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    AMV Reverse Transcriptase
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    AMV Reverse Transcriptase 1 000 units
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    New England Biolabs rna
    AMV Reverse Transcriptase
    AMV Reverse Transcriptase 1 000 units
    https://www.bioz.com/result/rna/product/New England Biolabs
    Average 93 stars, based on 959 article reviews
    Price from $9.99 to $1999.99
    rna - by Bioz Stars, 2020-08
    93/100 stars

    Images

    1) Product Images from "Histone variant H2A.Z deposition and acetylation directs the canonical Notch signaling response"

    Article Title: Histone variant H2A.Z deposition and acetylation directs the canonical Notch signaling response

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky551

    Acetylation of H2A.Z is required for upregulation of Hes1 Notch target gene. ( A ) Schematic representation of the Flag-RBP-J/Tip60 fusion proteins used in Figure 4B and C and in Supplementary Figures S9 and S10 . Amino acid numbering is accordingly to accession NP_033061.3 for RBP-J and NP_874368.1 for Tip60. RBP-J domain: LAG1-DNAbind, LAG1 DNA binding (CDD:255260); Tip60 domain: MOZ/SAS, MOZ/SAS family (CDD:250916). ( B ) RBP-J/Tip60 wildtype (wt) but not its catalytic dead (cd) mutant upregulates Hes1 expression in MT cells. MT cells were infected with retroviral particles delivering plasmids encoding Flag-tagged RBP-J/Tip60-wt, cd mutant or empty vector (Control). Total RNA was reverse transcribed into cDNA and analysed by qPCR using primers specific for Tbp or Hes1 . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD ([**] P
    Figure Legend Snippet: Acetylation of H2A.Z is required for upregulation of Hes1 Notch target gene. ( A ) Schematic representation of the Flag-RBP-J/Tip60 fusion proteins used in Figure 4B and C and in Supplementary Figures S9 and S10 . Amino acid numbering is accordingly to accession NP_033061.3 for RBP-J and NP_874368.1 for Tip60. RBP-J domain: LAG1-DNAbind, LAG1 DNA binding (CDD:255260); Tip60 domain: MOZ/SAS, MOZ/SAS family (CDD:250916). ( B ) RBP-J/Tip60 wildtype (wt) but not its catalytic dead (cd) mutant upregulates Hes1 expression in MT cells. MT cells were infected with retroviral particles delivering plasmids encoding Flag-tagged RBP-J/Tip60-wt, cd mutant or empty vector (Control). Total RNA was reverse transcribed into cDNA and analysed by qPCR using primers specific for Tbp or Hes1 . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD ([**] P

    Techniques Used: Binding Assay, Mutagenesis, Expressing, Infection, Plasmid Preparation, Real-time Polymerase Chain Reaction

    H2A.Z acetylation (H2A.Zac) but not H2A.Z occupancy positively correlates with activation of Notch target genes. ( A ) Schematic representation of the NICD-inducible system established in MT cells. The NICD was fused to the estrogen receptor binding domain (NICD-ER) and retrovirally introduced into MT cells. The NICD-ER fusion protein is retained into the cytoplasm unless cells are treated with ( Z )-4-hydroxytamoxifen (4-OHT) that induces its nuclear translocation and activation of Notch target genes. ( B ) Hes1 and Il2ra Notch target genes are induced upon 4-OHT treatment of MT NICD-ER cells. Total RNA from MT NICD-ER cells, treated for 24 h with 4-OHT or EtOH as control, was reverse transcribed into cDNA and analyzed by qPCR using primers specific for Tbp, Hes1 or Il2ra . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD of three independent experiments. ( C ) H2A.Z acetylation (H2A.Zac) but not H2A.Z occupancy positively correlates with activation of Notch target genes. MT NICD-ER cells were treated for 24 h with 4-OHT or EtOH as control and subjected to ChIP analysis using antibodies against H2A.Z, H2A.Zac, H3 or IgG as control. The qPCR analysis was focused at the Notch-dependent enhancers (red squares) represented on the left ( Hes1 +0.6 kb and Il2ra -26 kb ). Chrom X was used as negative control ( Control ). Data were normalized to the positive control ( GAPDH 0 kb ) and, in the case of H2A.Zac/H2A.Z, the H2A.Zac signals were further normalized to H2A.Z. Shown is the mean ± SD of two independent experiments.
    Figure Legend Snippet: H2A.Z acetylation (H2A.Zac) but not H2A.Z occupancy positively correlates with activation of Notch target genes. ( A ) Schematic representation of the NICD-inducible system established in MT cells. The NICD was fused to the estrogen receptor binding domain (NICD-ER) and retrovirally introduced into MT cells. The NICD-ER fusion protein is retained into the cytoplasm unless cells are treated with ( Z )-4-hydroxytamoxifen (4-OHT) that induces its nuclear translocation and activation of Notch target genes. ( B ) Hes1 and Il2ra Notch target genes are induced upon 4-OHT treatment of MT NICD-ER cells. Total RNA from MT NICD-ER cells, treated for 24 h with 4-OHT or EtOH as control, was reverse transcribed into cDNA and analyzed by qPCR using primers specific for Tbp, Hes1 or Il2ra . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD of three independent experiments. ( C ) H2A.Z acetylation (H2A.Zac) but not H2A.Z occupancy positively correlates with activation of Notch target genes. MT NICD-ER cells were treated for 24 h with 4-OHT or EtOH as control and subjected to ChIP analysis using antibodies against H2A.Z, H2A.Zac, H3 or IgG as control. The qPCR analysis was focused at the Notch-dependent enhancers (red squares) represented on the left ( Hes1 +0.6 kb and Il2ra -26 kb ). Chrom X was used as negative control ( Control ). Data were normalized to the positive control ( GAPDH 0 kb ) and, in the case of H2A.Zac/H2A.Z, the H2A.Zac signals were further normalized to H2A.Z. Shown is the mean ± SD of two independent experiments.

    Techniques Used: Activation Assay, Binding Assay, Translocation Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Negative Control, Positive Control

    Histone variant H2A.Z has a negative impact on the expression of Notch target genes. ( A ) Histone Variant H2A.Z is efficiently depleted by CRISPR/Cas9 in MT cells. Whole Cell Extract (WCE) was prepared from wildtype ( Control ) or H2A.Z depleted (clones sgH2afv/H2afz #12 and sgH2afv/H2afz #20 ) MT cells and analysed by Western blotting. GAPDH was used as loading control. ( B ) Hes1 and Il2ra Notch target genes are upregulated upon depletion of H2A.Z. Total RNA from wildtype ( Control ) or H2A.Z depleted (clones sgH2afv/H2afz #12 and sgH2afv/H2afz #20 ) MT cells was reverse transcribed into cDNA and analysed by qPCR using primers specific for Tbp, Hes1 or Il2ra . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD of five independent experiments ([*] P
    Figure Legend Snippet: Histone variant H2A.Z has a negative impact on the expression of Notch target genes. ( A ) Histone Variant H2A.Z is efficiently depleted by CRISPR/Cas9 in MT cells. Whole Cell Extract (WCE) was prepared from wildtype ( Control ) or H2A.Z depleted (clones sgH2afv/H2afz #12 and sgH2afv/H2afz #20 ) MT cells and analysed by Western blotting. GAPDH was used as loading control. ( B ) Hes1 and Il2ra Notch target genes are upregulated upon depletion of H2A.Z. Total RNA from wildtype ( Control ) or H2A.Z depleted (clones sgH2afv/H2afz #12 and sgH2afv/H2afz #20 ) MT cells was reverse transcribed into cDNA and analysed by qPCR using primers specific for Tbp, Hes1 or Il2ra . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD of five independent experiments ([*] P

    Techniques Used: Variant Assay, Expressing, CRISPR, Western Blot, Real-time Polymerase Chain Reaction

    2) Product Images from "Leptospira interrogans serovar Copenhageni Harbors Two lexA Genes Involved in SOS Response"

    Article Title: Leptospira interrogans serovar Copenhageni Harbors Two lexA Genes Involved in SOS Response

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0076419

    Genomic and transcriptional organization of the lexA2 region. (A) Schematic representation of the lexA2 genomic region from L. interrogans serovar Copenhageni (upper) compared to the equivalent region of serovar Lai (lower). Arrows represent predicted genes and transcription orientation. Light grey arrows represent genes orthologous between genomes, dark grey genes that are specific to Copenhageni and black arrows indicate genes encoding transposases. The white arrows represent genes with truncated versions in Lai genome (traced arrows) by insertion of IS elements. Remnants of a phage integrase are indicated by a traced line. The numbered bars below the genes indicate the amplified fragments corresponding to the primer pairs used in the RT-PCR analyses. (B) RT-PCR reactions, using either genomic DNA (gDNA), RNA (RT-) or cDNA (RT+) as templates, and primers flanking intergenic regions. The numbers refer to the respective fragments shown in (A).
    Figure Legend Snippet: Genomic and transcriptional organization of the lexA2 region. (A) Schematic representation of the lexA2 genomic region from L. interrogans serovar Copenhageni (upper) compared to the equivalent region of serovar Lai (lower). Arrows represent predicted genes and transcription orientation. Light grey arrows represent genes orthologous between genomes, dark grey genes that are specific to Copenhageni and black arrows indicate genes encoding transposases. The white arrows represent genes with truncated versions in Lai genome (traced arrows) by insertion of IS elements. Remnants of a phage integrase are indicated by a traced line. The numbered bars below the genes indicate the amplified fragments corresponding to the primer pairs used in the RT-PCR analyses. (B) RT-PCR reactions, using either genomic DNA (gDNA), RNA (RT-) or cDNA (RT+) as templates, and primers flanking intergenic regions. The numbers refer to the respective fragments shown in (A).

    Techniques Used: Amplification, Reverse Transcription Polymerase Chain Reaction

    Genomic and transcriptional organization of the lexA1 region. (A) Schematic representation of the lexA1 genomic region. The arrows indicate the direction of transcription. The fragments amplified by the primer pairs used for the RT-PCR analysis are indicated by numbered lines below the genes. (B) Composite image of agarose gels from resulting RT-PCR reactions, using either genomic DNA (DNA), RNA (RT-) or cDNA (RT+) as templates. The numbers refer to the respective fragments shown in (A).
    Figure Legend Snippet: Genomic and transcriptional organization of the lexA1 region. (A) Schematic representation of the lexA1 genomic region. The arrows indicate the direction of transcription. The fragments amplified by the primer pairs used for the RT-PCR analysis are indicated by numbered lines below the genes. (B) Composite image of agarose gels from resulting RT-PCR reactions, using either genomic DNA (DNA), RNA (RT-) or cDNA (RT+) as templates. The numbers refer to the respective fragments shown in (A).

    Techniques Used: Amplification, Reverse Transcription Polymerase Chain Reaction

    3) Product Images from "Transcriptional Regulation of Human Dual Specificity Protein Phosphatase 1 (DUSP1) Gene by Glucocorticoids"

    Article Title: Transcriptional Regulation of Human Dual Specificity Protein Phosphatase 1 (DUSP1) Gene by Glucocorticoids

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0013754

    Identification of DNA elements mediating glucocorticoid response in the human DUSP1 gene. A A549 cells were untreated, treated with DMSO for 90 min, or treated with DEX (0.5 µM) for 10 min, 30 min, and 90 min as shown. Nuclei were used for nuclear run-on experiments with biotin-UTP to monitor in vitro transcription. Newly synthesized RNA was isolated and used to make cDNA. qPCR was performed to monitor changes in transcription rates using primers specific to Dusp1 and Rpl19 (control). Data represent the standard error mean (SEM) of the fold induction (DMSO- or DEX-treated cells divided by untreated cells) from at least three experiments. B Identification of GR binding regions in human DUSP1 using ChIP scanning across the first 2 kb of the human DUSP1 promoter. A549 cells were treated with DMSO or DEX (0.5 µM) for 10 min, and ChIP was performed with GR-specific antibody as described. DUSP1 gene schematic shows the location of six primers used (black boxes labeled p1-p6) for qPCR analyses. The regions of the DUSP1 gene (relative to the TSS) amplified by these primers are: p1 (−1815 to −1717), p2 (−1421 to −1333), p3 (−1191 to −1118), p4 (−662 to −543), p5 (−160 to −54), and p6 (+98 to +215). Data represent the SEM of the fold enrichment (DEX-treated cells divided by DMSO-treated cells) from at least three experiments. C DUSP1 GR binding region mediates glucocorticoid response. −1672 to −959 (relative to the TSS) of the human DUSP1 genomic region was subcloned into pGL4-TATA reporter plasmid to create pDUSP1. pDUSP1 was transfected into A549 cells along with an expression vector for human GR. 24 h post-transfection, cells were treated with DMSO or DEX (0.5 µM) for 18–20 h. Luciferase assay data represents the SEM of the fold induction of luciferase activity (DEX-treated cells divided by DMSO-treated cells) from at least three experiments.
    Figure Legend Snippet: Identification of DNA elements mediating glucocorticoid response in the human DUSP1 gene. A A549 cells were untreated, treated with DMSO for 90 min, or treated with DEX (0.5 µM) for 10 min, 30 min, and 90 min as shown. Nuclei were used for nuclear run-on experiments with biotin-UTP to monitor in vitro transcription. Newly synthesized RNA was isolated and used to make cDNA. qPCR was performed to monitor changes in transcription rates using primers specific to Dusp1 and Rpl19 (control). Data represent the standard error mean (SEM) of the fold induction (DMSO- or DEX-treated cells divided by untreated cells) from at least three experiments. B Identification of GR binding regions in human DUSP1 using ChIP scanning across the first 2 kb of the human DUSP1 promoter. A549 cells were treated with DMSO or DEX (0.5 µM) for 10 min, and ChIP was performed with GR-specific antibody as described. DUSP1 gene schematic shows the location of six primers used (black boxes labeled p1-p6) for qPCR analyses. The regions of the DUSP1 gene (relative to the TSS) amplified by these primers are: p1 (−1815 to −1717), p2 (−1421 to −1333), p3 (−1191 to −1118), p4 (−662 to −543), p5 (−160 to −54), and p6 (+98 to +215). Data represent the SEM of the fold enrichment (DEX-treated cells divided by DMSO-treated cells) from at least three experiments. C DUSP1 GR binding region mediates glucocorticoid response. −1672 to −959 (relative to the TSS) of the human DUSP1 genomic region was subcloned into pGL4-TATA reporter plasmid to create pDUSP1. pDUSP1 was transfected into A549 cells along with an expression vector for human GR. 24 h post-transfection, cells were treated with DMSO or DEX (0.5 µM) for 18–20 h. Luciferase assay data represents the SEM of the fold induction of luciferase activity (DEX-treated cells divided by DMSO-treated cells) from at least three experiments.

    Techniques Used: In Vitro, Synthesized, Isolation, Real-time Polymerase Chain Reaction, Binding Assay, Chromatin Immunoprecipitation, Labeling, Amplification, Plasmid Preparation, Transfection, Expressing, Luciferase, Activity Assay

    p300 is a transcriptional coactivator for GR-regulated DUSP1 gene transcription. A Glucocorticoid treatment increased the level of p300 and CBP at the DUSP1 GLS2 region. A549 cells were treated with DMSO or DEX (0.5 µM) for 10 min, and ChIP was performed with antibodies against p300, CBP and IgG (control). Data is shown from qPCR analyses with primers specific to the DUSP1 GLS2. B RNAi knockdown causes substantial reduction of p300 protein and CBP protein. A549 cells reverse transfected with p300, CBP or scramble siRNA were harvested 48 h post-transfection, then probed by Western blot using an antibody against p300 or CBP, with β-actin as a loading control. C RNAi against p300 but not CBP decreased DEX-induced DUSP1 gene expression. p300, CBP or scramble (control) siRNA was transfected into A549 cells. 48 h post-transfection, cells were treated with DMSO or DEX (0.5 µM) for 5 h. Total RNA was isolated and converted to cDNA. qPCR was used to monitor the expression of DUSP1 gene. The expression of Rpl19 gene was used as an internal control. Data represents the SEM of DEX-induced DUSP1 gene expression (DEX-treated cells divided by DMSO-treated cells), as a percent of the control siRNA response from at least three experiments.
    Figure Legend Snippet: p300 is a transcriptional coactivator for GR-regulated DUSP1 gene transcription. A Glucocorticoid treatment increased the level of p300 and CBP at the DUSP1 GLS2 region. A549 cells were treated with DMSO or DEX (0.5 µM) for 10 min, and ChIP was performed with antibodies against p300, CBP and IgG (control). Data is shown from qPCR analyses with primers specific to the DUSP1 GLS2. B RNAi knockdown causes substantial reduction of p300 protein and CBP protein. A549 cells reverse transfected with p300, CBP or scramble siRNA were harvested 48 h post-transfection, then probed by Western blot using an antibody against p300 or CBP, with β-actin as a loading control. C RNAi against p300 but not CBP decreased DEX-induced DUSP1 gene expression. p300, CBP or scramble (control) siRNA was transfected into A549 cells. 48 h post-transfection, cells were treated with DMSO or DEX (0.5 µM) for 5 h. Total RNA was isolated and converted to cDNA. qPCR was used to monitor the expression of DUSP1 gene. The expression of Rpl19 gene was used as an internal control. Data represents the SEM of DEX-induced DUSP1 gene expression (DEX-treated cells divided by DMSO-treated cells), as a percent of the control siRNA response from at least three experiments.

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Expressing, Isolation

    4) Product Images from "Histidine-Rich Glycoprotein Can Prevent Development of Mouse Experimental Glioblastoma"

    Article Title: Histidine-Rich Glycoprotein Can Prevent Development of Mouse Experimental Glioblastoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0008536

    Presence and expression of viral transduced PDGF-B and HRG in tumors. ( A ) Insertion of the viral transduced human PDGF-B and HRG cDNA in genomic DNA prepared from PDGF-B+X (P+X) and PDGF-B+HRG (P+H) induced tumors. The tumor grade is given above each sample. Genomic DNA from U-706MG-a cells was used as positive control (+), and genomic DNA from an untreated mouse was used as negative control (−). ( B ) Expression of human PDGF-B and HRG mRNA in P+X and P+H tumors. RNA extracted from U-343MG and DF-1 RCAS-HRG cells were used as positive control for PDGF-B and HRG, respectively (+), and RNA from an untreated mouse brain was used as negative control (−).
    Figure Legend Snippet: Presence and expression of viral transduced PDGF-B and HRG in tumors. ( A ) Insertion of the viral transduced human PDGF-B and HRG cDNA in genomic DNA prepared from PDGF-B+X (P+X) and PDGF-B+HRG (P+H) induced tumors. The tumor grade is given above each sample. Genomic DNA from U-706MG-a cells was used as positive control (+), and genomic DNA from an untreated mouse was used as negative control (−). ( B ) Expression of human PDGF-B and HRG mRNA in P+X and P+H tumors. RNA extracted from U-343MG and DF-1 RCAS-HRG cells were used as positive control for PDGF-B and HRG, respectively (+), and RNA from an untreated mouse brain was used as negative control (−).

