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    Structured Review

    Thermo Fisher gapdh rna
    The 5-HT1A mRNA 3′-UTR is alternatively spliced in human cells to give rise to several mRNA isoforms. A , Schematic representation of the human 5-HT1A 3′-UTR construct used for 3′-RACE experiments, and of primers A–D used to quantify the resulting mRNA variants upon its transfection in human cells (unspliced, LS, and SS). Dashed lines represent the intron removed following splicing of the 3′-UTR. The relative location of the poly-adenylation site (polyA signal), miR135 site, and splice donor (gt dinucleotides) and acceptor (cag) sites are indicated. B , Top, RT-PCR analysis of the 5-HT1A 3′-UTR splice variants in HEK293 cells. <t>RNA</t> from HEK293 cells transfected with luciferase vector pGL3P or pGL3P 5-HT1A-3′-UTR was assessed by RT-PCR for the expression of unspliced (U, top, primers A/B) and spliced 3′UTR isoforms, short (SS) and long (LS) (bottom, primers C/D). M, DNA size markers; 1, negative control; 2, pGL3P; 3, pGL3P 5-HT1A-3′-UTR. Bottom, Real-time PCR quantification of unspliced (100%) and spliced HTR1A mRNA in HEK293 cells transfected with pGL3P 5-HT1A-3′-UTR using probes specific for unspliced, total spliced (S), or LS isoform 5-HT1A mRNA. C , Splicing of human 5-HT1A minigene in stably transfected SKN-SH cells. Top, RT-PCR: cDNA from uninfected cells (Lane 1) or cells infected with the 5-HT1A minigene (Lane 2) were analyzed for the unspliced 3′-UTR (U) or spliced isoforms (SS, LS) using primers shown in A . Below, real-time PCR: relative mRNA levels were normalized to <t>GAPDH</t> using the ΔΔCt method. Data are presented as mean ± SEM of the %unspliced from three independent experiments. D , Undetectable HTR1A RNA splicing in mouse brain. Full-length cDNA from human SKN-SH cells expressing the human 5-HT1A minigene or mouse PFC and hippocampus (HP) tissue was analyzed using two human primers C and D in A or the corresponding mouse 3′-UTR primers (521–2444 bp downstream of the stop codon). Spliced 5-HT1A mRNA isoforms (S) were only detected for the human transcript, while the unspliced form (U) was present in both. M, DNA size markers; 1, negative control. E ).
    Gapdh Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gapdh rna/product/Thermo Fisher
    Average 99 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    gapdh rna - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "A Novel Alternative Splicing Mechanism That Enhances Human 5-HT1A Receptor RNA Stability Is Altered in Major Depression"

    Article Title: A Novel Alternative Splicing Mechanism That Enhances Human 5-HT1A Receptor RNA Stability Is Altered in Major Depression

