i, and Ty1i Δ POL RNA from three biological replicate northern analyses. Graph bar colors correspond to WT strain and the congenic spt3Δ strain as a negative control (grey bars), Mediator head subunit gene deletion strain (red bars), middle subunit gene deletion strains (blue bars) and tail subunit gene deletion strains (yellow bars). Error bars represent s.d.

    Techniques Used: Polymerase Chain Reaction, Amplification, Quantitation Assay, Isolation, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Northern Blot, Negative Control

    20) Product Images from "The Mediator co-activator complex regulates Ty1 retromobility by controlling the balance between Ty1i and Ty1 promoters"

    Article Title: The Mediator co-activator complex regulates Ty1 retromobility by controlling the balance between Ty1i and Ty1 promoters

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007232

    Ty1i situated downstream of a repressed Ty1 element is repressed by Mediator tail module triad subunits. (A) Schematic of the GAL1 :Ty1 Δ POL cassette in pGTy1 Δ POL showing forward primer locations for detection of Ty1 RNA (blue) versus Ty1i RNA (red). Note that because Ty1i is contained within Ty1, the Ty1i primer reports both Ty1i and Ty1 transcripts. Both amplifications utilized the same reverse primer (purple), that crosses the deletion junction and contains sequences unique to the pGTy1 Δ POL element. A reverse primer specific for ACT1 mRNA was also used to synthesize cDNA used as a template for the PCR amplification. No PCR product was detected using the Δ POL reverse primer when RNA from yeast lacking pGTy1 Δ POL was used as template. (B) Quantitation of the products of Reverse Transciption-PCR reactions using polyA + RNA isolated from wild-type yeast bearing plasmid pGTy1 Δ ). Levels of RT-PCR amplification products using Ty1, Ty1i, and ACT1 primers are indicated. (C) Northern blot probed with a single-stranded riboprobe that hybridizes to Ty1, Ty1i, Ty1 Δ POL and Ty1i Δ POL RNA. Note that the full-length Ty1 Δ POL transcript cannot be distinguished from the rRNA band. Image is representative of three biological replicates. (D) Quantification for Ty1, Ty1i, and Ty1i Δ POL RNA from three biological replicate northern analyses. Graph bar colors correspond to WT strain and the congenic spt3Δ strain as a negative control (grey bars), Mediator head subunit gene deletion strain (red bars), middle subunit gene deletion strains (blue bars) and tail subunit gene deletion strains (yellow bars). Error bars represent s.d.
    Figure Legend Snippet: Ty1i situated downstream of a repressed Ty1 element is repressed by Mediator tail module triad subunits. (A) Schematic of the GAL1 :Ty1 Δ POL cassette in pGTy1 Δ POL showing forward primer locations for detection of Ty1 RNA (blue) versus Ty1i RNA (red). Note that because Ty1i is contained within Ty1, the Ty1i primer reports both Ty1i and Ty1 transcripts. Both amplifications utilized the same reverse primer (purple), that crosses the deletion junction and contains sequences unique to the pGTy1 Δ POL element. A reverse primer specific for ACT1 mRNA was also used to synthesize cDNA used as a template for the PCR amplification. No PCR product was detected using the Δ POL reverse primer when RNA from yeast lacking pGTy1 Δ POL was used as template. (B) Quantitation of the products of Reverse Transciption-PCR reactions using polyA + RNA isolated from wild-type yeast bearing plasmid pGTy1 Δ ). Levels of RT-PCR amplification products using Ty1, Ty1i, and ACT1 primers are indicated. (C) Northern blot probed with a single-stranded riboprobe that hybridizes to Ty1, Ty1i, Ty1 Δ POL and Ty1i Δ POL RNA. Note that the full-length Ty1 Δ POL transcript cannot be distinguished from the rRNA band. Image is representative of three biological replicates. (D) Quantification for Ty1, Ty1i, and Ty1i Δ POL RNA from three biological replicate northern analyses. Graph bar colors correspond to WT strain and the congenic spt3Δ strain as a negative control (grey bars), Mediator head subunit gene deletion strain (red bars), middle subunit gene deletion strains (blue bars) and tail subunit gene deletion strains (yellow bars). Error bars represent s.d.