    Techniques Used: Expressing, Positive Control, Negative Control

    5) Product Images from "The RNA Helicase Rm62 Cooperates with SU(VAR)3-9 to Re-Silence Active Transcription in Drosophila melanogaster"

    Article Title: The RNA Helicase Rm62 Cooperates with SU(VAR)3-9 to Re-Silence Active Transcription in Drosophila melanogaster

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0020761

    Prolonged RNA production from the hsp70 locus after reduction of SU(VAR)3-9 or Rm62. ( A ) RNAi against SU(VAR)3-9 and/or Rm62 specifically eliminates the respective proteins as well as it's mRNA from SL2 whole cell extract (WCE). SL2 cells were transfected with specific double stranded RNAs against SU(VAR)3-9, Rm62 or GST (control). WCEs and total RNA were prepared 6 days after transfection. Left : Proteins were analyzed by SDS-PAGE followed by western blotting with the indicated antibodies (GST: control RNAi, SU(VAR)3-9: RNAi against Su(var)3-9, Rm62: RNAi against Rm62, SU(VAR)3-9/Rm62: double knockdown of Su(var)3-9 and Rm62). Right : 1 µg oft total RNA, was reverse transcribed (Superscript™, Reverse Transcriptase, Invitrogen) using gene specific primers for Su(var)3-9 or Rm62, respectively. 10% of the obtained cDNA were analyzed by standard PCR using specific primers for Su(var)3-9 or Rm62, and separated by agarose gel electrophoresis. To discriminate between genomic and cDNA, we used intron spanning primer pairs in the PCR reaction. ( B ) Quantitative RT-PCR of the hsp70 m RNA from SL2 cells before (no HS), after heat shock (30 min HS) and a 180 min of recovery phase (+180 min recovery). SL2 cells, cultured under standard conditions, were subjected to RNAi against GST (control), Su(var)3-9 or Rm62. After 6 days of culturing, cells were either not treated (no HS) or treated with a heat shock (30 min HS) followed by a recovery for 180 min at 26°C (+180 min recovery), respectively. RNA from these cells was isolated, reverse transcribed and analyzed by quantitative real time PCR. Bars represent relative hsp70 RNA expression levels (in percent) normalized to an internal control (U6 snRNA), which does not respond to heat shock. Percent expression was calculated to the maximal amount of RNA measured after heat shock. The inlet graph shows an enlargement of the calculated values after 180 min recovery upon heat shock. The observed difference between the SU(VAR)3-9/GST and Rm62/GST is significant as calculated with an unpaired two sided Student's t-test (p = 0.041 and p = 0.014, respectively). Error bars indicate the standard deviation of two replicates. ( C ) Relative expression of hsp70 RNA from wild type (WT) Rm62 ( CBO2119/LipF ) and Su(var)3-9 heteroalleic flies ( Su(var)3-9 1 /Su(var)3-9 2 ) before (no HS) or after heat shock (30 min HS) followed by 120 min of recovery (+120 min recovery). RNAs were extracted from the flies either not treated or treated with a heat impulse and “recovered” for 120 min at 25°C and further subjected to quantitative real time PCR. Bars represent relative hsp70 RNA expression levels (in percent) normalized to an internal control (18S rRNA), which has a minimal effect on heat shock. The observed difference between Su(var)3-9 mutant and Rm62 mutants compared to wildtype flies is significant as calculated with an unpaired two sided Student's t-test (p = 0.047 and p = 0.003, respectively). Error bars indicate the standard deviation of three replicates.
    Figure Legend Snippet: Prolonged RNA production from the hsp70 locus after reduction of SU(VAR)3-9 or Rm62. ( A ) RNAi against SU(VAR)3-9 and/or Rm62 specifically eliminates the respective proteins as well as it's mRNA from SL2 whole cell extract (WCE). SL2 cells were transfected with specific double stranded RNAs against SU(VAR)3-9, Rm62 or GST (control). WCEs and total RNA were prepared 6 days after transfection. Left : Proteins were analyzed by SDS-PAGE followed by western blotting with the indicated antibodies (GST: control RNAi, SU(VAR)3-9: RNAi against Su(var)3-9, Rm62: RNAi against Rm62, SU(VAR)3-9/Rm62: double knockdown of Su(var)3-9 and Rm62). Right : 1 µg oft total RNA, was reverse transcribed (Superscript™, Reverse Transcriptase, Invitrogen) using gene specific primers for Su(var)3-9 or Rm62, respectively. 10% of the obtained cDNA were analyzed by standard PCR using specific primers for Su(var)3-9 or Rm62, and separated by agarose gel electrophoresis. To discriminate between genomic and cDNA, we used intron spanning primer pairs in the PCR reaction. ( B ) Quantitative RT-PCR of the hsp70 m RNA from SL2 cells before (no HS), after heat shock (30 min HS) and a 180 min of recovery phase (+180 min recovery). SL2 cells, cultured under standard conditions, were subjected to RNAi against GST (control), Su(var)3-9 or Rm62. After 6 days of culturing, cells were either not treated (no HS) or treated with a heat shock (30 min HS) followed by a recovery for 180 min at 26°C (+180 min recovery), respectively. RNA from these cells was isolated, reverse transcribed and analyzed by quantitative real time PCR. Bars represent relative hsp70 RNA expression levels (in percent) normalized to an internal control (U6 snRNA), which does not respond to heat shock. Percent expression was calculated to the maximal amount of RNA measured after heat shock. The inlet graph shows an enlargement of the calculated values after 180 min recovery upon heat shock. The observed difference between the SU(VAR)3-9/GST and Rm62/GST is significant as calculated with an unpaired two sided Student's t-test (p = 0.041 and p = 0.014, respectively). Error bars indicate the standard deviation of two replicates. ( C ) Relative expression of hsp70 RNA from wild type (WT) Rm62 ( CBO2119/LipF ) and Su(var)3-9 heteroalleic flies ( Su(var)3-9 1 /Su(var)3-9 2 ) before (no HS) or after heat shock (30 min HS) followed by 120 min of recovery (+120 min recovery). RNAs were extracted from the flies either not treated or treated with a heat impulse and “recovered” for 120 min at 25°C and further subjected to quantitative real time PCR. Bars represent relative hsp70 RNA expression levels (in percent) normalized to an internal control (18S rRNA), which has a minimal effect on heat shock. The observed difference between Su(var)3-9 mutant and Rm62 mutants compared to wildtype flies is significant as calculated with an unpaired two sided Student's t-test (p = 0.047 and p = 0.003, respectively). Error bars indicate the standard deviation of three replicates.

    Techniques Used: Transfection, SDS Page, Western Blot, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Quantitative RT-PCR, Cell Culture, Isolation, Real-time Polymerase Chain Reaction, RNA Expression, Expressing, Standard Deviation, Mutagenesis

    6) Product Images from "Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes"

    Article Title: Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw028

    Reverse transcription using AHP dUTP. ( A ) RNA template T5 with primer P3. ( B and C ) Twenty percent denaturing PAGE analysis of reactions using AMV and M-MuLV (RNase H-) reverse transcriptases at 42°C for 15 h.
    Figure Legend Snippet: Reverse transcription using AHP dUTP. ( A ) RNA template T5 with primer P3. ( B and C ) Twenty percent denaturing PAGE analysis of reactions using AMV and M-MuLV (RNase H-) reverse transcriptases at 42°C for 15 h.

    Techniques Used: Polyacrylamide Gel Electrophoresis

    7) Product Images from "Preferential Amplification of Pathogenic Sequences"

    Article Title: Preferential Amplification of Pathogenic Sequences

    Journal: Scientific Reports

    doi: 10.1038/srep11047

    Schematic representation of the PATHseq (Preferential Amplification of Pathogenic Sequences) method. ( 1 ) Total mRNAs from clinical sample, including human mRNAs and relatively scarce pathogenic mRNAs; ( 2 ) Total mRNAs are transcribed into first strand cDNAs with P1 primer; ( 3 ) RNase H cleaves RNAs in RNA-DNA duplex; ( 4 ) Reverse transcriptase (RT) synthesizes secondary cDNA strands with P2 primers; ( 5 ) T7 RNA polymerase synthesizes RNAs in the presence of T7 promoter; ( 6 ) Synthesized anti-sense RNAs; ( 7 ) Synthesized RNAs are hybridized to human reference (non-pathogenic) cDNA library coated on a solid phase. RNase H cleaves bound RNAs (human RNAs) in RNA-DNA duplex; ( 8 ) Pathogenic RNAs are enriched; ( 9 ) Reverse transcription; ( 10 ) RNase H cleaves RNAs in RNA-DNA duplex; ( 11 ) T7 RNA polymerase synthesizes RNAs; ( 12 ) New RNAs synthesized from enriched pathogenic RNAs are amplified 100-1000 fold.
    Figure Legend Snippet: Schematic representation of the PATHseq (Preferential Amplification of Pathogenic Sequences) method. ( 1 ) Total mRNAs from clinical sample, including human mRNAs and relatively scarce pathogenic mRNAs; ( 2 ) Total mRNAs are transcribed into first strand cDNAs with P1 primer; ( 3 ) RNase H cleaves RNAs in RNA-DNA duplex; ( 4 ) Reverse transcriptase (RT) synthesizes secondary cDNA strands with P2 primers; ( 5 ) T7 RNA polymerase synthesizes RNAs in the presence of T7 promoter; ( 6 ) Synthesized anti-sense RNAs; ( 7 ) Synthesized RNAs are hybridized to human reference (non-pathogenic) cDNA library coated on a solid phase. RNase H cleaves bound RNAs (human RNAs) in RNA-DNA duplex; ( 8 ) Pathogenic RNAs are enriched; ( 9 ) Reverse transcription; ( 10 ) RNase H cleaves RNAs in RNA-DNA duplex; ( 11 ) T7 RNA polymerase synthesizes RNAs; ( 12 ) New RNAs synthesized from enriched pathogenic RNAs are amplified 100-1000 fold.

    Techniques Used: Amplification, Synthesized, cDNA Library Assay

    8) Product Images from "Disruption of NAP1 genes supresses the fas1 mutant phenotype and enhances genome stability"

    Article Title: Disruption of NAP1 genes supresses the fas1 mutant phenotype and enhances genome stability

    Journal: bioRxiv

    doi: 10.1101/2020.01.03.894170

    NAP1 genes are overexpressed in fas1 . A) Transcript levels of NAP1;1, NAP1;2 and NAP1;3 genes were determined in 10-d-old seedlings of WT and mutant lines (fas1, m123-2 and fas1m123-2). The fas1 mutant shows higher expression of all NAP1 genes than WT plants. Error bars indicate standard deviations calculated from three biological replicates, ubiquitin 10 was used as a reference gene. B) Relative transcript levels of NAP1;2 gene were determined in leaves of 4-w-old plants of WT and mutant lines ( fas1, m123-2 and fas1m123-2). The fas1 mutant shows two-fold higher expression of NAP1:2 than WT plants, expression in m123-2 and fas1m123-2 mutants is negligible. Error bars indicate standard deviations calculated from three biological replicates, ubiquitin 10 was used as a reference gene.
    Figure Legend Snippet: NAP1 genes are overexpressed in fas1 . A) Transcript levels of NAP1;1, NAP1;2 and NAP1;3 genes were determined in 10-d-old seedlings of WT and mutant lines (fas1, m123-2 and fas1m123-2). The fas1 mutant shows higher expression of all NAP1 genes than WT plants. Error bars indicate standard deviations calculated from three biological replicates, ubiquitin 10 was used as a reference gene. B) Relative transcript levels of NAP1;2 gene were determined in leaves of 4-w-old plants of WT and mutant lines ( fas1, m123-2 and fas1m123-2). The fas1 mutant shows two-fold higher expression of NAP1:2 than WT plants, expression in m123-2 and fas1m123-2 mutants is negligible. Error bars indicate standard deviations calculated from three biological replicates, ubiquitin 10 was used as a reference gene.

    Techniques Used: Mutagenesis, Expressing

    BRCA1 and PARP1 mRNA levels are altered fas1m123-2. Transcript levels of DNA damage response genes BRCA1 and PARP1 in 10-d-old seedlings of WT, fasl, m123-2 and fas1m123-2 on ½ MS medium (control) and on medium supplemented with 10 nM CPT. Relative expression levels with WT set up as 100% −1 and using ubiquitin 10 as a reference are shown. Three biological replicates were analysed, three technical replicates each, error bars indicate standard deviations.
    Figure Legend Snippet: BRCA1 and PARP1 mRNA levels are altered fas1m123-2. Transcript levels of DNA damage response genes BRCA1 and PARP1 in 10-d-old seedlings of WT, fasl, m123-2 and fas1m123-2 on ½ MS medium (control) and on medium supplemented with 10 nM CPT. Relative expression levels with WT set up as 100% −1 and using ubiquitin 10 as a reference are shown. Three biological replicates were analysed, three technical replicates each, error bars indicate standard deviations.

    Techniques Used: Expressing

    fas1m123-2 plants are not sensitive to genotoxic agents A) 10-d-old WT, fas1, m123-2 and fas1m123-2 plants grown on plates with ½ MS medium (no treatment). B) 10-d-old WT, fas1, m123-2 and faslm123-2 plants grown for 3 days on plates with ½ MS medium and for additional 7 days on ½ MS medium containing 125 μΜ MMS. C) Absolute root lengths of WT, fas1, ml23-2 and fas1m123-2 plants on control ½ MS medium and on medium supplemented with 125 μΜ MMS; 40 plants of each line were used in this analysis. The results are summarised in the chart, error bars indicate standard deviations. D) Relative change of root lengths of WT, fas1, m 123-2 and fas1m123-2 plants grown on control ½ MS medium (100%) and on media supplemented with given concentrations of MMS, MMC, bleomycin, zeocin and camptothecin. Root lengths of 40 plants were measured on seedlings grown for 7 days on genotoxic agents. The results are summarised in the chart, error bars indicate standard deviations.
    Figure Legend Snippet: fas1m123-2 plants are not sensitive to genotoxic agents A) 10-d-old WT, fas1, m123-2 and fas1m123-2 plants grown on plates with ½ MS medium (no treatment). B) 10-d-old WT, fas1, m123-2 and faslm123-2 plants grown for 3 days on plates with ½ MS medium and for additional 7 days on ½ MS medium containing 125 μΜ MMS. C) Absolute root lengths of WT, fas1, ml23-2 and fas1m123-2 plants on control ½ MS medium and on medium supplemented with 125 μΜ MMS; 40 plants of each line were used in this analysis. The results are summarised in the chart, error bars indicate standard deviations. D) Relative change of root lengths of WT, fas1, m 123-2 and fas1m123-2 plants grown on control ½ MS medium (100%) and on media supplemented with given concentrations of MMS, MMC, bleomycin, zeocin and camptothecin. Root lengths of 40 plants were measured on seedlings grown for 7 days on genotoxic agents. The results are summarised in the chart, error bars indicate standard deviations.

    Techniques Used:

    9) Product Images from "Inhibition of mitochondrial respiration under hypoxia and increased antioxidant activity after reoxygenation of Tribolium castaneum"

    Article Title: Inhibition of mitochondrial respiration under hypoxia and increased antioxidant activity after reoxygenation of Tribolium castaneum

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0199056

    qRT-PCR analysis of selected transcripts to confirm expression profiles identified by RNA-seq. Tc ELOVL, elongation of very long chain fatty acids protein 7; Tc MRP, ATP-binding cassette subfamily C (CFTR/MRP) member 4; Tc HSP70, heat shock 70kDa protein; Tc MAPK, MAP kinase; Tc DUSP, Dual specificity MAP kinase phosphatase; Tc SD, superoxide dismutase, Cu-Zn family; Tc AG, alpha-glucosidase; Tc DACHS, DACHS, Hippo signling pathway; Tc FJBP Four-jointed box protein 1 (FJBP). Value represents mean ± SE of three independent PCR amplifications and quantifications.
    Figure Legend Snippet: qRT-PCR analysis of selected transcripts to confirm expression profiles identified by RNA-seq. Tc ELOVL, elongation of very long chain fatty acids protein 7; Tc MRP, ATP-binding cassette subfamily C (CFTR/MRP) member 4; Tc HSP70, heat shock 70kDa protein; Tc MAPK, MAP kinase; Tc DUSP, Dual specificity MAP kinase phosphatase; Tc SD, superoxide dismutase, Cu-Zn family; Tc AG, alpha-glucosidase; Tc DACHS, DACHS, Hippo signling pathway; Tc FJBP Four-jointed box protein 1 (FJBP). Value represents mean ± SE of three independent PCR amplifications and quantifications.

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

    Gene expression pattern of glycolytic (A) and Krebs (B) cycle enzymes of Tribolium castaneum larvae in response to hypoxia. Total RNA was isolated from the larvae after 12 hours’ hypoxia treatment. qRT-PCR was used to illustrate gene expression. Tc HK, hexokinase; Tc PGI, phosphoglucose isomerase; Tc PFKFB, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; Tc FBP, fructose 1,6-bisphosphate aldolase; Tc TPI, triosephosphate isomerase; Tc GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Tc PGK, phosphoglycerate kinase; Tc PGM, phosphoglycerate mutase; Tc ENO, Enolase; Tc PK, Pyruvate kinase; Tc LDH, L-lactate dehydrogenase; Tc PDC, pyruvate dehydrogenase; Tc ACO, aconitate hydratase, mitochondria; Tc IDH, isocitrate dehydrogenase; Tc SCSb, succinyl-CoA synthetase beta chain; Tc SDH, succinate dehydrogenase; Tc FH, fumarate hydratase; Tc MDH, malate dehydrogenase. Red color represents upregulate, green color represents downregulate and black color represents no change.
    Figure Legend Snippet: Gene expression pattern of glycolytic (A) and Krebs (B) cycle enzymes of Tribolium castaneum larvae in response to hypoxia. Total RNA was isolated from the larvae after 12 hours’ hypoxia treatment. qRT-PCR was used to illustrate gene expression. Tc HK, hexokinase; Tc PGI, phosphoglucose isomerase; Tc PFKFB, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; Tc FBP, fructose 1,6-bisphosphate aldolase; Tc TPI, triosephosphate isomerase; Tc GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Tc PGK, phosphoglycerate kinase; Tc PGM, phosphoglycerate mutase; Tc ENO, Enolase; Tc PK, Pyruvate kinase; Tc LDH, L-lactate dehydrogenase; Tc PDC, pyruvate dehydrogenase; Tc ACO, aconitate hydratase, mitochondria; Tc IDH, isocitrate dehydrogenase; Tc SCSb, succinyl-CoA synthetase beta chain; Tc SDH, succinate dehydrogenase; Tc FH, fumarate hydratase; Tc MDH, malate dehydrogenase. Red color represents upregulate, green color represents downregulate and black color represents no change.