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0902-18.2018

    The 5-HT1A mRNA 3′-UTR is alternatively spliced in human cells to give rise to several mRNA isoforms. A , Schematic representation of the human 5-HT1A 3′-UTR construct used for 3′-RACE experiments, and of primers A–D used to quantify the resulting mRNA variants upon its transfection in human cells (unspliced, LS, and SS). Dashed lines represent the intron removed following splicing of the 3′-UTR. The relative location of the poly-adenylation site (polyA signal), miR135 site, and splice donor (gt dinucleotides) and acceptor (cag) sites are indicated. B , Top, RT-PCR analysis of the 5-HT1A 3′-UTR splice variants in HEK293 cells. RNA from HEK293 cells transfected with luciferase vector pGL3P or pGL3P 5-HT1A-3′-UTR was assessed by RT-PCR for the expression of unspliced (U, top, primers A/B) and spliced 3′UTR isoforms, short (SS) and long (LS) (bottom, primers C/D). M, DNA size markers; 1, negative control; 2, pGL3P; 3, pGL3P 5-HT1A-3′-UTR. Bottom, Real-time PCR quantification of unspliced (100%) and spliced HTR1A mRNA in HEK293 cells transfected with pGL3P 5-HT1A-3′-UTR using probes specific for unspliced, total spliced (S), or LS isoform 5-HT1A mRNA. C , Splicing of human 5-HT1A minigene in stably transfected SKN-SH cells. Top, RT-PCR: cDNA from uninfected cells (Lane 1) or cells infected with the 5-HT1A minigene (Lane 2) were analyzed for the unspliced 3′-UTR (U) or spliced isoforms (SS, LS) using primers shown in A . Below, real-time PCR: relative mRNA levels were normalized to GAPDH using the ΔΔCt method. Data are presented as mean ± SEM of the %unspliced from three independent experiments. D , Undetectable HTR1A RNA splicing in mouse brain. Full-length cDNA from human SKN-SH cells expressing the human 5-HT1A minigene or mouse PFC and hippocampus (HP) tissue was analyzed using two human primers C and D in A or the corresponding mouse 3′-UTR primers (521–2444 bp downstream of the stop codon). Spliced 5-HT1A mRNA isoforms (S) were only detected for the human transcript, while the unspliced form (U) was present in both. M, DNA size markers; 1, negative control. E ).
    Figure Legend Snippet: The 5-HT1A mRNA 3′-UTR is alternatively spliced in human cells to give rise to several mRNA isoforms. A , Schematic representation of the human 5-HT1A 3′-UTR construct used for 3′-RACE experiments, and of primers A–D used to quantify the resulting mRNA variants upon its transfection in human cells (unspliced, LS, and SS). Dashed lines represent the intron removed following splicing of the 3′-UTR. The relative location of the poly-adenylation site (polyA signal), miR135 site, and splice donor (gt dinucleotides) and acceptor (cag) sites are indicated. B , Top, RT-PCR analysis of the 5-HT1A 3′-UTR splice variants in HEK293 cells. RNA from HEK293 cells transfected with luciferase vector pGL3P or pGL3P 5-HT1A-3′-UTR was assessed by RT-PCR for the expression of unspliced (U, top, primers A/B) and spliced 3′UTR isoforms, short (SS) and long (LS) (bottom, primers C/D). M, DNA size markers; 1, negative control; 2, pGL3P; 3, pGL3P 5-HT1A-3′-UTR. Bottom, Real-time PCR quantification of unspliced (100%) and spliced HTR1A mRNA in HEK293 cells transfected with pGL3P 5-HT1A-3′-UTR using probes specific for unspliced, total spliced (S), or LS isoform 5-HT1A mRNA. C , Splicing of human 5-HT1A minigene in stably transfected SKN-SH cells. Top, RT-PCR: cDNA from uninfected cells (Lane 1) or cells infected with the 5-HT1A minigene (Lane 2) were analyzed for the unspliced 3′-UTR (U) or spliced isoforms (SS, LS) using primers shown in A . Below, real-time PCR: relative mRNA levels were normalized to GAPDH using the ΔΔCt method. Data are presented as mean ± SEM of the %unspliced from three independent experiments. D , Undetectable HTR1A RNA splicing in mouse brain. Full-length cDNA from human SKN-SH cells expressing the human 5-HT1A minigene or mouse PFC and hippocampus (HP) tissue was analyzed using two human primers C and D in A or the corresponding mouse 3′-UTR primers (521–2444 bp downstream of the stop codon). Spliced 5-HT1A mRNA isoforms (S) were only detected for the human transcript, while the unspliced form (U) was present in both. M, DNA size markers; 1, negative control. E ).

    Techniques Used: Construct, Transfection, Reverse Transcription Polymerase Chain Reaction, Luciferase, Plasmid Preparation, Expressing, Negative Control, Real-time Polymerase Chain Reaction, Stable Transfection, Infection