    Techniques Used: Polymerase Chain Reaction, Amplification, Quantitation Assay, Isolation, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Northern Blot, Negative Control

    Related Articles

    Sequencing:

    Article Title: High through-put identification of miR-145 targets in human articular chondrocytes
    Article Snippet: .. mRNA-seq library construction, sequencing and analysis RNA quantity and integrity were assessed using Quant-IT RiboGreen Kit (Invitrogen) and Agilent Tapestation 2200R6K. mRNA enrichment was achieved from 400-900ng of total RNA using a Magnetic mRNA Isolation Kit (NEB). .. Generation of double stranded cDNA and library construction were performed using NEBNext® mRNA Sample Prep Reagent Set 1 (NEB).

    Isolation:

    Article Title: Human Survival Motor Neuron genes generate a vast repertoire of circular RNAs
    Article Snippet: .. Polyadenylated mRNA enrichment After isolation of total RNA, intact poly(A)+ RNA was isolated using a Magnetic mRNA Isolation kit (NEB) following the manufacturer's instructions. ..

    Article Title: Lysine-specific demethylase 1 promotes brown adipose tissue thermogenesis via repressing glucocorticoid activation
    Article Snippet: .. Total RNA was isolated from BAT with the Trizol reagent (ThermoFisher, 15596026). mRNA was purified with the Magenic mRNA isolation kit (New England Biolabs, S1550S), and a library was prepared with the NEBNext Ultra RNA library preparation kit for Illumina (New England Biolabs, E7530S). ..

    Article Title: High through-put identification of miR-145 targets in human articular chondrocytes
    Article Snippet: .. mRNA-seq library construction, sequencing and analysis RNA quantity and integrity were assessed using Quant-IT RiboGreen Kit (Invitrogen) and Agilent Tapestation 2200R6K. mRNA enrichment was achieved from 400-900ng of total RNA using a Magnetic mRNA Isolation Kit (NEB). .. Generation of double stranded cDNA and library construction were performed using NEBNext® mRNA Sample Prep Reagent Set 1 (NEB).

    Article Title: Kinesin-2 and kinesin-9 have atypical functions during ciliogenesis in the male gametophyte of Marsilea vestita
    Article Snippet: .. RNA was isolated from the developing gametophytes using the Magnetic mRNA Isolation Kit from NEB (#S1550S, New England Biolabs) according to manufacturer instructions. .. Primers Primers for MvKinesin-2, MvKinesin-9A, MvKinesin-9B, and MvCentrin were generated using sequence data from the gametophyte transcriptome (Additional file ).

    Article Title: Transcript-specific characteristics determine the contribution of endo- and exonucleolytic decay pathways during the degradation of nonsense-mediated decay substrates
    Article Snippet: .. siRNA-treated cells were used for poly(A)+ RNA isolation, utilizing the magnetic mRNA Isolation Kit (New England Biolabs). ..

    Article Title: Rbfox2 is Critical for Maintaining Alternative Polyadenylation and Mitochondrial Health in Myoblasts
    Article Snippet: .. For nanopore sequencing, poly(A)+ RNA was enriched by using magnetic mRNA isolation kit (New England biolabs S1550S). ..

    Article Title: Aphid feeding induces the relaxation of epigenetic control and the associated regulation of the defense response in Arabidopsis
    Article Snippet: .. Each library was barcoded and sequenced in one lane of an Illumina HiSeq 2000. mRNAs for RNA libraries were isolated using the NEB Magnetic mRNA Isolation Kit (New England Biolabs). .. RNA libraries were produced using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs).

    Article Title: Ongoing resolution of duplicate gene functions shapes the diversification of a metabolic network
    Article Snippet: .. Poly-A enrichment was performed with the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490, in the experiment to examine S. uvarum GAL network membership) or with the NEB Magnetic mRNA Isolation kit (NEB #S1550, in the experiment sampled during the TGA phase and at mid-log phase in galactose). .. Illumina libraries were constructed using the NEB Ultra Directional RNA Library Prep Kit for Illumina (NEB #E7420) and sequenced using the Illumina HiSeq 2500 platform.

    Purification:

    Article Title: Lysine-specific demethylase 1 promotes brown adipose tissue thermogenesis via repressing glucocorticoid activation
    Article Snippet: .. Total RNA was isolated from BAT with the Trizol reagent (ThermoFisher, 15596026). mRNA was purified with the Magenic mRNA isolation kit (New England Biolabs, S1550S), and a library was prepared with the NEBNext Ultra RNA library preparation kit for Illumina (New England Biolabs, E7530S). ..

    Nanopore Sequencing:

    Article Title: Rbfox2 is Critical for Maintaining Alternative Polyadenylation and Mitochondrial Health in Myoblasts
    Article Snippet: .. For nanopore sequencing, poly(A)+ RNA was enriched by using magnetic mRNA isolation kit (New England biolabs S1550S). ..