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR

    10) Product Images from "Inhibition of mitochondrial respiration under hypoxia and increased antioxidant activity after reoxygenation of Tribolium castaneum"

    Article Title: Inhibition of mitochondrial respiration under hypoxia and increased antioxidant activity after reoxygenation of Tribolium castaneum

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0199056

    qRT-PCR analysis of selected transcripts to confirm expression profiles identified by RNA-seq. Tc ELOVL, elongation of very long chain fatty acids protein 7; Tc MRP, ATP-binding cassette subfamily C (CFTR/MRP) member 4; Tc HSP70, heat shock 70kDa protein; Tc MAPK, MAP kinase; Tc DUSP, Dual specificity MAP kinase phosphatase; Tc SD, superoxide dismutase, Cu-Zn family; Tc AG, alpha-glucosidase; Tc DACHS, DACHS, Hippo signling pathway; Tc FJBP Four-jointed box protein 1 (FJBP). Value represents mean ± SE of three independent PCR amplifications and quantifications.
    Figure Legend Snippet: qRT-PCR analysis of selected transcripts to confirm expression profiles identified by RNA-seq. Tc ELOVL, elongation of very long chain fatty acids protein 7; Tc MRP, ATP-binding cassette subfamily C (CFTR/MRP) member 4; Tc HSP70, heat shock 70kDa protein; Tc MAPK, MAP kinase; Tc DUSP, Dual specificity MAP kinase phosphatase; Tc SD, superoxide dismutase, Cu-Zn family; Tc AG, alpha-glucosidase; Tc DACHS, DACHS, Hippo signling pathway; Tc FJBP Four-jointed box protein 1 (FJBP). Value represents mean ± SE of three independent PCR amplifications and quantifications.

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

    Gene expression pattern of glycolytic (A) and Krebs (B) cycle enzymes of Tribolium castaneum larvae in response to hypoxia. Total RNA was isolated from the larvae after 12 hours’ hypoxia treatment. qRT-PCR was used to illustrate gene expression. Tc HK, hexokinase; Tc PGI, phosphoglucose isomerase; Tc PFKFB, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; Tc FBP, fructose 1,6-bisphosphate aldolase; Tc TPI, triosephosphate isomerase; Tc GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Tc PGK, phosphoglycerate kinase; Tc PGM, phosphoglycerate mutase; Tc ENO, Enolase; Tc PK, Pyruvate kinase; Tc LDH, L-lactate dehydrogenase; Tc PDC, pyruvate dehydrogenase; Tc ACO, aconitate hydratase, mitochondria; Tc IDH, isocitrate dehydrogenase; Tc SCSb, succinyl-CoA synthetase beta chain; Tc SDH, succinate dehydrogenase; Tc FH, fumarate hydratase; Tc MDH, malate dehydrogenase. Red color represents upregulate, green color represents downregulate and black color represents no change.
    Figure Legend Snippet: Gene expression pattern of glycolytic (A) and Krebs (B) cycle enzymes of Tribolium castaneum larvae in response to hypoxia. Total RNA was isolated from the larvae after 12 hours’ hypoxia treatment. qRT-PCR was used to illustrate gene expression. Tc HK, hexokinase; Tc PGI, phosphoglucose isomerase; Tc PFKFB, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; Tc FBP, fructose 1,6-bisphosphate aldolase; Tc TPI, triosephosphate isomerase; Tc GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Tc PGK, phosphoglycerate kinase; Tc PGM, phosphoglycerate mutase; Tc ENO, Enolase; Tc PK, Pyruvate kinase; Tc LDH, L-lactate dehydrogenase; Tc PDC, pyruvate dehydrogenase; Tc ACO, aconitate hydratase, mitochondria; Tc IDH, isocitrate dehydrogenase; Tc SCSb, succinyl-CoA synthetase beta chain; Tc SDH, succinate dehydrogenase; Tc FH, fumarate hydratase; Tc MDH, malate dehydrogenase. Red color represents upregulate, green color represents downregulate and black color represents no change.

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR

    11) Product Images from "Encapsidated Host Factors in Alphavirus Particles Influence Midgut Infection of Aedes aegypti"

    Article Title: Encapsidated Host Factors in Alphavirus Particles Influence Midgut Infection of Aedes aegypti

    Journal: Viruses

    doi: 10.3390/v10050263

    Midgut immune response. Infected midguts were dissected out at 1 DPF and homogenized. RNA was extracted with TRIzol and cDNA was synthesized to quantify RNA levels of ( A ) nsP1 and indicator genes for Toll ( B ), IMD ( C ), JAK-STAT ( D ), apoptosis ( E ), and RNAi ( F ) pathways. P values were calculated by Mann–Whitney test. All error bars represent standard error of mean (SEM). The dashed line indicates gene expression levels in mosquitoes following a noninfectious blood meal.
    Figure Legend Snippet: Midgut immune response. Infected midguts were dissected out at 1 DPF and homogenized. RNA was extracted with TRIzol and cDNA was synthesized to quantify RNA levels of ( A ) nsP1 and indicator genes for Toll ( B ), IMD ( C ), JAK-STAT ( D ), apoptosis ( E ), and RNAi ( F ) pathways. P values were calculated by Mann–Whitney test. All error bars represent standard error of mean (SEM). The dashed line indicates gene expression levels in mosquitoes following a noninfectious blood meal.

    Techniques Used: Infection, Synthesized, Radial Immuno Diffusion, MANN-WHITNEY, Expressing

    12) Product Images from "A Simplified System to Express Circularized Inhibitors of miRNA for Stable and Potent Suppression of miRNA Functions"

    Article Title: A Simplified System to Express Circularized Inhibitors of miRNA for Stable and Potent Suppression of miRNA Functions

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2018.09.025

    Schematic Representation of the CimiR System for Expressing the Circularized AntamiRNAs or miRNA Antagonists (A and B) Overall structure (A) and the essential elements (B) of the CimiR system. Briefly, the consensus sequences of the RNA-splicing branch site (B) and polypyrimidine track (ppy) were constructed at the 5′ end of the antamiRNA locus, while the donor site sequence (D) and a 20-mer random sequence (R) were engineered at the 3′ end of AntamiR, which were flanked by the 100-bp inverted repeat sequence derived from mouse Rosa26 genomic sequence. (C) Formation of a circularized antamiR (or CimiR). Upon transcription driven by the hEFH promoter, the antamiR-containing pre-mRNA is processed through cis backsplicing mechanism to yield a circularized antamiR (CimiR) and a splicing by-product. The expression system was constructed on the basis of retroviral vector pSEBR, which expresses blasticidin selection marker and RFP-tracking marker. The same CimiR expression cassette was constructed in an adenoviral shuttle vector pAdTrace-CimiR as well.
    Figure Legend Snippet: Schematic Representation of the CimiR System for Expressing the Circularized AntamiRNAs or miRNA Antagonists (A and B) Overall structure (A) and the essential elements (B) of the CimiR system. Briefly, the consensus sequences of the RNA-splicing branch site (B) and polypyrimidine track (ppy) were constructed at the 5′ end of the antamiRNA locus, while the donor site sequence (D) and a 20-mer random sequence (R) were engineered at the 3′ end of AntamiR, which were flanked by the 100-bp inverted repeat sequence derived from mouse Rosa26 genomic sequence. (C) Formation of a circularized antamiR (or CimiR). Upon transcription driven by the hEFH promoter, the antamiR-containing pre-mRNA is processed through cis backsplicing mechanism to yield a circularized antamiR (CimiR) and a splicing by-product. The expression system was constructed on the basis of retroviral vector pSEBR, which expresses blasticidin selection marker and RFP-tracking marker. The same CimiR expression cassette was constructed in an adenoviral shuttle vector pAdTrace-CimiR as well.

    Techniques Used: Expressing, Construct, Sequencing, Derivative Assay, Plasmid Preparation, Selection, Marker

    13) Product Images from "Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes"

    Article Title: Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw028

    Primer extension using AHP dUTP (1.5 h). ( A ) Template T2 and primer P3. ( B ) Twenty percent denaturing PAGE analysis of reactions using Gotaq (Go, 72°C), Klenow (Kl, 37°C), KOD (KO, 72°C) and Therminator™ II (Th, 72°C) polymerases. Lane P: primer P3. ( C ) Reactions using Gotaq polymerase at 60°C. ( D ) Mass spectrum of AHP-modified fully extended product using Klenow (calculated mass: 10621).
    Figure Legend Snippet: Primer extension using AHP dUTP (1.5 h). ( A ) Template T2 and primer P3. ( B ) Twenty percent denaturing PAGE analysis of reactions using Gotaq (Go, 72°C), Klenow (Kl, 37°C), KOD (KO, 72°C) and Therminator™ II (Th, 72°C) polymerases. Lane P: primer P3. ( C ) Reactions using Gotaq polymerase at 60°C. ( D ) Mass spectrum of AHP-modified fully extended product using Klenow (calculated mass: 10621).

    Techniques Used: Polyacrylamide Gel Electrophoresis, Modification

    14) Product Images from "Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme"

    Article Title: Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0038371

    Reverse transcriptase assays. A. Fluorogenic assay. RT activity was measured by detection of RNA:DNA heteroduplex by fluorescence of EvaGreen binding. Oligo dT primed poly A was incubated at 37°C and 65°C in the presence of indicated Pol enzymes in manufacturer recommended buffers and dTTP. Fluorescence measurements were obtained every 6 seconds for 10 minutes. The initial slopes from a plot of RFU vs. time in seconds were determined by linear least square regression from 30 to 150 seconds at 37°C and from 30 to 90 seconds at 65°C. Error bars are standard error of regression slope. B. RT primer extension assay. HEX-labeled dT 20 primed poly A was incubated 10 minutes at 37°C and then 10 minutes at 65°C in the presence of indicated Pol enzymes and dTTP in manufacturer recommended buffers. Primer extension products were resolved by 10% denaturing PAGE and imaged on a Molecular Imager FX (Bio-Rad). Left facing triangle indicates migration of unextended dT20 primer and asterisk indicates bromophenol blue dye front. C. RT MS2-specific primer extension. 5′-labeled primer was annealed to MS2 RNA and incubated 10 minutes at 37°C and then 30 minutes at 65°C in the presence of indicated Pol enzymes with dNTPS (N = A,C,G,T) in manufacturer recommended buffers. Primer extension products were resolved by 5% denaturing PAGE. Lane 1 No RNA+MMLV RT; Lane 2: MS2 RNA No RT; Lane 3 MS2 RNA+MMLV RT, Lane 3 MS2 RNA+3173 Pol. Molecular weight in bases indicted. Red Arrow: ∼650 base MMLV extension product. Blue Arrow: ∼715 base PyroScript extension product. Green arrow: Non-templated MMLV reaction product.
    Figure Legend Snippet: Reverse transcriptase assays. A. Fluorogenic assay. RT activity was measured by detection of RNA:DNA heteroduplex by fluorescence of EvaGreen binding. Oligo dT primed poly A was incubated at 37°C and 65°C in the presence of indicated Pol enzymes in manufacturer recommended buffers and dTTP. Fluorescence measurements were obtained every 6 seconds for 10 minutes. The initial slopes from a plot of RFU vs. time in seconds were determined by linear least square regression from 30 to 150 seconds at 37°C and from 30 to 90 seconds at 65°C. Error bars are standard error of regression slope. B. RT primer extension assay. HEX-labeled dT 20 primed poly A was incubated 10 minutes at 37°C and then 10 minutes at 65°C in the presence of indicated Pol enzymes and dTTP in manufacturer recommended buffers. Primer extension products were resolved by 10% denaturing PAGE and imaged on a Molecular Imager FX (Bio-Rad). Left facing triangle indicates migration of unextended dT20 primer and asterisk indicates bromophenol blue dye front. C. RT MS2-specific primer extension. 5′-labeled primer was annealed to MS2 RNA and incubated 10 minutes at 37°C and then 30 minutes at 65°C in the presence of indicated Pol enzymes with dNTPS (N = A,C,G,T) in manufacturer recommended buffers. Primer extension products were resolved by 5% denaturing PAGE. Lane 1 No RNA+MMLV RT; Lane 2: MS2 RNA No RT; Lane 3 MS2 RNA+MMLV RT, Lane 3 MS2 RNA+3173 Pol. Molecular weight in bases indicted. Red Arrow: ∼650 base MMLV extension product. Blue Arrow: ∼715 base PyroScript extension product. Green arrow: Non-templated MMLV reaction product.

    Techniques Used: Activity Assay, Fluorescence, Binding Assay, Incubation, Primer Extension Assay, Labeling, Polyacrylamide Gel Electrophoresis, Migration, Molecular Weight

    RT-PCR detection of human transcript RNAs. Beta-actin, beta2-microglobulin and cyclophilin target sequences of the indicated sizes were amplified from human liver total RNA using the primers described in Table 1 . Shown are products of two step reactions where either MMLV RT or 3173 Pol were used for first strand cDNA synthesis, as indicated. Taq Pol was used for PCR. Products were resolved on a 1% agarose gel.
    Figure Legend Snippet: RT-PCR detection of human transcript RNAs. Beta-actin, beta2-microglobulin and cyclophilin target sequences of the indicated sizes were amplified from human liver total RNA using the primers described in Table 1 . Shown are products of two step reactions where either MMLV RT or 3173 Pol were used for first strand cDNA synthesis, as indicated. Taq Pol was used for PCR. Products were resolved on a 1% agarose gel.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Two step RT-PCR comparing 3173 Pol and MMLV RT. A. MS2 viral RNA and B., C. total human liver RNA were reverse transcribed using either 3173 Pol or MMLV RT and then PCR amplified using Taq Polymerase. Target amplicons : A. MS2 RNA phage 77 bp amplicon, 2% gel, B. Human beta-actin 144 bp amplicon, 2% gel, C. Human beta-actin 821 bp amplicon, 1% gel. Lanes : 1 , 11 : oligo dT primer; 2–4 , 12–14 : Gene specific primers; 5 , 15 : random hexamers; 6 , 16 : random nonamers; 7 , 17 : No primer plus RT; 8 : No RT enzyme; 9 : PCR No Target Control; 10 : Molecular Weight Marker (MW), 100 bp (50 bp lowest) for Panels A, B and 1000 bp (300, 500, 700 lowest) for Panel C. Correct PCR product size indicated by black triangle.
    Figure Legend Snippet: Two step RT-PCR comparing 3173 Pol and MMLV RT. A. MS2 viral RNA and B., C. total human liver RNA were reverse transcribed using either 3173 Pol or MMLV RT and then PCR amplified using Taq Polymerase. Target amplicons : A. MS2 RNA phage 77 bp amplicon, 2% gel, B. Human beta-actin 144 bp amplicon, 2% gel, C. Human beta-actin 821 bp amplicon, 1% gel. Lanes : 1 , 11 : oligo dT primer; 2–4 , 12–14 : Gene specific primers; 5 , 15 : random hexamers; 6 , 16 : random nonamers; 7 , 17 : No primer plus RT; 8 : No RT enzyme; 9 : PCR No Target Control; 10 : Molecular Weight Marker (MW), 100 bp (50 bp lowest) for Panels A, B and 1000 bp (300, 500, 700 lowest) for Panel C. Correct PCR product size indicated by black triangle.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Molecular Weight, Marker

    15) Product Images from "Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme"

    Article Title: Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0038371

    Reverse transcriptase assays. A. Fluorogenic assay. RT activity was measured by detection of RNA:DNA heteroduplex by fluorescence of EvaGreen binding. Oligo dT primed poly A was incubated at 37°C and 65°C in the presence of indicated Pol enzymes in manufacturer recommended buffers and dTTP. Fluorescence measurements were obtained every 6 seconds for 10 minutes. The initial slopes from a plot of RFU vs. time in seconds were determined by linear least square regression from 30 to 150 seconds at 37°C and from 30 to 90 seconds at 65°C. Error bars are standard error of regression slope. B. RT primer extension assay. HEX-labeled dT 20 primed poly A was incubated 10 minutes at 37°C and then 10 minutes at 65°C in the presence of indicated Pol enzymes and dTTP in manufacturer recommended buffers. Primer extension products were resolved by 10% denaturing PAGE and imaged on a Molecular Imager FX (Bio-Rad). Left facing triangle indicates migration of unextended dT20 primer and asterisk indicates bromophenol blue dye front. C. RT MS2-specific primer extension. 5′-labeled primer was annealed to MS2 RNA and incubated 10 minutes at 37°C and then 30 minutes at 65°C in the presence of indicated Pol enzymes with dNTPS (N = A,C,G,T) in manufacturer recommended buffers. Primer extension products were resolved by 5% denaturing PAGE. Lane 1 No RNA+MMLV RT; Lane 2: MS2 RNA No RT; Lane 3 MS2 RNA+MMLV RT, Lane 3 MS2 RNA+3173 Pol. Molecular weight in bases indicted. Red Arrow: ∼650 base MMLV extension product. Blue Arrow: ∼715 base PyroScript extension product. Green arrow: Non-templated MMLV reaction product.
    Figure Legend Snippet: Reverse transcriptase assays. A. Fluorogenic assay. RT activity was measured by detection of RNA:DNA heteroduplex by fluorescence of EvaGreen binding. Oligo dT primed poly A was incubated at 37°C and 65°C in the presence of indicated Pol enzymes in manufacturer recommended buffers and dTTP. Fluorescence measurements were obtained every 6 seconds for 10 minutes. The initial slopes from a plot of RFU vs. time in seconds were determined by linear least square regression from 30 to 150 seconds at 37°C and from 30 to 90 seconds at 65°C. Error bars are standard error of regression slope. B. RT primer extension assay. HEX-labeled dT 20 primed poly A was incubated 10 minutes at 37°C and then 10 minutes at 65°C in the presence of indicated Pol enzymes and dTTP in manufacturer recommended buffers. Primer extension products were resolved by 10% denaturing PAGE and imaged on a Molecular Imager FX (Bio-Rad). Left facing triangle indicates migration of unextended dT20 primer and asterisk indicates bromophenol blue dye front. C. RT MS2-specific primer extension. 5′-labeled primer was annealed to MS2 RNA and incubated 10 minutes at 37°C and then 30 minutes at 65°C in the presence of indicated Pol enzymes with dNTPS (N = A,C,G,T) in manufacturer recommended buffers. Primer extension products were resolved by 5% denaturing PAGE. Lane 1 No RNA+MMLV RT; Lane 2: MS2 RNA No RT; Lane 3 MS2 RNA+MMLV RT, Lane 3 MS2 RNA+3173 Pol. Molecular weight in bases indicted. Red Arrow: ∼650 base MMLV extension product. Blue Arrow: ∼715 base PyroScript extension product. Green arrow: Non-templated MMLV reaction product.