    Opposite regulation of 5-HT1A mRNA splicing and protein levels by splicing factors PTBP1 and nSR100. A–C , Effect of PTBP1 and PTBP2 knockdown on 5-HT1A RNA splicing. SKN-SH cells were infected with lentiviruses expressing shRNAs against PTBP1, PTBP2, or a nontargeting control (Ctrl) shRNA at multiplicity of infection of 10 and selected using puromycin. Puromycin-resistant cells were infected with the WT 5-HT1A minigene at multiplicity of infection of 20 for 72 h and RNA and protein were extracted and analyzed. Relative expression of PTBP1, PTBP2, and Flag-5-HT1A proteins was determined by Western blot ( A ), while the expression of the various 5-HT1A mRNA variants was assessed by RT-PCR ( B ) and quantified by real-time PCR ( C ) using probes specific for the spliced mRNA isoforms (total spliced (Tot spliced) or LS isoform), unspliced mRNA isoforms, or total 5-HT1A mRNA (Total 1A). Relative mRNA levels were normalized to GAPDH using the ΔΔCt method. D–F , Effect of Nova1, Nova2, and nSR100 overexpression on 5-HT1A RNA splicing. SKN-SH cells were infected with the WT 5-HT1A minigene at multiplicity of infection of 20 for 72 h, and then transfected with vector (pTRIEX) or plasmids encoding the S-tagged Nova1, Nova2, and nSR100 neuronal splice regulators. The relative expression of the S-tagged proteins (S-tag), Flag-5-HT1A receptor and alpha-tubulin was determined by Western blot ( D ), while the expression of the various 5-HT1A mRNA isoforms was assessed by RT-PCR ( E ) and quantified by real-time PCR ( F ). Data presented as the mean and SEM of 3–4 independent experiments ( C , F ). * p
    Figure Legend Snippet: Opposite regulation of 5-HT1A mRNA splicing and protein levels by splicing factors PTBP1 and nSR100. A–C , Effect of PTBP1 and PTBP2 knockdown on 5-HT1A RNA splicing. SKN-SH cells were infected with lentiviruses expressing shRNAs against PTBP1, PTBP2, or a nontargeting control (Ctrl) shRNA at multiplicity of infection of 10 and selected using puromycin. Puromycin-resistant cells were infected with the WT 5-HT1A minigene at multiplicity of infection of 20 for 72 h and RNA and protein were extracted and analyzed. Relative expression of PTBP1, PTBP2, and Flag-5-HT1A proteins was determined by Western blot ( A ), while the expression of the various 5-HT1A mRNA variants was assessed by RT-PCR ( B ) and quantified by real-time PCR ( C ) using probes specific for the spliced mRNA isoforms (total spliced (Tot spliced) or LS isoform), unspliced mRNA isoforms, or total 5-HT1A mRNA (Total 1A). Relative mRNA levels were normalized to GAPDH using the ΔΔCt method. D–F , Effect of Nova1, Nova2, and nSR100 overexpression on 5-HT1A RNA splicing. SKN-SH cells were infected with the WT 5-HT1A minigene at multiplicity of infection of 20 for 72 h, and then transfected with vector (pTRIEX) or plasmids encoding the S-tagged Nova1, Nova2, and nSR100 neuronal splice regulators. The relative expression of the S-tagged proteins (S-tag), Flag-5-HT1A receptor and alpha-tubulin was determined by Western blot ( D ), while the expression of the various 5-HT1A mRNA isoforms was assessed by RT-PCR ( E ) and quantified by real-time PCR ( F ). Data presented as the mean and SEM of 3–4 independent experiments ( C , F ). * p

    Techniques Used: Infection, Expressing, shRNA, Western Blot, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Over Expression, Transfection, Plasmid Preparation

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    Real-time Polymerase Chain Reaction:

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    Reverse Transcription Polymerase Chain Reaction:

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    Expressing:

    Article Title: Flutamide induces alterations in the cell-cell junction ultrastructure and reduces the expression of Cx43 at the blood-testis barrier with no disturbance in the rat seminiferous tubule morphology
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    Polymerase Chain Reaction:

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    Article Snippet: .. Real-time PCR was performed using TaqMan Universal PCR mastermix and preformulated primers for PDI and the endogenous controls β-2-microglobulin and GAPDH (Applied Biosystems, Foster City, CA, USA). .. The cycle threshold ( Ct ) values for PDI expression were normalized against two housekeeping genes, β-2-microglobulin and GAPDH.

    TaqMan Assay:

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    Control Assay:

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    Thermo Fisher silencer gapdh sirna
    The effects of p130Cas knockdown on E-cadherin and P-Src A. HCC1937 cells were transfected with a mock control or <t>siRNA</t> directed against <t>GAPDH</t> or p130Cas and whole cell lysates evaluated by immunoblotting for p130Cas, GAPDH, and E-cadherin 72 hours later. B. Mock treated as well as p130Cas and GAPDH siRNA-treated cells (p130Casi and GAPDHi respectively) were probed for p130Cas (green), E-cadherin (red), and Hoescht (blue) by indirect immunofluorescence. C. Mock treated as well as p130Cas and GAPDH siRNA-treated cells were probed for p130Cas (green), P-Y419 Src (red), and Hoescht (blue) by indirect immunofluorescence. Images are composed of deconvolved stacks.
    Silencer Gapdh Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/silencer gapdh sirna/product/Thermo Fisher
    Average 94 stars, based on 37 article reviews
    Price from $9.99 to $1999.99
    silencer gapdh sirna - by Bioz Stars, 2020-09
    94/100 stars
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    89
    Thermo Fisher gapdh rna
    The 5-HT1A mRNA 3′-UTR is alternatively spliced in human cells to give rise to several mRNA isoforms. A , Schematic representation of the human 5-HT1A 3′-UTR construct used for 3′-RACE experiments, and of primers A–D used to quantify the resulting mRNA variants upon its transfection in human cells (unspliced, LS, and SS). Dashed lines represent the intron removed following splicing of the 3′-UTR. The relative location of the poly-adenylation site (polyA signal), miR135 site, and splice donor (gt dinucleotides) and acceptor (cag) sites are indicated. B , Top, RT-PCR analysis of the 5-HT1A 3′-UTR splice variants in HEK293 cells. <t>RNA</t> from HEK293 cells transfected with luciferase vector pGL3P or pGL3P 5-HT1A-3′-UTR was assessed by RT-PCR for the expression of unspliced (U, top, primers A/B) and spliced 3′UTR isoforms, short (SS) and long (LS) (bottom, primers C/D). M, DNA size markers; 1, negative control; 2, pGL3P; 3, pGL3P 5-HT1A-3′-UTR. Bottom, Real-time PCR quantification of unspliced (100%) and spliced HTR1A mRNA in HEK293 cells transfected with pGL3P 5-HT1A-3′-UTR using probes specific for unspliced, total spliced (S), or LS isoform 5-HT1A mRNA. C , Splicing of human 5-HT1A minigene in stably transfected SKN-SH cells. Top, RT-PCR: cDNA from uninfected cells (Lane 1) or cells infected with the 5-HT1A minigene (Lane 2) were analyzed for the unspliced 3′-UTR (U) or spliced isoforms (SS, LS) using primers shown in A . Below, real-time PCR: relative mRNA levels were normalized to <t>GAPDH</t> using the ΔΔCt method. Data are presented as mean ± SEM of the %unspliced from three independent experiments. D , Undetectable HTR1A RNA splicing in mouse brain. Full-length cDNA from human SKN-SH cells expressing the human 5-HT1A minigene or mouse PFC and hippocampus (HP) tissue was analyzed using two human primers C and D in A or the corresponding mouse 3′-UTR primers (521–2444 bp downstream of the stop codon). Spliced 5-HT1A mRNA isoforms (S) were only detected for the human transcript, while the unspliced form (U) was present in both. M, DNA size markers; 1, negative control. E ).
    Gapdh Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gapdh rna/product/Thermo Fisher
    Average 89 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    gapdh rna - by Bioz Stars, 2020-09
    89/100 stars
      Buy from Supplier

    99
    Thermo Fisher rna interference rnai
    Observation of actin cytoskeleton and nucleus of MC3T3-E1 cells cultured on different material surfaces after 48h <t>RNA</t> interference treatment. Notes: ( A ) Plate; ( B ) CpTi; ( C ) 3Y-TZP; ( D ) NanoZr. MC3T3-E1 cells were transfected with a Negative Control siRNA or with siRNA that targets Syndecan-1 (#1, #2), Syndecan-2 (#1, #2), Syndecan-3 (#1, #2) and Syndecan-4 (#1, #2). Two different sequences of each Syndecan were used respectively. Magnification: 400×. The graph shows the MC3T3-E1 cell spread area ratio between si-Sdc group and si-NegCont group.*A statistical significant compared to the Negative Control group ( P
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    Image Search Results


    The effects of p130Cas knockdown on E-cadherin and P-Src A. HCC1937 cells were transfected with a mock control or siRNA directed against GAPDH or p130Cas and whole cell lysates evaluated by immunoblotting for p130Cas, GAPDH, and E-cadherin 72 hours later. B. Mock treated as well as p130Cas and GAPDH siRNA-treated cells (p130Casi and GAPDHi respectively) were probed for p130Cas (green), E-cadherin (red), and Hoescht (blue) by indirect immunofluorescence. C. Mock treated as well as p130Cas and GAPDH siRNA-treated cells were probed for p130Cas (green), P-Y419 Src (red), and Hoescht (blue) by indirect immunofluorescence. Images are composed of deconvolved stacks.