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    New England Biolabs magnetic mrna isolation kit
    RBFOX2-regulated tandem-APA modulates 3’UTR length and affects <t>mRNA</t> levels. (A) The number of downregulated and upregulated genes in RBFOX2 KD H9c2 cells. (B) Gene ontology analysis of downregulated or upregulated genes in RBFOX2 depleted H9c2 cells. (C) The number of genes undergoing tandem-APA generating shorter or longer 3’UTRs in RBFOX2 KD H9c2 cells. (D) The number of genes that undergo both APA and mRNA level changes in RBFOX2 KD H9c2 cells. (E) Hdlbp 3 ’ UTR shortening mediated via tandem-APA in RBFOX2 depleted H9c2 cells identified by PAC-seq (top panel) or by <t>nanopore</t> sequencing (bottom panel).
    Magnetic Mrna Isolation Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RBFOX2-regulated tandem-APA modulates 3’UTR length and affects mRNA levels. (A) The number of downregulated and upregulated genes in RBFOX2 KD H9c2 cells. (B) Gene ontology analysis of downregulated or upregulated genes in RBFOX2 depleted H9c2 cells. (C) The number of genes undergoing tandem-APA generating shorter or longer 3’UTRs in RBFOX2 KD H9c2 cells. (D) The number of genes that undergo both APA and mRNA level changes in RBFOX2 KD H9c2 cells. (E) Hdlbp 3 ’ UTR shortening mediated via tandem-APA in RBFOX2 depleted H9c2 cells identified by PAC-seq (top panel) or by nanopore sequencing (bottom panel).

    Journal: bioRxiv

    Article Title: Rbfox2 is Critical for Maintaining Alternative Polyadenylation and Mitochondrial Health in Myoblasts

    doi: 10.1101/2020.05.13.093013

    Figure Lengend Snippet: RBFOX2-regulated tandem-APA modulates 3’UTR length and affects mRNA levels. (A) The number of downregulated and upregulated genes in RBFOX2 KD H9c2 cells. (B) Gene ontology analysis of downregulated or upregulated genes in RBFOX2 depleted H9c2 cells. (C) The number of genes undergoing tandem-APA generating shorter or longer 3’UTRs in RBFOX2 KD H9c2 cells. (D) The number of genes that undergo both APA and mRNA level changes in RBFOX2 KD H9c2 cells. (E) Hdlbp 3 ’ UTR shortening mediated via tandem-APA in RBFOX2 depleted H9c2 cells identified by PAC-seq (top panel) or by nanopore sequencing (bottom panel).

    Article Snippet: For nanopore sequencing, poly(A)+ RNA was enriched by using magnetic mRNA isolation kit (New England biolabs S1550S).

    Techniques: Nanopore Sequencing

    RBFOX2 binding sites are enriched upstream of poly(A) sites in 3’UTRs that undergo APA changes. (A) Metagene analysis of RBFOX2 CLIP-binding distribution with respect to PASs in all detectable PASs, or in upregulated/downregulated PASs identified in RBFOX2 KD H9c2 cells. (B) Genome browser image of Rab7a 3’UTR displays tandem-APA changes determined by PAC-seq (left). Tandem-APA change in Rab7a in control vs RBFOX2 KD H9c2 cells determined by RT-qPCR (right). The ratio of “distal or long” to “common” mRNA expression levels of Rab7a by RT-qPCR in control cells was normalized to 1. Statistical significance was calculated using unpaired t-test to compare two different groups in three independent experiments (n=3). Data represent means ± SD. *p=0.0466. (C) RBFOX2 CLIP-seq reads mapped to the Rab7a 3’UTR. (D) FLAG western blot of FLAG-RBFOX2 pulled down with in-vitro transcribed and biotinylated Rab7a transcripts in induced or uninduced HEK293 cells. No RNA was used as a negative control for non-specific protein binding in induced or uninduced cells.