    Techniques Used: Activity Assay, Fluorescence, Binding Assay, Incubation, Primer Extension Assay, Labeling, Polyacrylamide Gel Electrophoresis, Migration, Molecular Weight

    RT-PCR detection of human transcript RNAs. Beta-actin, beta2-microglobulin and cyclophilin target sequences of the indicated sizes were amplified from human liver total RNA using the primers described in Table 1 . Shown are products of two step reactions where either MMLV RT or 3173 Pol were used for first strand cDNA synthesis, as indicated. Taq Pol was used for PCR. Products were resolved on a 1% agarose gel.
    Figure Legend Snippet: RT-PCR detection of human transcript RNAs. Beta-actin, beta2-microglobulin and cyclophilin target sequences of the indicated sizes were amplified from human liver total RNA using the primers described in Table 1 . Shown are products of two step reactions where either MMLV RT or 3173 Pol were used for first strand cDNA synthesis, as indicated. Taq Pol was used for PCR. Products were resolved on a 1% agarose gel.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Two step RT-PCR comparing 3173 Pol and MMLV RT. A. MS2 viral RNA and B., C. total human liver RNA were reverse transcribed using either 3173 Pol or MMLV RT and then PCR amplified using Taq Polymerase. Target amplicons : A. MS2 RNA phage 77 bp amplicon, 2% gel, B. Human beta-actin 144 bp amplicon, 2% gel, C. Human beta-actin 821 bp amplicon, 1% gel. Lanes : 1 , 11 : oligo dT primer; 2–4 , 12–14 : Gene specific primers; 5 , 15 : random hexamers; 6 , 16 : random nonamers; 7 , 17 : No primer plus RT; 8 : No RT enzyme; 9 : PCR No Target Control; 10 : Molecular Weight Marker (MW), 100 bp (50 bp lowest) for Panels A, B and 1000 bp (300, 500, 700 lowest) for Panel C. Correct PCR product size indicated by black triangle.
    Figure Legend Snippet: Two step RT-PCR comparing 3173 Pol and MMLV RT. A. MS2 viral RNA and B., C. total human liver RNA were reverse transcribed using either 3173 Pol or MMLV RT and then PCR amplified using Taq Polymerase. Target amplicons : A. MS2 RNA phage 77 bp amplicon, 2% gel, B. Human beta-actin 144 bp amplicon, 2% gel, C. Human beta-actin 821 bp amplicon, 1% gel. Lanes : 1 , 11 : oligo dT primer; 2–4 , 12–14 : Gene specific primers; 5 , 15 : random hexamers; 6 , 16 : random nonamers; 7 , 17 : No primer plus RT; 8 : No RT enzyme; 9 : PCR No Target Control; 10 : Molecular Weight Marker (MW), 100 bp (50 bp lowest) for Panels A, B and 1000 bp (300, 500, 700 lowest) for Panel C. Correct PCR product size indicated by black triangle.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Molecular Weight, Marker

    16) Product Images from "Integrated microRNA and mRNA analysis in the pathogenic filamentous fungus Trichophyton rubrum"

    Article Title: Integrated microRNA and mRNA analysis in the pathogenic filamentous fungus Trichophyton rubrum

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-5316-3

    Validation of RNA-Seq results by qRT-PCR. Three biological replicates were performed. * indicates significant difference of milRNA/mRNA expression level in conidial vs. mycelial stages (*: P
    Figure Legend Snippet: Validation of RNA-Seq results by qRT-PCR. Three biological replicates were performed. * indicates significant difference of milRNA/mRNA expression level in conidial vs. mycelial stages (*: P

    Techniques Used: RNA Sequencing Assay, Quantitative RT-PCR, Expressing

    17) Product Images from "Transcriptome-Wide Analysis of Hepatitis B Virus-Mediated Changes to Normal Hepatocyte Gene Expression"

    Article Title: Transcriptome-Wide Analysis of Hepatitis B Virus-Mediated Changes to Normal Hepatocyte Gene Expression

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005438

    Confirmation of differentially expressed genes by qRT-PCR. Expression of genes from the
    Figure Legend Snippet: Confirmation of differentially expressed genes by qRT-PCR. Expression of genes from the "HBV-only" and "HBV-specific" was confirmed by qRT-PCR (bars labeled qPCR). Expression is presented as fold change of AdGFP-HBV-infected cells compared to AdGFP-infected cells at the indicated time point. Fold change using the RPKM values described in the primary RNA-seq dataset was included for comparison of expression patterns (bars labeled RPKM).

    Techniques Used: Quantitative RT-PCR, Expressing, Labeling, Real-time Polymerase Chain Reaction, Infection, RNA Sequencing Assay

    18) Product Images from "Conditionally Immortalized Mouse Embryonic Fibroblasts Retain Proliferative Activity without Compromising Multipotent Differentiation Potential"

    Article Title: Conditionally Immortalized Mouse Embryonic Fibroblasts Retain Proliferative Activity without Compromising Multipotent Differentiation Potential

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0032428

    Induction of osteogenic, chondrogenic, and adipogenic lineage markers in iMEFs. ( A ) Expression of lineage-specific regulators in iMEFs stimulated by BMP9. Subconfluent iMEF cells were infected with AdBMP9 or AdGFP. Total RNA was isolated at the indicated time points and subjected to RT-PCR reactions. The cDNA products were used as templates for semi-quantitative amplification of mouse Runx2, Sox9 and PPARγ2 transcripts. All samples were normalized with GAPDH expression levels. ( B ) and ( C ) Induction of early osteogenic marker alkaline phosphatase (ALP) in primary MEFs and iMEFs. Subconfluent primary MEFs and iMEFs were infected with AdBMP9 or AdGFP. ALP activity was histochemically stained on day 5 (B) or quantitatively determined at days 3, 5 and 7 (C). The ALP activity was normalized with total cellular protein (TCP) (C). ( D ) Matrix mineralization assessed with Alizarin Red S staining. Subconfluent MEFs and iMEFs were infected with AdBMP9 or AdGFP for 14 days. Cells were fixed and stained with Alizarin Red S. ( E ) Adipogenic differentiation assessed with Oil Red O staining. Subconfluent iMEFs were infected with AdBMP9, AdPPARγ2, or AdGFP for 10 days. Cells were fixed and stained with Oil Red O staining ( panels a and b ), or stained for ALP activity, followed by Oil-Red O staining ( panels c and d ). ( F ) Osteogenic and adipogenic differentiation of immortalized human bone marrow stromal stem cells. Subconfluent cells were infected with AdBMP9 or AdGFP. ALP staining was carried out at day 7 ( a ) while Oil-red O staining was done at day 14 ( b ). Each assay condition was done in triplicate. The assays were repeated in at least two independent batches. Representative results are shown.
    Figure Legend Snippet: Induction of osteogenic, chondrogenic, and adipogenic lineage markers in iMEFs. ( A ) Expression of lineage-specific regulators in iMEFs stimulated by BMP9. Subconfluent iMEF cells were infected with AdBMP9 or AdGFP. Total RNA was isolated at the indicated time points and subjected to RT-PCR reactions. The cDNA products were used as templates for semi-quantitative amplification of mouse Runx2, Sox9 and PPARγ2 transcripts. All samples were normalized with GAPDH expression levels. ( B ) and ( C ) Induction of early osteogenic marker alkaline phosphatase (ALP) in primary MEFs and iMEFs. Subconfluent primary MEFs and iMEFs were infected with AdBMP9 or AdGFP. ALP activity was histochemically stained on day 5 (B) or quantitatively determined at days 3, 5 and 7 (C). The ALP activity was normalized with total cellular protein (TCP) (C). ( D ) Matrix mineralization assessed with Alizarin Red S staining. Subconfluent MEFs and iMEFs were infected with AdBMP9 or AdGFP for 14 days. Cells were fixed and stained with Alizarin Red S. ( E ) Adipogenic differentiation assessed with Oil Red O staining. Subconfluent iMEFs were infected with AdBMP9, AdPPARγ2, or AdGFP for 10 days. Cells were fixed and stained with Oil Red O staining ( panels a and b ), or stained for ALP activity, followed by Oil-Red O staining ( panels c and d ). ( F ) Osteogenic and adipogenic differentiation of immortalized human bone marrow stromal stem cells. Subconfluent cells were infected with AdBMP9 or AdGFP. ALP staining was carried out at day 7 ( a ) while Oil-red O staining was done at day 14 ( b ). Each assay condition was done in triplicate. The assays were repeated in at least two independent batches. Representative results are shown.

    Techniques Used: Expressing, Infection, Isolation, Reverse Transcription Polymerase Chain Reaction, Amplification, Marker, ALP Assay, Activity Assay, Staining

    19) Product Images from "Late steps of ribosome assembly in E. coli are sensitive to a severe heat stress but are assisted by the HSP70 chaperone machine †"

    Article Title: Late steps of ribosome assembly in E. coli are sensitive to a severe heat stress but are assisted by the HSP70 chaperone machine †

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1049

    ( A ) Schematic processing of the p1 16S rRNA. The extra-sequences of 115 nt and 33 nt, flanking the m 16S rRNA at its 5′ and 3′ ends, respectively, are shown on a grey background. RA and FA are the primers used for 3′5′ RACE analysis. The site of annealing of RA to m 16S rRNA, and that of FA to the reverse complement of the m 16S rRNA, are indicated by arrows. Figure not drawn to scale. ( B ) Expected sizes in bp of the RT-PCR products (amplicons) obtained from the different species of 16S rRNA ( p1 , p2 , p3 and m ) by 3′5′ RACE. ( C–F ) Agarose gel electrophoresis of RT-PCR products obtained by 3′5′ RACE from total RNA isolated from MC4100 bacteria grown at 30°C (C), or 44°C (D), or 45°C (E) or 46°C (F). Each RNA sample was thermo-denatured (lanes b), or not (lanes a) prior to the 3′5′ ligation. The sizes (in bp) of the molecular weight markers are indicated to the left of each gel (M). ( G ) The thermodenaturation step dissociates the complementary sequences present at the 3′ and 5′ends of the p1 16S rRNA, and therefore offers to all the 16S rRNA species an equal chance to access to the T4 RNA ligase.
    Figure Legend Snippet: ( A ) Schematic processing of the p1 16S rRNA. The extra-sequences of 115 nt and 33 nt, flanking the m 16S rRNA at its 5′ and 3′ ends, respectively, are shown on a grey background. RA and FA are the primers used for 3′5′ RACE analysis. The site of annealing of RA to m 16S rRNA, and that of FA to the reverse complement of the m 16S rRNA, are indicated by arrows. Figure not drawn to scale. ( B ) Expected sizes in bp of the RT-PCR products (amplicons) obtained from the different species of 16S rRNA ( p1 , p2 , p3 and m ) by 3′5′ RACE. ( C–F ) Agarose gel electrophoresis of RT-PCR products obtained by 3′5′ RACE from total RNA isolated from MC4100 bacteria grown at 30°C (C), or 44°C (D), or 45°C (E) or 46°C (F). Each RNA sample was thermo-denatured (lanes b), or not (lanes a) prior to the 3′5′ ligation. The sizes (in bp) of the molecular weight markers are indicated to the left of each gel (M). ( G ) The thermodenaturation step dissociates the complementary sequences present at the 3′ and 5′ends of the p1 16S rRNA, and therefore offers to all the 16S rRNA species an equal chance to access to the T4 RNA ligase.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Isolation, Ligation, Molecular Weight

    20) Product Images from "Alternative promoters of Peg3 with maternal specificity"

    Article Title: Alternative promoters of Peg3 with maternal specificity

    Journal: Scientific Reports

    doi: 10.1038/srep24438

    Upstream alternative promoter of human PEG3 . ( A ) A schematic of the human PEG3 locus with RT-PCR primer combinations. Gray and black boxes indicate the exons of MIMT1 and PEG3 , respectively. Transcriptional direction for each gene is represented using arrows with corresponding colors. The black arrow indicates the directionality of Ex2-R1, the primer used for cDNA synthesis. The open oval represents the alternative 1 st exon U1. ( B ) 5′ RACE analysis for upstream exons of PEG3 . The Ex2-R2 indicates the anchoring primer used for nested PCR after 5′ RACE. This primer was coupled with U1 and E1 specific primers to amplify the respective exons. The percentage of transcripts preferred by the adult human brain was calculated by counting the sequences specific for U1 and E1. ( C ) RT-PCR analysis of upstream alternative exons in human tissues. The RT-PCR panel shows the expression patterns of PEG3 using cDNA from AB (adult brain), NB (neonate brain), HT (adult heart), LV (adult liver), KD (adult kidney) and PC (adult placenta). U1-Ex2R2 and E1-Ex2R2 primer combinations amplified the upstream exons of PEG3 to show the expression profile preferred by each 1 st exon. The Ex1-Ex2 primer combination amplified the expression pattern of MIMT1 in the AB and NB. Equal amounts of total RNA were used for RT-PCR, which were normalized by β-actin expression levels.
    Figure Legend Snippet: Upstream alternative promoter of human PEG3 . ( A ) A schematic of the human PEG3 locus with RT-PCR primer combinations. Gray and black boxes indicate the exons of MIMT1 and PEG3 , respectively. Transcriptional direction for each gene is represented using arrows with corresponding colors. The black arrow indicates the directionality of Ex2-R1, the primer used for cDNA synthesis. The open oval represents the alternative 1 st exon U1. ( B ) 5′ RACE analysis for upstream exons of PEG3 . The Ex2-R2 indicates the anchoring primer used for nested PCR after 5′ RACE. This primer was coupled with U1 and E1 specific primers to amplify the respective exons. The percentage of transcripts preferred by the adult human brain was calculated by counting the sequences specific for U1 and E1. ( C ) RT-PCR analysis of upstream alternative exons in human tissues. The RT-PCR panel shows the expression patterns of PEG3 using cDNA from AB (adult brain), NB (neonate brain), HT (adult heart), LV (adult liver), KD (adult kidney) and PC (adult placenta). U1-Ex2R2 and E1-Ex2R2 primer combinations amplified the upstream exons of PEG3 to show the expression profile preferred by each 1 st exon. The Ex1-Ex2 primer combination amplified the expression pattern of MIMT1 in the AB and NB. Equal amounts of total RNA were used for RT-PCR, which were normalized by β-actin expression levels.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Nested PCR, Expressing, Amplification

    21) Product Images from "The Chromatin Remodeling Factor CHD5 Is a Transcriptional Repressor of WEE1"

    Article Title: The Chromatin Remodeling Factor CHD5 Is a Transcriptional Repressor of WEE1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0108066

    CHD5 expression leads to repression of WEE1 . (A) The neuroblastoma line KELLY or (B) 293T-derived cells were transiently transfected with vector (vec.) or the vector containing the human CHD5 cDNA. In the case of the 293T-derived cells, two amounts of CHD5 cDNA were transfected (see Methods ). Two days after transfection, the cells were harvested and total RNA was prepared. Quantitative RT-PCR was performed to monitor the levels of the CHD5 and WEE1 transcripts. The values shown are normalized to a 5S rRNA housekeeping transcript. (C) CHD5 levels were reduced siRNA-mediated knockdown in PANC-1 cells. As a control (ctrl), a scramble siRNA was transfected in parallel. The levels of CHD5 protein were detected by western blotting (left) and the levels of WEE1 mRNA were analyzed by qRT-PCR. Error bars represent SD [n = 3].
    Figure Legend Snippet: CHD5 expression leads to repression of WEE1 . (A) The neuroblastoma line KELLY or (B) 293T-derived cells were transiently transfected with vector (vec.) or the vector containing the human CHD5 cDNA. In the case of the 293T-derived cells, two amounts of CHD5 cDNA were transfected (see Methods ). Two days after transfection, the cells were harvested and total RNA was prepared. Quantitative RT-PCR was performed to monitor the levels of the CHD5 and WEE1 transcripts. The values shown are normalized to a 5S rRNA housekeeping transcript. (C) CHD5 levels were reduced siRNA-mediated knockdown in PANC-1 cells. As a control (ctrl), a scramble siRNA was transfected in parallel. The levels of CHD5 protein were detected by western blotting (left) and the levels of WEE1 mRNA were analyzed by qRT-PCR. Error bars represent SD [n = 3].

    Techniques Used: Expressing, Derivative Assay, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot

    CHD5 co-purifies with the NuRD transcriptional repressor complex. (A) FLAG-tagged CHD4 and CHD5 were immunoprecipitated from a transiently transfected HEK293T-derived cell line, and the samples were analyzed by western blotting using the indicated antibodies. The empty vector was also transfected in parallel [Control]. (B) The HEK293T-derived line was transiently transfected with increasing amounts of the CHD5-expression plasmid. An empty vector was included where necessary to keep the final amount (15 µg) of transfected plasmid constant. Following transfection, cell extracts and RNA were prepared to measure CHD4 levels by western blotting and CHD4 and CHD5 mRNA levels by qRT-PCR.
    Figure Legend Snippet: CHD5 co-purifies with the NuRD transcriptional repressor complex. (A) FLAG-tagged CHD4 and CHD5 were immunoprecipitated from a transiently transfected HEK293T-derived cell line, and the samples were analyzed by western blotting using the indicated antibodies. The empty vector was also transfected in parallel [Control]. (B) The HEK293T-derived line was transiently transfected with increasing amounts of the CHD5-expression plasmid. An empty vector was included where necessary to keep the final amount (15 µg) of transfected plasmid constant. Following transfection, cell extracts and RNA were prepared to measure CHD4 levels by western blotting and CHD4 and CHD5 mRNA levels by qRT-PCR.