    Journal: Oncotarget

    Article Title: Regulation of E-cadherin localization by microtubule targeting agents: rapid promotion of cortical E-cadherin through p130Cas/Src inhibition by eribulin

    doi: 10.18632/oncotarget.23798

    Figure Lengend Snippet: The effects of p130Cas knockdown on E-cadherin and P-Src A. HCC1937 cells were transfected with a mock control or siRNA directed against GAPDH or p130Cas and whole cell lysates evaluated by immunoblotting for p130Cas, GAPDH, and E-cadherin 72 hours later. B. Mock treated as well as p130Cas and GAPDH siRNA-treated cells (p130Casi and GAPDHi respectively) were probed for p130Cas (green), E-cadherin (red), and Hoescht (blue) by indirect immunofluorescence. C. Mock treated as well as p130Cas and GAPDH siRNA-treated cells were probed for p130Cas (green), P-Y419 Src (red), and Hoescht (blue) by indirect immunofluorescence. Images are composed of deconvolved stacks.

    Article Snippet: siRNA transfection HCC1937 cells were transfected using Lipofectamine RNAiMAX as recommended by the manufacturer (ThermoFisher). siRNAs against GAPDH (AM4605 ThermoFischer) and p130Cas (SASI_Hs01_00184840 and SASI_Hs02_00345830 (Sigma Aldrich) were used.

    Techniques: Transfection, Immunofluorescence

    The 5-HT1A mRNA 3′-UTR is alternatively spliced in human cells to give rise to several mRNA isoforms. A , Schematic representation of the human 5-HT1A 3′-UTR construct used for 3′-RACE experiments, and of primers A–D used to quantify the resulting mRNA variants upon its transfection in human cells (unspliced, LS, and SS). Dashed lines represent the intron removed following splicing of the 3′-UTR. The relative location of the poly-adenylation site (polyA signal), miR135 site, and splice donor (gt dinucleotides) and acceptor (cag) sites are indicated. B , Top, RT-PCR analysis of the 5-HT1A 3′-UTR splice variants in HEK293 cells. RNA from HEK293 cells transfected with luciferase vector pGL3P or pGL3P 5-HT1A-3′-UTR was assessed by RT-PCR for the expression of unspliced (U, top, primers A/B) and spliced 3′UTR isoforms, short (SS) and long (LS) (bottom, primers C/D). M, DNA size markers; 1, negative control; 2, pGL3P; 3, pGL3P 5-HT1A-3′-UTR. Bottom, Real-time PCR quantification of unspliced (100%) and spliced HTR1A mRNA in HEK293 cells transfected with pGL3P 5-HT1A-3′-UTR using probes specific for unspliced, total spliced (S), or LS isoform 5-HT1A mRNA. C , Splicing of human 5-HT1A minigene in stably transfected SKN-SH cells. Top, RT-PCR: cDNA from uninfected cells (Lane 1) or cells infected with the 5-HT1A minigene (Lane 2) were analyzed for the unspliced 3′-UTR (U) or spliced isoforms (SS, LS) using primers shown in A . Below, real-time PCR: relative mRNA levels were normalized to GAPDH using the ΔΔCt method. Data are presented as mean ± SEM of the %unspliced from three independent experiments. D , Undetectable HTR1A RNA splicing in mouse brain. Full-length cDNA from human SKN-SH cells expressing the human 5-HT1A minigene or mouse PFC and hippocampus (HP) tissue was analyzed using two human primers C and D in A or the corresponding mouse 3′-UTR primers (521–2444 bp downstream of the stop codon). Spliced 5-HT1A mRNA isoforms (S) were only detected for the human transcript, while the unspliced form (U) was present in both. M, DNA size markers; 1, negative control. E ).

    Journal: The Journal of Neuroscience

    Article Title: A Novel Alternative Splicing Mechanism That Enhances Human 5-HT1A Receptor RNA Stability Is Altered in Major Depression