    Journal: bioRxiv

    Article Title: Rbfox2 is Critical for Maintaining Alternative Polyadenylation and Mitochondrial Health in Myoblasts

    doi: 10.1101/2020.05.13.093013

    Figure Lengend Snippet: RBFOX2 binding sites are enriched upstream of poly(A) sites in 3’UTRs that undergo APA changes. (A) Metagene analysis of RBFOX2 CLIP-binding distribution with respect to PASs in all detectable PASs, or in upregulated/downregulated PASs identified in RBFOX2 KD H9c2 cells. (B) Genome browser image of Rab7a 3’UTR displays tandem-APA changes determined by PAC-seq (left). Tandem-APA change in Rab7a in control vs RBFOX2 KD H9c2 cells determined by RT-qPCR (right). The ratio of “distal or long” to “common” mRNA expression levels of Rab7a by RT-qPCR in control cells was normalized to 1. Statistical significance was calculated using unpaired t-test to compare two different groups in three independent experiments (n=3). Data represent means ± SD. *p=0.0466. (C) RBFOX2 CLIP-seq reads mapped to the Rab7a 3’UTR. (D) FLAG western blot of FLAG-RBFOX2 pulled down with in-vitro transcribed and biotinylated Rab7a transcripts in induced or uninduced HEK293 cells. No RNA was used as a negative control for non-specific protein binding in induced or uninduced cells.

    Article Snippet: For nanopore sequencing, poly(A)+ RNA was enriched by using magnetic mRNA isolation kit (New England biolabs S1550S).

    Techniques: Binding Assay, Cross-linking Immunoprecipitation, Quantitative RT-PCR, Expressing, Western Blot, In Vitro, Negative Control, Protein Binding

    The regulation of PGM1 by galactose was inferred as the ancestral state. ( A ) mRNA levels of S. uvarum PGM1 and PGM2 during mid-log phase in SC + 2% galactose, SC + 5% glycerol, and SC + 2% glucose. Note that PGM2 , which encodes the major isoform of phosphoglucomutase, has long been known to be transcriptionally induced by ~three–four fold in galactose, but it lacks a Gal4 binding site and does not appear to be a direct target in S. cerevisiae ( Oh and Hopper, 1990 ; Rubio-Texeira, 2005 ). These features are broadly shared with S. uvarum PGM2 , which is transcriptionally induced two-fold by galactose relative to glycerol but is not transcriptionally up-regulated in the gal80∆ gal80b∆ mutant; nor does it have a consensus Gal4 site. ( B ) Conservation of putative Gal4 binding sites upstream of PGM1 in S. uvarum , S. eubayanus , S. arboricola , and two outgroup species. The orange dot indicates the inferred loss of direct regulation of PGM1 by Gal4 based on the presence or absence of putative Gal4 binding sites (CGGN 11 CCG). The distances upstream from the start codon are shown at the right. The putative sites are shown as red boxes at the relative position of the upstream sequences of PGM1 . Note that, in Kazachstania nagansihii , the upstream intergenic region of PGM1 ortholog is 1958 bp, an unusually long intergenic region for yeasts, and contains a divergent promoter that also drives expression of the PMU1 ortholog. DOI: http://dx.doi.org/10.7554/eLife.19027.015

    Journal: eLife

    Article Title: Ongoing resolution of duplicate gene functions shapes the diversification of a metabolic network

    doi: 10.7554/eLife.19027

    Figure Lengend Snippet: The regulation of PGM1 by galactose was inferred as the ancestral state. ( A ) mRNA levels of S. uvarum PGM1 and PGM2 during mid-log phase in SC + 2% galactose, SC + 5% glycerol, and SC + 2% glucose. Note that PGM2 , which encodes the major isoform of phosphoglucomutase, has long been known to be transcriptionally induced by ~three–four fold in galactose, but it lacks a Gal4 binding site and does not appear to be a direct target in S. cerevisiae ( Oh and Hopper, 1990 ; Rubio-Texeira, 2005 ). These features are broadly shared with S. uvarum PGM2 , which is transcriptionally induced two-fold by galactose relative to glycerol but is not transcriptionally up-regulated in the gal80∆ gal80b∆ mutant; nor does it have a consensus Gal4 site. ( B ) Conservation of putative Gal4 binding sites upstream of PGM1 in S. uvarum , S. eubayanus , S. arboricola , and two outgroup species. The orange dot indicates the inferred loss of direct regulation of PGM1 by Gal4 based on the presence or absence of putative Gal4 binding sites (CGGN 11 CCG). The distances upstream from the start codon are shown at the right. The putative sites are shown as red boxes at the relative position of the upstream sequences of PGM1 . Note that, in Kazachstania nagansihii , the upstream intergenic region of PGM1 ortholog is 1958 bp, an unusually long intergenic region for yeasts, and contains a divergent promoter that also drives expression of the PMU1 ortholog. DOI: http://dx.doi.org/10.7554/eLife.19027.015

    Article Snippet: Poly-A enrichment was performed with the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490, in the experiment to examine S. uvarum GAL network membership) or with the NEB Magnetic mRNA Isolation kit (NEB #S1550, in the experiment sampled during the TGA phase and at mid-log phase in galactose).