    Techniques Used: Immunoprecipitation, Transfection, Derivative Assay, Western Blot, Plasmid Preparation, Expressing, Quantitative RT-PCR

    22) Product Images from "The role of G-density in switch region repeats for immunoglobulin class switch recombination"

    Article Title: The role of G-density in switch region repeats for immunoglobulin class switch recombination

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku1100

    Overall G-density is important for efficient CSR. (A) 1F7 cells, which are resistant to puromycin and sensitive to ganciclovir, are transfected with both an exchange vector containing the sequence to be integrated (sequence of interest) and a CRE-expression vector. Cre mediates recombination between the exchange vector and the 1F7 chromosome at the corresponding LoxP sites. Cells that have successfully replaced the selection cassette by the sequence of interest would survive during ganciclovir selection, which are further screened with a puromycin sensitivity test and PCR. (B) Nine repeats of 80 bp oligonucleotides (top strand of duplex shown below the RIZ/REZ diagram) with 4 AGCT sites (orange) and different G-densities were used for the REZ in the synthetic switch regions. The top panel has 29% G-density on the non-template DNA strand, whereas the bottom panel has 46% G-density. The RIZ contains 5 G-clusters. (C) Fluorescence-activated cell sorting (FACS) analysis of CSR is shown. Each data symbol represents an independent clone (after subtraction of the IgA+ level for the same clone without CIT stimulation). The bars represent the mean of each group. (D) Germ-line transcription (GLT) Quantification. Healthy cells grown to 10 6 /ml were treated with 1 μg/ml anti-CD40, 5 ng/ml IL-4 and 0.5 ng/ml TGF-β1 (CIT) for 6 h. Total RNA was extracted, reverse-transcribed into cDNA and quantified by real-time PCR. β-Actin was used as an internal control. In parallel, the same cellular clones without CIT treatment were used to measure the background of GLT. The error bar represents the SEM of 3–5 independent clones.
    Figure Legend Snippet: Overall G-density is important for efficient CSR. (A) 1F7 cells, which are resistant to puromycin and sensitive to ganciclovir, are transfected with both an exchange vector containing the sequence to be integrated (sequence of interest) and a CRE-expression vector. Cre mediates recombination between the exchange vector and the 1F7 chromosome at the corresponding LoxP sites. Cells that have successfully replaced the selection cassette by the sequence of interest would survive during ganciclovir selection, which are further screened with a puromycin sensitivity test and PCR. (B) Nine repeats of 80 bp oligonucleotides (top strand of duplex shown below the RIZ/REZ diagram) with 4 AGCT sites (orange) and different G-densities were used for the REZ in the synthetic switch regions. The top panel has 29% G-density on the non-template DNA strand, whereas the bottom panel has 46% G-density. The RIZ contains 5 G-clusters. (C) Fluorescence-activated cell sorting (FACS) analysis of CSR is shown. Each data symbol represents an independent clone (after subtraction of the IgA+ level for the same clone without CIT stimulation). The bars represent the mean of each group. (D) Germ-line transcription (GLT) Quantification. Healthy cells grown to 10 6 /ml were treated with 1 μg/ml anti-CD40, 5 ng/ml IL-4 and 0.5 ng/ml TGF-β1 (CIT) for 6 h. Total RNA was extracted, reverse-transcribed into cDNA and quantified by real-time PCR. β-Actin was used as an internal control. In parallel, the same cellular clones without CIT treatment were used to measure the background of GLT. The error bar represents the SEM of 3–5 independent clones.

    Techniques Used: Transfection, Plasmid Preparation, Sequencing, Expressing, Selection, Polymerase Chain Reaction, Fluorescence, FACS, Real-time Polymerase Chain Reaction, Clone Assay

    23) Product Images from "Mechanistic Consequences of hnRNP C Binding to Both RNA Termini of Poliovirus Negative-Strand RNA Intermediates ▿"

    Article Title: Mechanistic Consequences of hnRNP C Binding to Both RNA Termini of Poliovirus Negative-Strand RNA Intermediates ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02198-09

    Poliovirus growth kinetics and viral-RNA accumulation in hnRNP C-depleted HeLa cells. Knockdown of hnRNP C via retrovirus infection and selection of infected cells is described in Materials and Methods. Plaque assays were carried out as described previously
    Figure Legend Snippet: Poliovirus growth kinetics and viral-RNA accumulation in hnRNP C-depleted HeLa cells. Knockdown of hnRNP C via retrovirus infection and selection of infected cells is described in Materials and Methods. Plaque assays were carried out as described previously

    Techniques Used: Infection, Selection

    The cellular protein hnRNP C binds the 5′ end of poliovirus negative-strand RNA. (A) Increased concentrations of hnRNP C proteins in HeLa S10 cytoplasmic extracts generated from poliovirus-infected cells. Cytoplasmic extracts of HeLa cells were
    Figure Legend Snippet: The cellular protein hnRNP C binds the 5′ end of poliovirus negative-strand RNA. (A) Increased concentrations of hnRNP C proteins in HeLa S10 cytoplasmic extracts generated from poliovirus-infected cells. Cytoplasmic extracts of HeLa cells were

    Techniques Used: Generated, Infection

    24) Product Images from "RNA-Binding Protein RBP-P Is Required for Glutelin and Prolamine mRNA Localization in Rice Endosperm Cells [OPEN]"

    Article Title: RNA-Binding Protein RBP-P Is Required for Glutelin and Prolamine mRNA Localization in Rice Endosperm Cells [OPEN]

    Journal: The Plant Cell

    doi: 10.1105/tpc.18.00321

    Hypothetical Function and Structure of the Multiple Domains of RBP-P. (A) Predicted structure of RBP-P. Right panel is a 90° left rotation view of the left panel. The two RRM motifs, RRM1 and RRM2, are labeled. The residues Gly-401, Gly-373, and Ala-252 are indicated by arrows. (B) Schematic representation of the structure and function of the RRM domain and the N and C terminus in RBP-P. The N terminus of RBP-P contains the Ala-rich and Glu-rich motifs of unknown function. The RNA binding module consists of two RRM domains, ∼80 amino acids in length, the interdomain linker, and a short sequence in the C terminus. This protein module is responsible for RNA binding activity as well as dimerization with another RBP-P molecule or other proteins, such as RBP-L. The glycine-rich C terminus is involved in protein-protein interactions.
    Figure Legend Snippet: Hypothetical Function and Structure of the Multiple Domains of RBP-P. (A) Predicted structure of RBP-P. Right panel is a 90° left rotation view of the left panel. The two RRM motifs, RRM1 and RRM2, are labeled. The residues Gly-401, Gly-373, and Ala-252 are indicated by arrows. (B) Schematic representation of the structure and function of the RRM domain and the N and C terminus in RBP-P. The N terminus of RBP-P contains the Ala-rich and Glu-rich motifs of unknown function. The RNA binding module consists of two RRM domains, ∼80 amino acids in length, the interdomain linker, and a short sequence in the C terminus. This protein module is responsible for RNA binding activity as well as dimerization with another RBP-P molecule or other proteins, such as RBP-L. The glycine-rich C terminus is involved in protein-protein interactions.

    Techniques Used: Labeling, RNA Binding Assay, Sequencing, Activity Assay

    Mutations of RBP-P Confer Partial Mislocalization of Prolamine and Glutelin mRNAs. (A) Schematic representation of RBP-P point mutation sites in P1MH, P2MH, and P3MH mutants. A/E, alanine (A) and glutamic acid (E) enrichment in the N-terminal domain; G, glycine (G) enrichment in the C-terminal domain. aa, amino acids. (B) and (C) Results of in situ RT-PCR to assess the location of prolamine (B) and glutelin (C) mRNAs to subdomains of the cortical-ER in wild-type and RBP-P mutant lines. In situ RT-PCR was performed directly on rice grain sections in the presence of Alexa-488-UTP (green) to label mRNAs. PB-ER was stained using Rhodamine B dye (red). Note that prolamine and glutelin mRNAs are localized to the PB-ER and Cis-ER, respectively, in the wild type. In P1MH and P3MH, the location of both mRNA species is disrupted and instead distributed to both PB-ER and Cis-ER. Bar = 5 µm.
    Figure Legend Snippet: Mutations of RBP-P Confer Partial Mislocalization of Prolamine and Glutelin mRNAs. (A) Schematic representation of RBP-P point mutation sites in P1MH, P2MH, and P3MH mutants. A/E, alanine (A) and glutamic acid (E) enrichment in the N-terminal domain; G, glycine (G) enrichment in the C-terminal domain. aa, amino acids. (B) and (C) Results of in situ RT-PCR to assess the location of prolamine (B) and glutelin (C) mRNAs to subdomains of the cortical-ER in wild-type and RBP-P mutant lines. In situ RT-PCR was performed directly on rice grain sections in the presence of Alexa-488-UTP (green) to label mRNAs. PB-ER was stained using Rhodamine B dye (red). Note that prolamine and glutelin mRNAs are localized to the PB-ER and Cis-ER, respectively, in the wild type. In P1MH and P3MH, the location of both mRNA species is disrupted and instead distributed to both PB-ER and Cis-ER. Bar = 5 µm.

    Techniques Used: Mutagenesis, In Situ, Reverse Transcription Polymerase Chain Reaction, Staining

    Identification of RBP-L and RBP-208 as Interacting Partners of RBP-P. (A) Schematic structure of RBP-P, RBP-L, and RBP-208. A/E rich, alanine and glutamic acid-rich domain; G-rich, glycine-rich domain; P-rich, proline-rich domain; Q-rich, glutamine-rich domain. a.a., amino acids. (B) Interactions of RBP-P with RBP-L and with RBP-208 as revealed by Y2H assay. Yeast cells were cotransfected with pGBK and pGAD constructs carrying the corresponding genes and grown on SD/-Leu/-Trp or SD/-Leu/-Trp/-His/-Ade/+ 3-AT. (C) Co - IP analyses using affinity-purified antibodies specific against each RBP. Input, starting rice grain lysate; Ub, unbound fraction; B, elution of bound fraction from IP. (D) In vivo interactions among RBP-P, RBP-L, and RBP-208 as revealed by BiFC analysis in BY-2 protoplasts. —, Original empty vector as negative control. Bars = 20 μm.
    Figure Legend Snippet: Identification of RBP-L and RBP-208 as Interacting Partners of RBP-P. (A) Schematic structure of RBP-P, RBP-L, and RBP-208. A/E rich, alanine and glutamic acid-rich domain; G-rich, glycine-rich domain; P-rich, proline-rich domain; Q-rich, glutamine-rich domain. a.a., amino acids. (B) Interactions of RBP-P with RBP-L and with RBP-208 as revealed by Y2H assay. Yeast cells were cotransfected with pGBK and pGAD constructs carrying the corresponding genes and grown on SD/-Leu/-Trp or SD/-Leu/-Trp/-His/-Ade/+ 3-AT. (C) Co - IP analyses using affinity-purified antibodies specific against each RBP. Input, starting rice grain lysate; Ub, unbound fraction; B, elution of bound fraction from IP. (D) In vivo interactions among RBP-P, RBP-L, and RBP-208 as revealed by BiFC analysis in BY-2 protoplasts. —, Original empty vector as negative control. Bars = 20 μm.

    Techniques Used: Y2H Assay, Construct, Co-Immunoprecipitation Assay, Affinity Purification, In Vivo, Bimolecular Fluorescence Complementation Assay, Plasmid Preparation, Negative Control

    Phenotype of P2MH Carrying a G373E Mutation in the RBP-P Gene. (A) Morphology of the wild type, P2MH (homozygous P2 mutant), and P2N (background genotype carrying normal RBP-P isolated from the heterozygous P2 mutant) during vegetative growth. The P2MH mutant is dwarf, has chlorophyll-deficient leaves, and produces fewer tillers than the wild type. (B) Direct comparison of the leaf blades of P2MH and P2N with the wild type. Box shows an enlarged image of a region of the P2MH leaf. (C) P2MH mutant showed late development of reproductive organs. When grains produced by wild-type and P2NH plants started to mature (turn brown), P2MH just began to “flower.” (D) and (E) Comparison of the tassels from the wild type, P2N, and P2MH. (E) is an enlarged view of the area enclosed in a rectangle in (D) . (F) Severe flowering defect in P2MH. Due to the abnormal development of flower structure, P2MH failed to produce grains. Bar = 1 cm.
    Figure Legend Snippet: Phenotype of P2MH Carrying a G373E Mutation in the RBP-P Gene. (A) Morphology of the wild type, P2MH (homozygous P2 mutant), and P2N (background genotype carrying normal RBP-P isolated from the heterozygous P2 mutant) during vegetative growth. The P2MH mutant is dwarf, has chlorophyll-deficient leaves, and produces fewer tillers than the wild type. (B) Direct comparison of the leaf blades of P2MH and P2N with the wild type. Box shows an enlarged image of a region of the P2MH leaf. (C) P2MH mutant showed late development of reproductive organs. When grains produced by wild-type and P2NH plants started to mature (turn brown), P2MH just began to “flower.” (D) and (E) Comparison of the tassels from the wild type, P2N, and P2MH. (E) is an enlarged view of the area enclosed in a rectangle in (D) . (F) Severe flowering defect in P2MH. Due to the abnormal development of flower structure, P2MH failed to produce grains. Bar = 1 cm.

    Techniques Used: Mutagenesis, Isolation, Produced

    Expression Level of Prolamine and Glutelin in Developing Rice Grains. (A) and (B) Expression level of prolamine (A) and glutelin (B) transcripts in wild-type, P1MH, and P3MH grains as revealed by RPKM values. (C) Total protein profile to reveal the expression of prolamine and glutelin proteins in wild-type, P1MH, and P3MH grains. Glutelin precursor, acidic and basic subunits, and prolamine are indicated. Note that the total accumulation levels of glutelin and prolamine are not altered in either RBP-P mutant.
    Figure Legend Snippet: Expression Level of Prolamine and Glutelin in Developing Rice Grains. (A) and (B) Expression level of prolamine (A) and glutelin (B) transcripts in wild-type, P1MH, and P3MH grains as revealed by RPKM values. (C) Total protein profile to reveal the expression of prolamine and glutelin proteins in wild-type, P1MH, and P3MH grains. Glutelin precursor, acidic and basic subunits, and prolamine are indicated. Note that the total accumulation levels of glutelin and prolamine are not altered in either RBP-P mutant.

    Techniques Used: Expressing, Mutagenesis

    Differentially Expressed Genes in RBP-P Mutants as Revealed by RNA-Seq Studies. (A) and (B) Venn diagrams showing the number of differentially expressed genes (A) and down- or upregulated genes (B) in P1MH and P3MH compared with the wild type. (C) .
    Figure Legend Snippet: Differentially Expressed Genes in RBP-P Mutants as Revealed by RNA-Seq Studies. (A) and (B) Venn diagrams showing the number of differentially expressed genes (A) and down- or upregulated genes (B) in P1MH and P3MH compared with the wild type. (C) .

    Techniques Used: RNA Sequencing Assay

    Gene Expression Profile Presented as a Clustered Heat Map and Volcano Plot. (A) Clustered heat map showing the genotype grouping of wild type and RBP-P mutants, P1MH and P3MH. (B) and (C) Volcano plots of differentially expressed genes in P1MH (B) and P3MH (C) compared with the wild type. The horizontal lines represent log 2 fold changes, and the vertical lines represent P value (−log 10 ). Differentially expressed genes were defined as having a log 2 fold change of > 1 and a P value of
    Figure Legend Snippet: Gene Expression Profile Presented as a Clustered Heat Map and Volcano Plot. (A) Clustered heat map showing the genotype grouping of wild type and RBP-P mutants, P1MH and P3MH. (B) and (C) Volcano plots of differentially expressed genes in P1MH (B) and P3MH (C) compared with the wild type. The horizontal lines represent log 2 fold changes, and the vertical lines represent P value (−log 10 ). Differentially expressed genes were defined as having a log 2 fold change of > 1 and a P value of

    Techniques Used: Expressing

    In Vivo Binding Activities of Wild-Type and Mutant RBP-P to Glutelin and Prolamine mRNAs. (A) Simplified representation of RNA-IP procedure. (B) Agarose gel-resolved cDNA products synthesized from RNAs associated with IPs generated by anti-RBP-P (α-RBPP) or anti-GFP (α-GFP). Total: PCR using cDNA synthesized from starting material for IP. −CT: Negative control using water as template. (C) Immunoblot of RNA-IP products from the wild type, P1MH, and P3MH using anti-RBP-P. Note that similar amounts of RBP-P were captured by IP of wild-type, P1MH, and P3MH grain extracts. Ip, input; Ub, unbound fraction; B, elution of bound fraction from IP. Arrowhead indicates the position of RBP-P on SDS-PAGE gel. (D) The relative enrichment of glutelin and prolamine mRNAs in IPs generated by anti-RBP-P antibodies of wild-type, P1, and P3 developing grain extracts. *P value of two-tailed t test
    Figure Legend Snippet: In Vivo Binding Activities of Wild-Type and Mutant RBP-P to Glutelin and Prolamine mRNAs. (A) Simplified representation of RNA-IP procedure. (B) Agarose gel-resolved cDNA products synthesized from RNAs associated with IPs generated by anti-RBP-P (α-RBPP) or anti-GFP (α-GFP). Total: PCR using cDNA synthesized from starting material for IP. −CT: Negative control using water as template. (C) Immunoblot of RNA-IP products from the wild type, P1MH, and P3MH using anti-RBP-P. Note that similar amounts of RBP-P were captured by IP of wild-type, P1MH, and P3MH grain extracts. Ip, input; Ub, unbound fraction; B, elution of bound fraction from IP. Arrowhead indicates the position of RBP-P on SDS-PAGE gel. (D) The relative enrichment of glutelin and prolamine mRNAs in IPs generated by anti-RBP-P antibodies of wild-type, P1, and P3 developing grain extracts. *P value of two-tailed t test

    Techniques Used: In Vivo, Binding Assay, Mutagenesis, Agarose Gel Electrophoresis, Synthesized, Generated, Polymerase Chain Reaction, Negative Control, SDS Page, Two Tailed Test

    25) Product Images from "Histone variant H2A.Z deposition and acetylation directs the canonical Notch signaling response"

    Article Title: Histone variant H2A.Z deposition and acetylation directs the canonical Notch signaling response