    doi: 10.1523/JNEUROSCI.0902-18.2018

    Figure Lengend Snippet: The 5-HT1A mRNA 3′-UTR is alternatively spliced in human cells to give rise to several mRNA isoforms. A , Schematic representation of the human 5-HT1A 3′-UTR construct used for 3′-RACE experiments, and of primers A–D used to quantify the resulting mRNA variants upon its transfection in human cells (unspliced, LS, and SS). Dashed lines represent the intron removed following splicing of the 3′-UTR. The relative location of the poly-adenylation site (polyA signal), miR135 site, and splice donor (gt dinucleotides) and acceptor (cag) sites are indicated. B , Top, RT-PCR analysis of the 5-HT1A 3′-UTR splice variants in HEK293 cells. RNA from HEK293 cells transfected with luciferase vector pGL3P or pGL3P 5-HT1A-3′-UTR was assessed by RT-PCR for the expression of unspliced (U, top, primers A/B) and spliced 3′UTR isoforms, short (SS) and long (LS) (bottom, primers C/D). M, DNA size markers; 1, negative control; 2, pGL3P; 3, pGL3P 5-HT1A-3′-UTR. Bottom, Real-time PCR quantification of unspliced (100%) and spliced HTR1A mRNA in HEK293 cells transfected with pGL3P 5-HT1A-3′-UTR using probes specific for unspliced, total spliced (S), or LS isoform 5-HT1A mRNA. C , Splicing of human 5-HT1A minigene in stably transfected SKN-SH cells. Top, RT-PCR: cDNA from uninfected cells (Lane 1) or cells infected with the 5-HT1A minigene (Lane 2) were analyzed for the unspliced 3′-UTR (U) or spliced isoforms (SS, LS) using primers shown in A . Below, real-time PCR: relative mRNA levels were normalized to GAPDH using the ΔΔCt method. Data are presented as mean ± SEM of the %unspliced from three independent experiments. D , Undetectable HTR1A RNA splicing in mouse brain. Full-length cDNA from human SKN-SH cells expressing the human 5-HT1A minigene or mouse PFC and hippocampus (HP) tissue was analyzed using two human primers C and D in A or the corresponding mouse 3′-UTR primers (521–2444 bp downstream of the stop codon). Spliced 5-HT1A mRNA isoforms (S) were only detected for the human transcript, while the unspliced form (U) was present in both. M, DNA size markers; 1, negative control. E ).

    Article Snippet: Real-time PCR quantifications of total 5-HT1A, PTBP1/2, nSR100, and GAPDH RNA were performed using TaqMan Gene expression assay kits (Thermo Fisher Scientific) for human 5-HT1A (Hs00265014_s1), PTBP1 (Hs00243060p_m1), PTBP2 (Hs00221842_m1), nSR100 (Hs00916552_m1), and GAPDH (Hs02758991_g1) in a 20 μl reaction.

    Techniques: Construct, Transfection, Reverse Transcription Polymerase Chain Reaction, Luciferase, Plasmid Preparation, Expressing, Negative Control, Real-time Polymerase Chain Reaction, Stable Transfection, Infection

    Opposite regulation of 5-HT1A mRNA splicing and protein levels by splicing factors PTBP1 and nSR100. A–C , Effect of PTBP1 and PTBP2 knockdown on 5-HT1A RNA splicing. SKN-SH cells were infected with lentiviruses expressing shRNAs against PTBP1, PTBP2, or a nontargeting control (Ctrl) shRNA at multiplicity of infection of 10 and selected using puromycin. Puromycin-resistant cells were infected with the WT 5-HT1A minigene at multiplicity of infection of 20 for 72 h and RNA and protein were extracted and analyzed. Relative expression of PTBP1, PTBP2, and Flag-5-HT1A proteins was determined by Western blot ( A ), while the expression of the various 5-HT1A mRNA variants was assessed by RT-PCR ( B ) and quantified by real-time PCR ( C ) using probes specific for the spliced mRNA isoforms (total spliced (Tot spliced) or LS isoform), unspliced mRNA isoforms, or total 5-HT1A mRNA (Total 1A). Relative mRNA levels were normalized to GAPDH using the ΔΔCt method. D–F , Effect of Nova1, Nova2, and nSR100 overexpression on 5-HT1A RNA splicing. SKN-SH cells were infected with the WT 5-HT1A minigene at multiplicity of infection of 20 for 72 h, and then transfected with vector (pTRIEX) or plasmids encoding the S-tagged Nova1, Nova2, and nSR100 neuronal splice regulators. The relative expression of the S-tagged proteins (S-tag), Flag-5-HT1A receptor and alpha-tubulin was determined by Western blot ( D ), while the expression of the various 5-HT1A mRNA isoforms was assessed by RT-PCR ( E ) and quantified by real-time PCR ( F ). Data presented as the mean and SEM of 3–4 independent experiments ( C , F ). * p