    Techniques: Binding Assay, Mutagenesis, Expressing

    Analysis of the effect of miR-145 over-expression on mRNA levels of selected genes in freshly isolated HACs. A) 16 randomly selected mRNAs from CD-IP and CD2DB1 from both CD-IP and DT-RNA. B) 3 genes only present in DT-RNA and COL2A1. Control (C) or miR-145 mimics were transfected in freshly isolated HACs and RNA was extracted 48h after transfection. Values are presented as relative to that obtained in cells transfected with control mimics for each patient and normalized to RPLP0. Data represents average ± S.E.M. from 7 different experiments, each performed with different donor cells. * P

    Journal: bioRxiv

    Article Title: High through-put identification of miR-145 targets in human articular chondrocytes

    doi: 10.1101/2020.02.03.931683

    Figure Lengend Snippet: Analysis of the effect of miR-145 over-expression on mRNA levels of selected genes in freshly isolated HACs. A) 16 randomly selected mRNAs from CD-IP and CD2DB1 from both CD-IP and DT-RNA. B) 3 genes only present in DT-RNA and COL2A1. Control (C) or miR-145 mimics were transfected in freshly isolated HACs and RNA was extracted 48h after transfection. Values are presented as relative to that obtained in cells transfected with control mimics for each patient and normalized to RPLP0. Data represents average ± S.E.M. from 7 different experiments, each performed with different donor cells. * P

    Article Snippet: mRNA-seq library construction, sequencing and analysis RNA quantity and integrity were assessed using Quant-IT RiboGreen Kit (Invitrogen) and Agilent Tapestation 2200R6K. mRNA enrichment was achieved from 400-900ng of total RNA using a Magnetic mRNA Isolation Kit (NEB).

    Techniques: Over Expression, Isolation, Transfection

    Changes induced at TE expression by aphid feeding. A. Volcano plot depicting TE mRNA-seq expression in the comparison aphid vs control RNA samples. Dots colored in red indicated genes with significant upregulation. B. Volcano plot depicting gene expression in the comparison aphid vs control PARE samples. Dots colored in red indicated genes with significant upregulation. C. Global sRNAs profiles of control and stressed samples. D. TE-derived sRNA profiles of control and stressed samples. E. Relative accumulation of 21,22 and 24 nt sRNAs in control (C) and aphid infested samples (Mper) for TEs of different sizes. Values shown are relative to control, where accumulation values for each sRNA category were set to 1. F. Venn diagram showing the overlap between the TE populations identified from each of the different RNA sequencing analyses. G. Venn diagram depicting the overlap of TEs upregulated two fold in the PARE sequencing data and TEs losing or gaining two fold 24 nt sRNAs. G. Screenshot of a genome browser showing the accumulation of PARE reads and 24 nt sRNAs in control and aphid samples for two of the TEs upregulated in the PARE libraries and showing a decrease of 24 nt sRNA accumulation.

    Journal: bioRxiv

    Article Title: Aphid feeding induces the relaxation of epigenetic control and the associated regulation of the defense response in Arabidopsis

    doi: 10.1101/2020.01.24.916783

    Figure Lengend Snippet: Changes induced at TE expression by aphid feeding. A. Volcano plot depicting TE mRNA-seq expression in the comparison aphid vs control RNA samples. Dots colored in red indicated genes with significant upregulation. B. Volcano plot depicting gene expression in the comparison aphid vs control PARE samples. Dots colored in red indicated genes with significant upregulation. C. Global sRNAs profiles of control and stressed samples. D. TE-derived sRNA profiles of control and stressed samples. E. Relative accumulation of 21,22 and 24 nt sRNAs in control (C) and aphid infested samples (Mper) for TEs of different sizes. Values shown are relative to control, where accumulation values for each sRNA category were set to 1. F. Venn diagram showing the overlap between the TE populations identified from each of the different RNA sequencing analyses. G. Venn diagram depicting the overlap of TEs upregulated two fold in the PARE sequencing data and TEs losing or gaining two fold 24 nt sRNAs. G. Screenshot of a genome browser showing the accumulation of PARE reads and 24 nt sRNAs in control and aphid samples for two of the TEs upregulated in the PARE libraries and showing a decrease of 24 nt sRNA accumulation.

    Article Snippet: Each library was barcoded and sequenced in one lane of an Illumina HiSeq 2000. mRNAs for RNA libraries were isolated using the NEB Magnetic mRNA Isolation Kit (New England Biolabs).

    Techniques: Expressing, Derivative Assay, RNA Sequencing Assay, Sequencing