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky551

    H2A.Z acetylation (H2A.Zac) but not H2A.Z occupancy positively correlates with activation of Notch target genes. ( A ) Schematic representation of the NICD-inducible system established in MT cells. The NICD was fused to the estrogen receptor binding domain (NICD-ER) and retrovirally introduced into MT cells. The NICD-ER fusion protein is retained into the cytoplasm unless cells are treated with ( Z )-4-hydroxytamoxifen (4-OHT) that induces its nuclear translocation and activation of Notch target genes. ( B ) Hes1 and Il2ra Notch target genes are induced upon 4-OHT treatment of MT NICD-ER cells. Total RNA from MT NICD-ER cells, treated for 24 h with 4-OHT or EtOH as control, was reverse transcribed into cDNA and analyzed by qPCR using primers specific for Tbp, Hes1 or Il2ra . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD of three independent experiments. ( C ) H2A.Z acetylation (H2A.Zac) but not H2A.Z occupancy positively correlates with activation of Notch target genes. MT NICD-ER cells were treated for 24 h with 4-OHT or EtOH as control and subjected to ChIP analysis using antibodies against H2A.Z, H2A.Zac, H3 or IgG as control. The qPCR analysis was focused at the Notch-dependent enhancers (red squares) represented on the left ( Hes1 +0.6 kb and Il2ra -26 kb ). Chrom X was used as negative control ( Control ). Data were normalized to the positive control ( GAPDH 0 kb ) and, in the case of H2A.Zac/H2A.Z, the H2A.Zac signals were further normalized to H2A.Z. Shown is the mean ± SD of two independent experiments.
    Figure Legend Snippet: H2A.Z acetylation (H2A.Zac) but not H2A.Z occupancy positively correlates with activation of Notch target genes. ( A ) Schematic representation of the NICD-inducible system established in MT cells. The NICD was fused to the estrogen receptor binding domain (NICD-ER) and retrovirally introduced into MT cells. The NICD-ER fusion protein is retained into the cytoplasm unless cells are treated with ( Z )-4-hydroxytamoxifen (4-OHT) that induces its nuclear translocation and activation of Notch target genes. ( B ) Hes1 and Il2ra Notch target genes are induced upon 4-OHT treatment of MT NICD-ER cells. Total RNA from MT NICD-ER cells, treated for 24 h with 4-OHT or EtOH as control, was reverse transcribed into cDNA and analyzed by qPCR using primers specific for Tbp, Hes1 or Il2ra . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD of three independent experiments. ( C ) H2A.Z acetylation (H2A.Zac) but not H2A.Z occupancy positively correlates with activation of Notch target genes. MT NICD-ER cells were treated for 24 h with 4-OHT or EtOH as control and subjected to ChIP analysis using antibodies against H2A.Z, H2A.Zac, H3 or IgG as control. The qPCR analysis was focused at the Notch-dependent enhancers (red squares) represented on the left ( Hes1 +0.6 kb and Il2ra -26 kb ). Chrom X was used as negative control ( Control ). Data were normalized to the positive control ( GAPDH 0 kb ) and, in the case of H2A.Zac/H2A.Z, the H2A.Zac signals were further normalized to H2A.Z. Shown is the mean ± SD of two independent experiments.

    Techniques Used: Activation Assay, Binding Assay, Translocation Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Negative Control, Positive Control

    Histone variant H2A.Z has a negative impact on the expression of Notch target genes. ( A ) Histone Variant H2A.Z is efficiently depleted by CRISPR/Cas9 in MT cells. Whole Cell Extract (WCE) was prepared from wildtype ( Control ) or H2A.Z depleted (clones sgH2afv/H2afz #12 and sgH2afv/H2afz #20 ) MT cells and analysed by Western blotting. GAPDH was used as loading control. ( B ) Hes1 and Il2ra Notch target genes are upregulated upon depletion of H2A.Z. Total RNA from wildtype ( Control ) or H2A.Z depleted (clones sgH2afv/H2afz #12 and sgH2afv/H2afz #20 ) MT cells was reverse transcribed into cDNA and analysed by qPCR using primers specific for Tbp, Hes1 or Il2ra . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD of five independent experiments ([*] P
    Figure Legend Snippet: Histone variant H2A.Z has a negative impact on the expression of Notch target genes. ( A ) Histone Variant H2A.Z is efficiently depleted by CRISPR/Cas9 in MT cells. Whole Cell Extract (WCE) was prepared from wildtype ( Control ) or H2A.Z depleted (clones sgH2afv/H2afz #12 and sgH2afv/H2afz #20 ) MT cells and analysed by Western blotting. GAPDH was used as loading control. ( B ) Hes1 and Il2ra Notch target genes are upregulated upon depletion of H2A.Z. Total RNA from wildtype ( Control ) or H2A.Z depleted (clones sgH2afv/H2afz #12 and sgH2afv/H2afz #20 ) MT cells was reverse transcribed into cDNA and analysed by qPCR using primers specific for Tbp, Hes1 or Il2ra . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD of five independent experiments ([*] P

    Techniques Used: Variant Assay, Expressing, CRISPR, Western Blot, Real-time Polymerase Chain Reaction

    26) Product Images from "TLR4/NF-κB axis signaling pathway-dependent up-regulation of miR-625-5p contributes to human intervertebral disc degeneration by targeting COL1A1"

    Article Title: TLR4/NF-κB axis signaling pathway-dependent up-regulation of miR-625-5p contributes to human intervertebral disc degeneration by targeting COL1A1

    Journal: American Journal of Translational Research

    doi:

    Inhibition of LPS and pro-inflammatory cytokines repressed miR-625-5p levels but increased COL1A1 levels. hNPC cells were treated with 100 ng/mL LPS, 10 ng/mL recombinant TNF-α or 20 ng/mL recombinant IL-6, followed by treatment with 1 mM CLI095 (LPS inhibitor), 1 mg/mL siltuximab (IL-6 inhibitor) or 1 mg/mL D2E7 (TNF-α inhibitor), respectively. The obtained cells were subjected to RNA and protein extraction to determine the miR-625-5p, COL1A1 mRNA and protein levels. (A-C) The relative levels of (A) miR-625-5p, (B) COL1A1 mRNA and (C) COL1A1 protein following LPS and CLI095 treatment. *** P
    Figure Legend Snippet: Inhibition of LPS and pro-inflammatory cytokines repressed miR-625-5p levels but increased COL1A1 levels. hNPC cells were treated with 100 ng/mL LPS, 10 ng/mL recombinant TNF-α or 20 ng/mL recombinant IL-6, followed by treatment with 1 mM CLI095 (LPS inhibitor), 1 mg/mL siltuximab (IL-6 inhibitor) or 1 mg/mL D2E7 (TNF-α inhibitor), respectively. The obtained cells were subjected to RNA and protein extraction to determine the miR-625-5p, COL1A1 mRNA and protein levels. (A-C) The relative levels of (A) miR-625-5p, (B) COL1A1 mRNA and (C) COL1A1 protein following LPS and CLI095 treatment. *** P

    Techniques Used: Inhibition, Recombinant, Protein Extraction

    COL1A1 is a direct target of miR-625-5p. (A) Schematic representation of the 3’-UTRs of COL1A1 containing two putative miR-625-5p binding sites. These two binding sites are indicated as 1 and 2 and are shown with red lines. The seed locations of miR-625-5p are indicated with red font, while the mutated locations are indicated with blue font. (B-D) COL1A1 can be targeted by miR-625-5p at site 1. The coding sequences of COL1A1 and its wild-type (WT) and mutated (Mut1 and 2) 3’-UTRs were cloned into the pCDNA3 vector. Then, the following combinations of plasmids were transfected into hNPC cells: miR-NC; miR-NC + COL1A1-3’-UTR WT ; miR-NC + COL1A1-3’-UTR Mut-1 ; miR-NC + COL1A1-3’-UTR Mut-2 ; miR-625-5p-mimic; miR-625-5p-mimic + COL1A1-3’-UTR WT ; miR-625-5p-mimic + COL1A1-3’-UTR Mut-1 ; anti-miR-625-5p; anti-miR-625-5p + COL1A1-3’-UTR WT ; anti-miR-625-5p + COL1A1-3’-UTR Mut-1 ; and anti-miR-625-5p + COL1A1-3’-UTR Mut-2 . After transfection for 48 hr, cells were collected to detect (B) miR-625-5p, (C) COL1A1 mRNA and (D) COL1A1 protein levels. * P
    Figure Legend Snippet: COL1A1 is a direct target of miR-625-5p. (A) Schematic representation of the 3’-UTRs of COL1A1 containing two putative miR-625-5p binding sites. These two binding sites are indicated as 1 and 2 and are shown with red lines. The seed locations of miR-625-5p are indicated with red font, while the mutated locations are indicated with blue font. (B-D) COL1A1 can be targeted by miR-625-5p at site 1. The coding sequences of COL1A1 and its wild-type (WT) and mutated (Mut1 and 2) 3’-UTRs were cloned into the pCDNA3 vector. Then, the following combinations of plasmids were transfected into hNPC cells: miR-NC; miR-NC + COL1A1-3’-UTR WT ; miR-NC + COL1A1-3’-UTR Mut-1 ; miR-NC + COL1A1-3’-UTR Mut-2 ; miR-625-5p-mimic; miR-625-5p-mimic + COL1A1-3’-UTR WT ; miR-625-5p-mimic + COL1A1-3’-UTR Mut-1 ; anti-miR-625-5p; anti-miR-625-5p + COL1A1-3’-UTR WT ; anti-miR-625-5p + COL1A1-3’-UTR Mut-1 ; and anti-miR-625-5p + COL1A1-3’-UTR Mut-2 . After transfection for 48 hr, cells were collected to detect (B) miR-625-5p, (C) COL1A1 mRNA and (D) COL1A1 protein levels. * P

    Techniques Used: Binding Assay, Clone Assay, Plasmid Preparation, Transfection

    27) Product Images from "Insertion of a homing endonuclease creates a genes-in-pieces ribonucleotide reductase that retains function"

    Article Title: Insertion of a homing endonuclease creates a genes-in-pieces ribonucleotide reductase that retains function

    Journal:

    doi: 10.1073/pnas.0609915104

    The Aeh1 mobE insertion is not a self-splicing intron or intein. ( A ) Schematic of the Aeh1 mobE insertion indicating the approximate position of primers. ( B ) RT-PCR with primers DE-25/DE-26. Shown is a 1% agarose gel of aliquots of RT-PCRs using total
    Figure Legend Snippet: The Aeh1 mobE insertion is not a self-splicing intron or intein. ( A ) Schematic of the Aeh1 mobE insertion indicating the approximate position of primers. ( B ) RT-PCR with primers DE-25/DE-26. Shown is a 1% agarose gel of aliquots of RT-PCRs using total

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis

    28) Product Images from "Potential Mechanisms of AtNPR1 Mediated Resistance against Huanglongbing (HLB) in Citrus"

    Article Title: Potential Mechanisms of AtNPR1 Mediated Resistance against Huanglongbing (HLB) in Citrus

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21062009

    RNAseq analysis ( A ) Correlation between RNA-Seq samples. NPR1_1/2/3 represent three replicates of the NPR1 overexpressing line (NPR1-2), Val_1/2/3 represent three replicates of the control non-transgenic ‘Valencia’ line, heat maps of the correlation coefficient between samples, R 2 means the square of the Pearson coefficient; ( B ) Venn diagram of expressed genes in transgenic NPR1 overexpressing line (NPR1-2) and control non-transgenic ‘Valencia’ line. FPKM > 1 is the expression threshold, NPR1 and Val represent the AtNPR1 -transgenic line (NPR1-2) and non-transgenic ‘Valencia’ line, respectively.
    Figure Legend Snippet: RNAseq analysis ( A ) Correlation between RNA-Seq samples. NPR1_1/2/3 represent three replicates of the NPR1 overexpressing line (NPR1-2), Val_1/2/3 represent three replicates of the control non-transgenic ‘Valencia’ line, heat maps of the correlation coefficient between samples, R 2 means the square of the Pearson coefficient; ( B ) Venn diagram of expressed genes in transgenic NPR1 overexpressing line (NPR1-2) and control non-transgenic ‘Valencia’ line. FPKM > 1 is the expression threshold, NPR1 and Val represent the AtNPR1 -transgenic line (NPR1-2) and non-transgenic ‘Valencia’ line, respectively.

    Techniques Used: RNA Sequencing Assay, Transgenic Assay, Expressing

    29) Product Images from "Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells"

    Article Title: Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004712

    Silencing of HPV18 E6/E7 expression by RNA interference. (A) qRT-PCR analysis of HPV18 E6/E7 (left panel) and p21 (right panel) mRNA expression, 72 h after transfection of HeLa cells with si18E6/E7, control siRNA siContr-1, or upon mock treatment. mRNA levels were normalized to ACTB and calculated relative to the mock control. Data represent mean ± SEM (n = 4). Asterisks above columns indicate statistically significant differences from siContr-1-treated cells (p ≤ 0.05 (*), p ≤ 0.001 (***)). (B) Immunoblot analysis of HPV18 E6, p53, and p21 protein levels, 72 h after transfection of HeLa cells with si18E6/E7 or siContr-1. α-Tubulin: loading control. (C) Immunoblot analysis of HPV18 E7, total pRb (pRb), phosphorylated pRb (pRb-P), and Cyclin A1 protein levels, 72 h after transfection of HeLa cells with si18E6/E7 or siContr-1. α-Tubulin: loading control.
    Figure Legend Snippet: Silencing of HPV18 E6/E7 expression by RNA interference. (A) qRT-PCR analysis of HPV18 E6/E7 (left panel) and p21 (right panel) mRNA expression, 72 h after transfection of HeLa cells with si18E6/E7, control siRNA siContr-1, or upon mock treatment. mRNA levels were normalized to ACTB and calculated relative to the mock control. Data represent mean ± SEM (n = 4). Asterisks above columns indicate statistically significant differences from siContr-1-treated cells (p ≤ 0.05 (*), p ≤ 0.001 (***)). (B) Immunoblot analysis of HPV18 E6, p53, and p21 protein levels, 72 h after transfection of HeLa cells with si18E6/E7 or siContr-1. α-Tubulin: loading control. (C) Immunoblot analysis of HPV18 E7, total pRb (pRb), phosphorylated pRb (pRb-P), and Cyclin A1 protein levels, 72 h after transfection of HeLa cells with si18E6/E7 or siContr-1. α-Tubulin: loading control.

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection

    Effects of miRNAs of the miR-17~92 cluster on p21 expression in HeLa cells. (A) qRT-PCR analyses of cellular miRNA levels, 72 h after transfection of HeLa cells with the indicated vectors or upon mock treatment. miR-17~92: vector coding for the mir -17~92 cluster; “control”: repective empty expression vector. miRNA levels were normalized to snRNA RNU6–2 and calculated relative to the mock control. miR-17–5p, miR-20a-5p, miR-19b-3p, miR-92a-3p: encoded by the mir -17~92 expression vector; miR-34a-5p: negative control (not encoded by the vector). Data represent mean ± SEM (n = 3). Asterisks above columns indicate statistically significant differences from vector control-treated cells (p ≤ 0.05 (*)). (B) qRT-PCR analysis of p21 mRNA expression, 72 h after transfection of HeLa cells with the indicated vectors or upon mock treatment. mRNA levels were normalized to ACTB and calculated relative to the mock control. Data represent mean ± SEM (n = 4). Asterisks above columns indicate statistically significant differences from vector control-treated cells (p ≤ 0.05 (*)). (C) Immunoblot analysis of p53 and p21 protein levels, 72 h after transfection with the indicated vectors. α-Tubulin: loading control. A representative image is shown with corresponding densitometrically quantified band intensities of p21, normalized to α-Tubulin and calculated relative to mock. (D) miRNA inhibitors against miR-17–5p and miR-20a-5p increase the expression of p21 in HeLa cells. Left panel: Immunoblot analysis of p53 and p21 protein levels, 72 h after transfection of HeLa cells with the indicated miRNA inhibitors, an inhibitor control (‘Inhib. control’), or upon mock treatment. α-Tubulin: loading control. A representative image is shown. Numbers below individual lanes correspond to densitometrically quantified band intensities for p21, normalized to α-Tubulin and calculated relative to the Inhib. control. Right panel: Summary of densitometric quantification of p21 protein signal intensities. Data represent mean ± SEM (n = 3). Asterisks above columns indicate statistically significant differences from Inhib. control-treated cells (p ≤ 0.05 (*), p ≤ 0.01 (**)).
    Figure Legend Snippet: Effects of miRNAs of the miR-17~92 cluster on p21 expression in HeLa cells. (A) qRT-PCR analyses of cellular miRNA levels, 72 h after transfection of HeLa cells with the indicated vectors or upon mock treatment. miR-17~92: vector coding for the mir -17~92 cluster; “control”: repective empty expression vector. miRNA levels were normalized to snRNA RNU6–2 and calculated relative to the mock control. miR-17–5p, miR-20a-5p, miR-19b-3p, miR-92a-3p: encoded by the mir -17~92 expression vector; miR-34a-5p: negative control (not encoded by the vector). Data represent mean ± SEM (n = 3). Asterisks above columns indicate statistically significant differences from vector control-treated cells (p ≤ 0.05 (*)). (B) qRT-PCR analysis of p21 mRNA expression, 72 h after transfection of HeLa cells with the indicated vectors or upon mock treatment. mRNA levels were normalized to ACTB and calculated relative to the mock control. Data represent mean ± SEM (n = 4). Asterisks above columns indicate statistically significant differences from vector control-treated cells (p ≤ 0.05 (*)). (C) Immunoblot analysis of p53 and p21 protein levels, 72 h after transfection with the indicated vectors. α-Tubulin: loading control. A representative image is shown with corresponding densitometrically quantified band intensities of p21, normalized to α-Tubulin and calculated relative to mock. (D) miRNA inhibitors against miR-17–5p and miR-20a-5p increase the expression of p21 in HeLa cells. Left panel: Immunoblot analysis of p53 and p21 protein levels, 72 h after transfection of HeLa cells with the indicated miRNA inhibitors, an inhibitor control (‘Inhib. control’), or upon mock treatment. α-Tubulin: loading control. A representative image is shown. Numbers below individual lanes correspond to densitometrically quantified band intensities for p21, normalized to α-Tubulin and calculated relative to the Inhib. control. Right panel: Summary of densitometric quantification of p21 protein signal intensities. Data represent mean ± SEM (n = 3). Asterisks above columns indicate statistically significant differences from Inhib. control-treated cells (p ≤ 0.05 (*), p ≤ 0.01 (**)).