    Journal: The Journal of Neuroscience

    Article Title: A Novel Alternative Splicing Mechanism That Enhances Human 5-HT1A Receptor RNA Stability Is Altered in Major Depression

    doi: 10.1523/JNEUROSCI.0902-18.2018

    Figure Lengend Snippet: Opposite regulation of 5-HT1A mRNA splicing and protein levels by splicing factors PTBP1 and nSR100. A–C , Effect of PTBP1 and PTBP2 knockdown on 5-HT1A RNA splicing. SKN-SH cells were infected with lentiviruses expressing shRNAs against PTBP1, PTBP2, or a nontargeting control (Ctrl) shRNA at multiplicity of infection of 10 and selected using puromycin. Puromycin-resistant cells were infected with the WT 5-HT1A minigene at multiplicity of infection of 20 for 72 h and RNA and protein were extracted and analyzed. Relative expression of PTBP1, PTBP2, and Flag-5-HT1A proteins was determined by Western blot ( A ), while the expression of the various 5-HT1A mRNA variants was assessed by RT-PCR ( B ) and quantified by real-time PCR ( C ) using probes specific for the spliced mRNA isoforms (total spliced (Tot spliced) or LS isoform), unspliced mRNA isoforms, or total 5-HT1A mRNA (Total 1A). Relative mRNA levels were normalized to GAPDH using the ΔΔCt method. D–F , Effect of Nova1, Nova2, and nSR100 overexpression on 5-HT1A RNA splicing. SKN-SH cells were infected with the WT 5-HT1A minigene at multiplicity of infection of 20 for 72 h, and then transfected with vector (pTRIEX) or plasmids encoding the S-tagged Nova1, Nova2, and nSR100 neuronal splice regulators. The relative expression of the S-tagged proteins (S-tag), Flag-5-HT1A receptor and alpha-tubulin was determined by Western blot ( D ), while the expression of the various 5-HT1A mRNA isoforms was assessed by RT-PCR ( E ) and quantified by real-time PCR ( F ). Data presented as the mean and SEM of 3–4 independent experiments ( C , F ). * p

    Article Snippet: Real-time PCR quantifications of total 5-HT1A, PTBP1/2, nSR100, and GAPDH RNA were performed using TaqMan Gene expression assay kits (Thermo Fisher Scientific) for human 5-HT1A (Hs00265014_s1), PTBP1 (Hs00243060p_m1), PTBP2 (Hs00221842_m1), nSR100 (Hs00916552_m1), and GAPDH (Hs02758991_g1) in a 20 μl reaction.

    Techniques: Infection, Expressing, shRNA, Western Blot, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Over Expression, Transfection, Plasmid Preparation

    Observation of actin cytoskeleton and nucleus of MC3T3-E1 cells cultured on different material surfaces after 48h RNA interference treatment. Notes: ( A ) Plate; ( B ) CpTi; ( C ) 3Y-TZP; ( D ) NanoZr. MC3T3-E1 cells were transfected with a Negative Control siRNA or with siRNA that targets Syndecan-1 (#1, #2), Syndecan-2 (#1, #2), Syndecan-3 (#1, #2) and Syndecan-4 (#1, #2). Two different sequences of each Syndecan were used respectively. Magnification: 400×. The graph shows the MC3T3-E1 cell spread area ratio between si-Sdc group and si-NegCont group.*A statistical significant compared to the Negative Control group ( P

    Journal: International Journal of Nanomedicine

    Article Title: The Effects of Syndecan on Osteoblastic Cell Adhesion Onto Nano-Zirconia Surface

    doi: 10.2147/IJN.S263053

    Figure Lengend Snippet: Observation of actin cytoskeleton and nucleus of MC3T3-E1 cells cultured on different material surfaces after 48h RNA interference treatment. Notes: ( A ) Plate; ( B ) CpTi; ( C ) 3Y-TZP; ( D ) NanoZr. MC3T3-E1 cells were transfected with a Negative Control siRNA or with siRNA that targets Syndecan-1 (#1, #2), Syndecan-2 (#1, #2), Syndecan-3 (#1, #2) and Syndecan-4 (#1, #2). Two different sequences of each Syndecan were used respectively. Magnification: 400×. The graph shows the MC3T3-E1 cell spread area ratio between si-Sdc group and si-NegCont group.*A statistical significant compared to the Negative Control group ( P

    Article Snippet: After 48 hours of transfection, the efficiency of RNA interference (RNAi) was detected by quantitative RT-PCR analysis.