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Negative Control, Inhibition

    Influence of combined silencing of p21 and HPV18 E6/E7 expression on the senescent phenotype of HPV-positive cancer cells. (A) qRT-PCR analysis of HPV18 E6/E7 (left panel) and p21 (right panel) mRNA expression, 72 h after transfection of HeLa cells with the indicated siRNAs or in mock-treated cells. mRNA levels were normalized to ACTB and calculated relative to the mock control. Data represent mean ± SEM (n = 2 or 3). Asterisks above columns indicate statistically significant differences between the indicated treatments (p ≤ 0.05 (*), p ≤ 0.01 (**)). (B) Immunoblot analysis of HPV18 E7, p53, and p21 protein levels, 72 h after transfection of HeLa cells with the indicated siRNAs or upon mock-treatment. α-Tubulin: loading control. (C + D) Cell cycle distribution analyzed by FACS, 72 h after transfection of HeLa cells with the indicated siRNAs or upon mock treatment. Percentage of cells in the G 1 , S and G 2 cell cycle phases are indicated. Representative samples of one experiment are shown as well as a summary of multiple biological replicates. Data represent mean ± SEM (n = 3). (E) HeLa cells were stained for expression of the senescence marker SA-β-Gal, 168 h after transfection with the indicated siRNAs. Visualization by bright field microscopy.
    Figure Legend Snippet: Influence of combined silencing of p21 and HPV18 E6/E7 expression on the senescent phenotype of HPV-positive cancer cells. (A) qRT-PCR analysis of HPV18 E6/E7 (left panel) and p21 (right panel) mRNA expression, 72 h after transfection of HeLa cells with the indicated siRNAs or in mock-treated cells. mRNA levels were normalized to ACTB and calculated relative to the mock control. Data represent mean ± SEM (n = 2 or 3). Asterisks above columns indicate statistically significant differences between the indicated treatments (p ≤ 0.05 (*), p ≤ 0.01 (**)). (B) Immunoblot analysis of HPV18 E7, p53, and p21 protein levels, 72 h after transfection of HeLa cells with the indicated siRNAs or upon mock-treatment. α-Tubulin: loading control. (C + D) Cell cycle distribution analyzed by FACS, 72 h after transfection of HeLa cells with the indicated siRNAs or upon mock treatment. Percentage of cells in the G 1 , S and G 2 cell cycle phases are indicated. Representative samples of one experiment are shown as well as a summary of multiple biological replicates. Data represent mean ± SEM (n = 3). (E) HeLa cells were stained for expression of the senescence marker SA-β-Gal, 168 h after transfection with the indicated siRNAs. Visualization by bright field microscopy.

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection, FACS, Staining, Marker, Microscopy

    Effects of the p53 status on the E6/E7 -dependent modulation of intracellular miRNAs. (A) qRT-PCR analysis of HPV18 E6/E7 (left panel) and p21 (right panel) mRNA expression, 72 h after transfection of parental or “p53-null” HeLa cells with si18E6/E7, control siRNA (siContr-1), or upon mock treatment. mRNA levels were normalized to ACTB and calculated relative to the mock control (mock). Data represent mean ± SEM (n = 3). Asterisks above columns indicate statistically significant differences from siContr-1-treated cells (p ≤ 0.05 (*), p ≤ 0.001 (***)). (B) Immunoblot analysis of HPV18 E6, p53 and p21 protein levels, 72 h after transfection of parental or “p53-null” HeLa cells with si18E6/E7 or siContr-1, or upon mock treatment. α-Tubulin: loading control. (C) qRT-PCR analyses of selected cellular miRNAs, 72 h after transfection of parental or “p53-null” HeLa cells with si18E6/E7 or siContr-1. miR-34a-3p, positive control miRNA (p53-inducible). Cellular miRNA levels were normalized to snRNA RNU6–2 and calculated relative to siContr-1 (log 2 display). Dashed lines: 1.5-fold up- or downregulation (log 2 (1.5) = 0.585). Data represent mean ± SEM (n = 3). Asterisks indicate statistically significant differences (p ≤ 0.05 (*), p ≤ 0.01 (**) and p ≤ 0.001 (***)).
    Figure Legend Snippet: Effects of the p53 status on the E6/E7 -dependent modulation of intracellular miRNAs. (A) qRT-PCR analysis of HPV18 E6/E7 (left panel) and p21 (right panel) mRNA expression, 72 h after transfection of parental or “p53-null” HeLa cells with si18E6/E7, control siRNA (siContr-1), or upon mock treatment. mRNA levels were normalized to ACTB and calculated relative to the mock control (mock). Data represent mean ± SEM (n = 3). Asterisks above columns indicate statistically significant differences from siContr-1-treated cells (p ≤ 0.05 (*), p ≤ 0.001 (***)). (B) Immunoblot analysis of HPV18 E6, p53 and p21 protein levels, 72 h after transfection of parental or “p53-null” HeLa cells with si18E6/E7 or siContr-1, or upon mock treatment. α-Tubulin: loading control. (C) qRT-PCR analyses of selected cellular miRNAs, 72 h after transfection of parental or “p53-null” HeLa cells with si18E6/E7 or siContr-1. miR-34a-3p, positive control miRNA (p53-inducible). Cellular miRNA levels were normalized to snRNA RNU6–2 and calculated relative to siContr-1 (log 2 display). Dashed lines: 1.5-fold up- or downregulation (log 2 (1.5) = 0.585). Data represent mean ± SEM (n = 3). Asterisks indicate statistically significant differences (p ≤ 0.05 (*), p ≤ 0.01 (**) and p ≤ 0.001 (***)).

    Techniques Used: Quantitative RT-PCR, Expressing, Transfection, Positive Control

    30) Product Images from "Efficient targeted DNA methylation with chimeric dCas9–Dnmt3a–Dnmt3L methyltransferase"

    Article Title: Efficient targeted DNA methylation with chimeric dCas9–Dnmt3a–Dnmt3L methyltransferase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1112

    Targeted methylation of the endogenous human EpCAM promoter in SKOV-3 cells with dCas9–Dnmt3a3L. ( A ) The dCas9–Dnmt3a3L fusion protein was targeted either to separate or multiple sites within the CpG island in the EpCAM promoter (Chr. 2: 47368894–47370157). Methylation was investigated in four consecutive amplicons covering the CpG island (indicated with green bars). For details cf. the legend to Fig. 1B . The heat map shows targeted DNA methylation within the EpCAM promoter CpG island when the dCas9–Dnmt3a3L fusion protein was targeted to single or multiple locations within the island. ( B ) Average methylation level observed over the whole analyzed EpCAM promoter region containing 131 CpG sites. The guide RNAs that were used in the targeting experiments are indicated. Error bars represent SEM of average methylation from two biological replicates. Color of the bars denotes experimental sets: green—control experiments, light blue—targeting of the dCas9 fusions with single gRNAs, dark blue—targeting with multiple gRNAs and yellow corresponds to targeting of C706A catalytic mutant. ( C ) Relative EpCAM mRNA expression levels measured with RT-qPCR. The numbers below the bars denote gRNAs that were used to target the dCas9–Dnmt3a3L. The error-bars denote SEM from at least two biological repeats performed in technical triplicate). * P
    Figure Legend Snippet: Targeted methylation of the endogenous human EpCAM promoter in SKOV-3 cells with dCas9–Dnmt3a3L. ( A ) The dCas9–Dnmt3a3L fusion protein was targeted either to separate or multiple sites within the CpG island in the EpCAM promoter (Chr. 2: 47368894–47370157). Methylation was investigated in four consecutive amplicons covering the CpG island (indicated with green bars). For details cf. the legend to Fig. 1B . The heat map shows targeted DNA methylation within the EpCAM promoter CpG island when the dCas9–Dnmt3a3L fusion protein was targeted to single or multiple locations within the island. ( B ) Average methylation level observed over the whole analyzed EpCAM promoter region containing 131 CpG sites. The guide RNAs that were used in the targeting experiments are indicated. Error bars represent SEM of average methylation from two biological replicates. Color of the bars denotes experimental sets: green—control experiments, light blue—targeting of the dCas9 fusions with single gRNAs, dark blue—targeting with multiple gRNAs and yellow corresponds to targeting of C706A catalytic mutant. ( C ) Relative EpCAM mRNA expression levels measured with RT-qPCR. The numbers below the bars denote gRNAs that were used to target the dCas9–Dnmt3a3L. The error-bars denote SEM from at least two biological repeats performed in technical triplicate). * P

    Techniques Used: Methylation, DNA Methylation Assay, Mutagenesis, Expressing, Quantitative RT-PCR

    Spreading of DNA methylation is dependent on Dnmt3a3L multimerization. ( A ) Heat map of DNA methylation deposited within the EpCAM promoter region in untransfected SKOV-3 cells and cells co-transfected with a pool of guide RNAs (EpCAM gRNAs 6, 7, 9 and 10) and the dCas9–Dnmt3a3L wild type, catalytically inactive mutant C706A, non-multimerizing R832E mutant or a C706A R832E double mutant. ( B ) Model illustrating the mechanism of DNA methylation setting and spreading. Binding of the effector domain of targeted dCas9–Dnmt3a3L can be achieved both in cis and in trans relative to the DNA strand bound by dCas9 (here shown for two different guide RNAs). This leads to the deposition of DNA methylation (black filled circles) in the direct vicinity of the bound site or on a DNA strand that comes into spatial proximity to the dCas9 bound site. The Dnmt3a3L dimers can multimerize along the DNA via the R832 interaction interface (top). The R832E mutant with a disrupted multimerization interface cannot form fibers, leading to more locally defined DNA methylation either directly next to the dCas9 binding site or further away via DNA looping (middle). In a native situation (bottom), a transcription factor (brown) can recruit native Dnmt3a (green) or in complex with Dnmt3L (blue) to a specific site; this in turn can serve as a nucleation point to elongate the formed fiber causing cooperative deposition of DNA methylation in a larger genomic region.
    Figure Legend Snippet: Spreading of DNA methylation is dependent on Dnmt3a3L multimerization. ( A ) Heat map of DNA methylation deposited within the EpCAM promoter region in untransfected SKOV-3 cells and cells co-transfected with a pool of guide RNAs (EpCAM gRNAs 6, 7, 9 and 10) and the dCas9–Dnmt3a3L wild type, catalytically inactive mutant C706A, non-multimerizing R832E mutant or a C706A R832E double mutant. ( B ) Model illustrating the mechanism of DNA methylation setting and spreading. Binding of the effector domain of targeted dCas9–Dnmt3a3L can be achieved both in cis and in trans relative to the DNA strand bound by dCas9 (here shown for two different guide RNAs). This leads to the deposition of DNA methylation (black filled circles) in the direct vicinity of the bound site or on a DNA strand that comes into spatial proximity to the dCas9 bound site. The Dnmt3a3L dimers can multimerize along the DNA via the R832 interaction interface (top). The R832E mutant with a disrupted multimerization interface cannot form fibers, leading to more locally defined DNA methylation either directly next to the dCas9 binding site or further away via DNA looping (middle). In a native situation (bottom), a transcription factor (brown) can recruit native Dnmt3a (green) or in complex with Dnmt3L (blue) to a specific site; this in turn can serve as a nucleation point to elongate the formed fiber causing cooperative deposition of DNA methylation in a larger genomic region.

    Techniques Used: DNA Methylation Assay, Transfection, Mutagenesis, Binding Assay

    31) Product Images from ""

    Article Title:

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    doi: 10.1124/jpet.115.225631

    Cellular stability and RNase susceptibility of chimeric tRNA/mir-34a. (A) Chimeric tRNA/mir-34a was processed to mature miR-34a in various types of human carcinoma cells, as determined by selective stem-loop reverse transcription qRT-PCR analyses. Values
    Figure Legend Snippet: Cellular stability and RNase susceptibility of chimeric tRNA/mir-34a. (A) Chimeric tRNA/mir-34a was processed to mature miR-34a in various types of human carcinoma cells, as determined by selective stem-loop reverse transcription qRT-PCR analyses. Values

    Techniques Used: Quantitative RT-PCR

    32) Product Images from "Establishment and Characterization of the Reversibly Immortalized Mouse Fetal Heart Progenitors"

    Article Title: Establishment and Characterization of the Reversibly Immortalized Mouse Fetal Heart Progenitors

    Journal: International Journal of Medical Sciences

    doi: 10.7150/ijms.6639

    Characterization of iCP15 clones. Total RNA was isolated from 20 chosen iCP15 clones and subjected to reverse transcriptase reactions. The cDNA products were used as templates for sqPCR analysis of marker expression, including the common stem cell markers Oct3/4, Nanog, and Sox2 ( A ), the early cardiomyogenic progenitor markers, ISL-1, Sca1 and c-kit ( B ), the immediate early cardiomyogenic progenitor markers Nkx2.5, Tbx5, and Hand2 ( C ), and the late cardiomyogenic markers, cardiac α-actin (Actc1), α-myosin heavy chain (α-MyHC), and Atrial natriuretic factor (ANF) ( D ). All samples were normalized for their GAPDH expression. All sqPCR reactions were repeated at least in three independent experiments. Representative results are shown.
    Figure Legend Snippet: Characterization of iCP15 clones. Total RNA was isolated from 20 chosen iCP15 clones and subjected to reverse transcriptase reactions. The cDNA products were used as templates for sqPCR analysis of marker expression, including the common stem cell markers Oct3/4, Nanog, and Sox2 ( A ), the early cardiomyogenic progenitor markers, ISL-1, Sca1 and c-kit ( B ), the immediate early cardiomyogenic progenitor markers Nkx2.5, Tbx5, and Hand2 ( C ), and the late cardiomyogenic markers, cardiac α-actin (Actc1), α-myosin heavy chain (α-MyHC), and Atrial natriuretic factor (ANF) ( D ). All samples were normalized for their GAPDH expression. All sqPCR reactions were repeated at least in three independent experiments. Representative results are shown.

    Techniques Used: Clone Assay, Isolation, Marker, Expressing

    33) Product Images from "Efficient Cellular Release of Rift Valley Fever Virus Requires Genomic RNA"

    Article Title: Efficient Cellular Release of Rift Valley Fever Virus Requires Genomic RNA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018070

    Genomic RNA is packaged into RVF-VLPs that lack RdRp. BSR-T7/5 cells were transfected with genome and all of the structural proteins (WT), or one or more of the components was replaced with an equivalent amount of empty vector (-Gn/Gc, -N, -RdRp, -genome, -Gn and -Gc) or with plasmids expressing mutant alleles of Gn or Gc (GnK48 or GcW1). At 24 h the media was replaced with fresh media containing benzonase nuclease. At 48 h the media was removed, clarified and the RVF-VLPs were harvested by ultracentrifugation. The RNA was isolated from the RVF-VLPs and cDNA was generated with primers that recognize the genomic termini and reverse transcriptase (+RT). Duplicate samples were also run without reverse transcriptase (-RT). PCR was performed using primers that flank the intergenic region. The numbers on the left of the gel image indicate size standards.
    Figure Legend Snippet: Genomic RNA is packaged into RVF-VLPs that lack RdRp. BSR-T7/5 cells were transfected with genome and all of the structural proteins (WT), or one or more of the components was replaced with an equivalent amount of empty vector (-Gn/Gc, -N, -RdRp, -genome, -Gn and -Gc) or with plasmids expressing mutant alleles of Gn or Gc (GnK48 or GcW1). At 24 h the media was replaced with fresh media containing benzonase nuclease. At 48 h the media was removed, clarified and the RVF-VLPs were harvested by ultracentrifugation. The RNA was isolated from the RVF-VLPs and cDNA was generated with primers that recognize the genomic termini and reverse transcriptase (+RT). Duplicate samples were also run without reverse transcriptase (-RT). PCR was performed using primers that flank the intergenic region. The numbers on the left of the gel image indicate size standards.

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Mutagenesis, Isolation, Generated, Reverse Transcription Polymerase Chain Reaction

    34) Product Images from "A Re-Examination of Global Suppression of RNA Interference by HIV-1"

    Article Title: A Re-Examination of Global Suppression of RNA Interference by HIV-1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017246

    The effect of Tat over-expression on the silencing potency of miEGFP. (A) Immunoblot analysis of protein extracts obtained from P4R5 cells 2 days post-transfection with indicated plasmids. The blot was analyzed using antibodies specific to EGFP and β-actin, which serves as a loading control. (B) Spot-densitometry analysis of two individual experiments, as described in (A). The data are shown as the ratio of EGFP to β-actin and presented as the percentage of control (no miEGFP, no Tat). Error bars represents standard deviation from 3 replicates. (C) Total RNA was extracted from cells transfected in (A) and, following DNase treatment, qRT-PCR was performed to quantitate EGFP mRNA. The data are normalized to β-actin and presented as fold change over control (no miEGFP). Error bars represent standard deviation from 3 replicates. (D) Lower panel: the RNA preparation from (C) was subjected to primer extension reaction to detect mature miEGFP. Upper panel: U6 RNA was detected by northern blotting to confirm RNA integrity and quantification.
    Figure Legend Snippet: The effect of Tat over-expression on the silencing potency of miEGFP. (A) Immunoblot analysis of protein extracts obtained from P4R5 cells 2 days post-transfection with indicated plasmids. The blot was analyzed using antibodies specific to EGFP and β-actin, which serves as a loading control. (B) Spot-densitometry analysis of two individual experiments, as described in (A). The data are shown as the ratio of EGFP to β-actin and presented as the percentage of control (no miEGFP, no Tat). Error bars represents standard deviation from 3 replicates. (C) Total RNA was extracted from cells transfected in (A) and, following DNase treatment, qRT-PCR was performed to quantitate EGFP mRNA. The data are normalized to β-actin and presented as fold change over control (no miEGFP). Error bars represent standard deviation from 3 replicates. (D) Lower panel: the RNA preparation from (C) was subjected to primer extension reaction to detect mature miEGFP. Upper panel: U6 RNA was detected by northern blotting to confirm RNA integrity and quantification.