    Techniques: Cell Culture, Transfection, Negative Control

    SEM observation of cell morphology of MC3T3-E1 cells cultured on different surfaces after 48h RNA interference treatment. Notes: ( A ) Plate; ( B ) CpTi; ( C ) 3Y-TZP; ( D ) NanoZr. MC3T3-E1 cells were transfected with a Negative Control siRNA or with siRNA that targets Syndecan-1 (#1, #2), Syndecan-2 (#1, #2), Syndecan-3 (#1, #2) and Syndecan-4 (#1, #2). Two different sequences of each Syndecan were used respectively. Magnification: 100× and 1000×. Abbreviations: CpTi, commercial pure titanium; 3Y-TZP, 3mol% yttria-stabilized tetragonal zirconia polycrystalline; NanoZr, nano-zirconia; siRNA, small interfering RNA; si-Sdc, small interfering-Syndecan; NC, negative control; SEM, scanning electron microscopy.

    Journal: International Journal of Nanomedicine

    Article Title: The Effects of Syndecan on Osteoblastic Cell Adhesion Onto Nano-Zirconia Surface

    doi: 10.2147/IJN.S263053

    Figure Lengend Snippet: SEM observation of cell morphology of MC3T3-E1 cells cultured on different surfaces after 48h RNA interference treatment. Notes: ( A ) Plate; ( B ) CpTi; ( C ) 3Y-TZP; ( D ) NanoZr. MC3T3-E1 cells were transfected with a Negative Control siRNA or with siRNA that targets Syndecan-1 (#1, #2), Syndecan-2 (#1, #2), Syndecan-3 (#1, #2) and Syndecan-4 (#1, #2). Two different sequences of each Syndecan were used respectively. Magnification: 100× and 1000×. Abbreviations: CpTi, commercial pure titanium; 3Y-TZP, 3mol% yttria-stabilized tetragonal zirconia polycrystalline; NanoZr, nano-zirconia; siRNA, small interfering RNA; si-Sdc, small interfering-Syndecan; NC, negative control; SEM, scanning electron microscopy.

    Article Snippet: After 48 hours of transfection, the efficiency of RNA interference (RNAi) was detected by quantitative RT-PCR analysis.

    Techniques: Cell Culture, Transfection, Negative Control, Small Interfering RNA, Electron Microscopy

    Organization of actin stress fibers and focal adhesion of MC3T3-E1 cells cultured on different material surfaces after 48h RNA interference treatment. Notes: ( A ) Plate; ( B ) CpTi; ( C ) 3Y-TZP; ( D ) NanoZr. MC3T3-E1 cells were transfected with a Negative Control siRNA or with siRNA that targets Syndecan-1 (#1, #2), Syndecan-2 (#1, #2), Syndecan-3 (#1, #2) and Syndecan-4 (#1, #2). Two different sequences of each Syndecan were used respectively. Magnification: 400×. Abbreviations: CpTi, commercial pure titanium; 3Y-TZP, 3mol% yttria-stabilized tetragonal zirconia polycrystalline; NanoZr, nano-zirconia; siRNA, small interfering RNA; si-Sdc, small interfering-Syndecan; NC, negative control.

    Journal: International Journal of Nanomedicine

    Article Title: The Effects of Syndecan on Osteoblastic Cell Adhesion Onto Nano-Zirconia Surface

    doi: 10.2147/IJN.S263053

    Figure Lengend Snippet: Organization of actin stress fibers and focal adhesion of MC3T3-E1 cells cultured on different material surfaces after 48h RNA interference treatment. Notes: ( A ) Plate; ( B ) CpTi; ( C ) 3Y-TZP; ( D ) NanoZr. MC3T3-E1 cells were transfected with a Negative Control siRNA or with siRNA that targets Syndecan-1 (#1, #2), Syndecan-2 (#1, #2), Syndecan-3 (#1, #2) and Syndecan-4 (#1, #2). Two different sequences of each Syndecan were used respectively. Magnification: 400×. Abbreviations: CpTi, commercial pure titanium; 3Y-TZP, 3mol% yttria-stabilized tetragonal zirconia polycrystalline; NanoZr, nano-zirconia; siRNA, small interfering RNA; si-Sdc, small interfering-Syndecan; NC, negative control.

    Article Snippet: After 48 hours of transfection, the efficiency of RNA interference (RNAi) was detected by quantitative RT-PCR analysis.

    Techniques: Cell Culture, Transfection, Negative Control, Small Interfering RNA