    Techniques Used: Over Expression, Transfection, Standard Deviation, Quantitative RT-PCR, Northern Blot

    Efficacy of EGFP silencing by miEGFP in the presence of HIV-1 replication. (A) Protein extracts were prepared 2d post-transfection from 293T cells transfected with pCMV-dsEGFP and the indicated plasmids, and then analyzed by immunoblotting with antibodies specific to EGFP and β-actin. Results show two replicates from the same experiment. (B) Total RNA was isolated from cells transfected in (A) and, following DNase treatment, qRT-PCR was performed to determine the level of EGFP mRNA. The data are normalized to β-actin mRNA and presented as fold change over control. Two individual replicates from the same experiment are shown. Error bars represent standard deviation from three qPCR replicates of the same sample.
    Figure Legend Snippet: Efficacy of EGFP silencing by miEGFP in the presence of HIV-1 replication. (A) Protein extracts were prepared 2d post-transfection from 293T cells transfected with pCMV-dsEGFP and the indicated plasmids, and then analyzed by immunoblotting with antibodies specific to EGFP and β-actin. Results show two replicates from the same experiment. (B) Total RNA was isolated from cells transfected in (A) and, following DNase treatment, qRT-PCR was performed to determine the level of EGFP mRNA. The data are normalized to β-actin mRNA and presented as fold change over control. Two individual replicates from the same experiment are shown. Error bars represent standard deviation from three qPCR replicates of the same sample.

    Techniques Used: Transfection, Isolation, Quantitative RT-PCR, Standard Deviation, Real-time Polymerase Chain Reaction

    35) Product Images from "MicroRNA-182 Regulates Neurite Outgrowth Involving the PTEN/AKT Pathway"

    Article Title: MicroRNA-182 Regulates Neurite Outgrowth Involving the PTEN/AKT Pathway

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2017.00096

    MiR-182 promotes axon outgrowth. (A,B) A schematic diagram showing scramble microRNA and miR-182 mimics plus GFP -encoding plasmid that were transfected into cortical neurons at 1 DIV and imaged at 3 DIV. (C) Quantification of axon length. Data were presented as mean ± SEM. ∗ p
    Figure Legend Snippet: MiR-182 promotes axon outgrowth. (A,B) A schematic diagram showing scramble microRNA and miR-182 mimics plus GFP -encoding plasmid that were transfected into cortical neurons at 1 DIV and imaged at 3 DIV. (C) Quantification of axon length. Data were presented as mean ± SEM. ∗ p

    Techniques Used: Plasmid Preparation, Transfection

    PTEN/AKT pathway is involved in regulating axon outgrowth. (A) Cellular fractions overexpressing miR-182 were analyzed by western blot using antibodies against P-AKT S473, P-AKT T308, AKT, PTEN, and P-PTEN S380. β-actin was used as the loading control, and the data represented the results of at least three different experiments. (B) Phosphorylation of AKT and PTEN was analyzed when miR-182 was downregulated. (C–F) Representative cortical neurons transfected with microRNA scramble plus DMSO, microRNA scramble plus LY294002, miR-182 plus DMSO and miR-182 plus LY294002. (G) Quantification of axon length ( ∗ p
    Figure Legend Snippet: PTEN/AKT pathway is involved in regulating axon outgrowth. (A) Cellular fractions overexpressing miR-182 were analyzed by western blot using antibodies against P-AKT S473, P-AKT T308, AKT, PTEN, and P-PTEN S380. β-actin was used as the loading control, and the data represented the results of at least three different experiments. (B) Phosphorylation of AKT and PTEN was analyzed when miR-182 was downregulated. (C–F) Representative cortical neurons transfected with microRNA scramble plus DMSO, microRNA scramble plus LY294002, miR-182 plus DMSO and miR-182 plus LY294002. (G) Quantification of axon length ( ∗ p

    Techniques Used: Western Blot, Transfection

    MiR-182 promotes dendrite branching out. (A,B) Cortical neurons were transfected with scramble mimics and miR-182 mimics (60 nM) at 5 DIV. After 48 h, neurons were harvested and images were recorded. A representative image is shown each for neurons transfected with scramble microRNA mimics and miR-182 mimics. (C) Representative picture of the Sholl analysis. (D) Quantitative results of the number of dendrite process intersections by Sholl analysis. MiR-182 increased dendritic branching at the distance of 130 and 145 μm from the soma. One-way ANOVA, Tukey’s post-test ( ∗ p
    Figure Legend Snippet: MiR-182 promotes dendrite branching out. (A,B) Cortical neurons were transfected with scramble mimics and miR-182 mimics (60 nM) at 5 DIV. After 48 h, neurons were harvested and images were recorded. A representative image is shown each for neurons transfected with scramble microRNA mimics and miR-182 mimics. (C) Representative picture of the Sholl analysis. (D) Quantitative results of the number of dendrite process intersections by Sholl analysis. MiR-182 increased dendritic branching at the distance of 130 and 145 μm from the soma. One-way ANOVA, Tukey’s post-test ( ∗ p

    Techniques Used: Transfection

    36) Product Images from "Distinct cellular toxicity of two mutant huntingtin mRNA variants due to translation regulation"

    Article Title: Distinct cellular toxicity of two mutant huntingtin mRNA variants due to translation regulation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0177610

    Effects of HTT 3′UTRs on protein translation. (A) Schematic of constructs with either the short or long HTT 3′UTR for expression of Kaede-tagged normal and mutant HTT N-terminal fragment. (B) Representative images of transfected HEK293 cells, showing photoconversion of Kaede upon UV irradiation. With 2 minutes of UV irradiation, Kaede undergoes irreversible conversion from green fluorescence to red fluorescence. One hour after UV irradiation, newly synthesized Kaede was indicated by the return of green fluorescence. The scale bar represents 100 μm. (C) Levels of newly synthesized Kaede-tagged normal Htt N-terminal fragment in HEK293. Newly synthesized Kaede in a cell was monitored by time-lapse confocal imaging. Its level at time t was quantified as ( F t − F o )/ F o , where F t and F o are green fluorescence intensity at time t and immediately after UV irradiation. There is a significant effect from constructs ( F (1,102) = 6.013, p = 0.0159; n = 52 cells for each construct). (D) Levels of newly synthesized Kaede-tagged mHtt N-terminal fragment in HEK293. There is a significant effect from constructs ( F (1,99) = 6.019, p = 0.0159; n = 49 cells for each construct).
    Figure Legend Snippet: Effects of HTT 3′UTRs on protein translation. (A) Schematic of constructs with either the short or long HTT 3′UTR for expression of Kaede-tagged normal and mutant HTT N-terminal fragment. (B) Representative images of transfected HEK293 cells, showing photoconversion of Kaede upon UV irradiation. With 2 minutes of UV irradiation, Kaede undergoes irreversible conversion from green fluorescence to red fluorescence. One hour after UV irradiation, newly synthesized Kaede was indicated by the return of green fluorescence. The scale bar represents 100 μm. (C) Levels of newly synthesized Kaede-tagged normal Htt N-terminal fragment in HEK293. Newly synthesized Kaede in a cell was monitored by time-lapse confocal imaging. Its level at time t was quantified as ( F t − F o )/ F o , where F t and F o are green fluorescence intensity at time t and immediately after UV irradiation. There is a significant effect from constructs ( F (1,102) = 6.013, p = 0.0159; n = 52 cells for each construct). (D) Levels of newly synthesized Kaede-tagged mHtt N-terminal fragment in HEK293. There is a significant effect from constructs ( F (1,99) = 6.019, p = 0.0159; n = 49 cells for each construct).

    Techniques Used: Construct, Expressing, Mutagenesis, Transfection, Irradiation, Fluorescence, Synthesized, Imaging

    HTT 3′UTRs do not affect distribution of normal Htt N-terminal fragment in HEK293 cells. (A) Schematic of normal HTT Exon1 constructs with genomic sequences coding for either the short HTT 3′UTR (A) or long HTT 3′ TR (A*B). Symbol * indicates that first polyadenylation site has been disrupted, such that sequence A*B will only produce the long 3′UTR. Exon1 contains a tract of 23 glutamine-coding codons (Q23). (B) Expression of normal Htt N-terminal fragment from the short 3′UTR construct (pExon1Q23-Myc-A) in HEK293 cells. Transfected cells were stained with antibodies against Myc and huntingtin 48 and 72 h after transfection. Nuclear DNA was stained with DAPI. The experiment was repeated at least 3 times with consistent results. The scale bar represents 100 μm. (C) Expression of normal Htt N-terminal fragment from the long 3′UTR construct (pExon1Q23-Myc-A*B) in HEK293 cells. Transfected cells were stained with antibodies against Myc and huntingtin 48 and 72 h after transfection. Nuclear DNA was stained with DAPI. The experiment was repeated at least 3 times with consistent results. The scale bar represents 100 μm.
    Figure Legend Snippet: HTT 3′UTRs do not affect distribution of normal Htt N-terminal fragment in HEK293 cells. (A) Schematic of normal HTT Exon1 constructs with genomic sequences coding for either the short HTT 3′UTR (A) or long HTT 3′ TR (A*B). Symbol * indicates that first polyadenylation site has been disrupted, such that sequence A*B will only produce the long 3′UTR. Exon1 contains a tract of 23 glutamine-coding codons (Q23). (B) Expression of normal Htt N-terminal fragment from the short 3′UTR construct (pExon1Q23-Myc-A) in HEK293 cells. Transfected cells were stained with antibodies against Myc and huntingtin 48 and 72 h after transfection. Nuclear DNA was stained with DAPI. The experiment was repeated at least 3 times with consistent results. The scale bar represents 100 μm. (C) Expression of normal Htt N-terminal fragment from the long 3′UTR construct (pExon1Q23-Myc-A*B) in HEK293 cells. Transfected cells were stained with antibodies against Myc and huntingtin 48 and 72 h after transfection. Nuclear DNA was stained with DAPI. The experiment was repeated at least 3 times with consistent results. The scale bar represents 100 μm.

    Techniques Used: Construct, Genomic Sequencing, Sequencing, Expressing, Transfection, Staining

    Effects of HTT 3′UTRs on formation of protein aggregates in HEK293 cells. (A) Schematic of mutant HTT Exon1 constructs. Exon1 contains a tract of 145 glutamine-coding codons (Q145). (B) Expression of mHtt N-terminal fragment from the short 3′UTR construct (pExon1Q145-Myc-A) or the long 3′UTR construct (pExon1Q145-Myc-A*B) in HEK293 cells. Transfected cells were stained with antibodies against Myc and huntingtin 48 and 72 h after transfection. Nuclear DNA was stained with DAPI. Scale bars represent 100 μm. Arrows denote aggregates. (C) Percentage of cells containing mHtt aggregates in transfected cells (n = 4 replicates, 410–700 cells analyzed in each replicate). (D) Size of mHtt aggregates in transfected cells (n = 3 replicates, more than 50 aggregates analyzed in each replicate). Error bars indicate standard errors. Student’s t test: ** p
    Figure Legend Snippet: Effects of HTT 3′UTRs on formation of protein aggregates in HEK293 cells. (A) Schematic of mutant HTT Exon1 constructs. Exon1 contains a tract of 145 glutamine-coding codons (Q145). (B) Expression of mHtt N-terminal fragment from the short 3′UTR construct (pExon1Q145-Myc-A) or the long 3′UTR construct (pExon1Q145-Myc-A*B) in HEK293 cells. Transfected cells were stained with antibodies against Myc and huntingtin 48 and 72 h after transfection. Nuclear DNA was stained with DAPI. Scale bars represent 100 μm. Arrows denote aggregates. (C) Percentage of cells containing mHtt aggregates in transfected cells (n = 4 replicates, 410–700 cells analyzed in each replicate). (D) Size of mHtt aggregates in transfected cells (n = 3 replicates, more than 50 aggregates analyzed in each replicate). Error bars indicate standard errors. Student’s t test: ** p

    Techniques Used: Mutagenesis, Construct, Expressing, Transfection, Staining

    Distribution and stability of mRNA with different HTT 3′UTRs. (A) Representative images of FISH, showing distribution of GFP mRNA with the short and long HTT 3′UTR in HEK293 cells. After GFP FISH (green) was completed, cells were counter stained with phalloidin for actin cytoskeleton (red) and DAPI for nuclei (blue). The scale bar represents 50 μm. (B) Levels of GFP mRNA in HEK293 cells. Error bars indicate standard errors (n = 40 cells for each construct). (C) Relative levels of HTT exon1 mRNA in HEK293 cells transfected with either pExon1Q145-Myc-A (Q145-A) or pExon1Q145-Myc-A*B (Q145-A*B) (n = 3 for each construct).
    Figure Legend Snippet: Distribution and stability of mRNA with different HTT 3′UTRs. (A) Representative images of FISH, showing distribution of GFP mRNA with the short and long HTT 3′UTR in HEK293 cells. After GFP FISH (green) was completed, cells were counter stained with phalloidin for actin cytoskeleton (red) and DAPI for nuclei (blue). The scale bar represents 50 μm. (B) Levels of GFP mRNA in HEK293 cells. Error bars indicate standard errors (n = 40 cells for each construct). (C) Relative levels of HTT exon1 mRNA in HEK293 cells transfected with either pExon1Q145-Myc-A (Q145-A) or pExon1Q145-Myc-A*B (Q145-A*B) (n = 3 for each construct).

    Techniques Used: Fluorescence In Situ Hybridization, Staining, Construct, Transfection

    Effects of the HTT 3′UTRs on formation of mHtt aggregates in neurons. (A) Diagrams of four lentivirus constructs for expression of a Myc-tagged normal (Q23) or mutant (Q145) Htt N-terminal fragment encoded by Exon1 of the HTT gene. The constructs contain genomic sequences encoding the short HTT 3′UTR (termed “A”) or the segment of the long HTT 3′UTR that lacks the sequence for the short 3′UTR (termed “B”). (B and C) Confocal images of cultured rat cortical neurons, showing Myc-immunoreactive protein aggregates. Neurons were infected with lentivirus at DIV5 and fixed at DIV25 for immunocytochemistry with antibodies against Myc and MAP2. Nuclear DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI). Arrows denote aggregates. Scale bars represent 100 μm. (D) Percentage of neurons containing mHtt aggregates in infected neurons at DIV25 (n = 3 replicates, 340–370 cells analyzed in each replicate). (E) Size of mHTT aggregates in infected neurons at DIV25 (n = 3 replicates, more than 50 aggregates analyzed in each replicate). Error bars indicate standard errors. Student’s t test: ** p
    Figure Legend Snippet: Effects of the HTT 3′UTRs on formation of mHtt aggregates in neurons. (A) Diagrams of four lentivirus constructs for expression of a Myc-tagged normal (Q23) or mutant (Q145) Htt N-terminal fragment encoded by Exon1 of the HTT gene. The constructs contain genomic sequences encoding the short HTT 3′UTR (termed “A”) or the segment of the long HTT 3′UTR that lacks the sequence for the short 3′UTR (termed “B”). (B and C) Confocal images of cultured rat cortical neurons, showing Myc-immunoreactive protein aggregates. Neurons were infected with lentivirus at DIV5 and fixed at DIV25 for immunocytochemistry with antibodies against Myc and MAP2. Nuclear DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI). Arrows denote aggregates. Scale bars represent 100 μm. (D) Percentage of neurons containing mHtt aggregates in infected neurons at DIV25 (n = 3 replicates, 340–370 cells analyzed in each replicate). (E) Size of mHTT aggregates in infected neurons at DIV25 (n = 3 replicates, more than 50 aggregates analyzed in each replicate). Error bars indicate standard errors. Student’s t test: ** p

    Techniques Used: Construct, Expressing, Mutagenesis, Genomic Sequencing, Sequencing, Cell Culture, Infection, Immunocytochemistry, Staining

    37) Product Images from "Circular RNA identified from Peg3 and Igf2r"

    Article Title: Circular RNA identified from Peg3 and Igf2r

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0203850

    Circular RNA identified from 5’RACE experiments. Schematic representation of 5’RACE experiment. Total RNA isolated from tissues were first reverse-transcribed with the gene-specific primers that are derived from the 2nd exon of individual genes (grey arrows). The subsequent cDNA were further modified with G-tailing (arrowhead). These cDNA were amplified and used for NGS runs. Inspection of the sequence reads identified several different types of transcripts: normal transcripts with a proper joining of two exons (E1, E2), unspliced transcripts, and alternative transcripts driven by upstream alternative promoters/exons (U1). Detailed examination of the sequence reads also revealed the presence of circular RNAs: E2 + circE and E2 + E3.
    Figure Legend Snippet: Circular RNA identified from 5’RACE experiments. Schematic representation of 5’RACE experiment. Total RNA isolated from tissues were first reverse-transcribed with the gene-specific primers that are derived from the 2nd exon of individual genes (grey arrows). The subsequent cDNA were further modified with G-tailing (arrowhead). These cDNA were amplified and used for NGS runs. Inspection of the sequence reads identified several different types of transcripts: normal transcripts with a proper joining of two exons (E1, E2), unspliced transcripts, and alternative transcripts driven by upstream alternative promoters/exons (U1). Detailed examination of the sequence reads also revealed the presence of circular RNAs: E2 + circE and E2 + E3.

    Techniques Used: Isolation, Derivative Assay, Modification, Amplification, Next-Generation Sequencing, Sequencing

    Circular RNA from the Peg3 locus, circPeg3. ( A ) The two gel images on top represents the results of a nested PCR amplifying circPeg3 from two independent sets of cDNA that have been derived from the total RNA of various mouse tissues; the third image on middle shows the expression profile of Peg 3; and the fourth image on bottom indicates the relative amount of cDNA between different tissues based on the expression levels of β-actin. ( B ) Schematic representation of the exon structure of the mouse Peg3 locus. The black vertical lines indicate individual exons and the green vertical line indicates the circE exon. The arrows within the box indicate the two sets of primers that have been used for a nested PCR scheme to amplify circPeg3.
    Figure Legend Snippet: Circular RNA from the Peg3 locus, circPeg3. ( A ) The two gel images on top represents the results of a nested PCR amplifying circPeg3 from two independent sets of cDNA that have been derived from the total RNA of various mouse tissues; the third image on middle shows the expression profile of Peg 3; and the fourth image on bottom indicates the relative amount of cDNA between different tissues based on the expression levels of β-actin. ( B ) Schematic representation of the exon structure of the mouse Peg3 locus. The black vertical lines indicate individual exons and the green vertical line indicates the circE exon. The arrows within the box indicate the two sets of primers that have been used for a nested PCR scheme to amplify circPeg3.

    Techniques Used: Nested PCR, Derivative Assay, Expressing

    Circular RNA from the Igf2r locus, circIgf2r. ( A ) The gel image on top represents the results of the first PCR amplifying circIgf2r from a set of cDNA that have been derived from the total RNA of various mouse tissues and the image on bottom shows the expression profile of Igf2r . ( B ) Schematic representation of the exon structure of the mouse Igf2r locus. The black vertical lines indicate individual exons and the vertical lines within a parenthesis indicate the simplified version of the remaining exons, Exon 11 through 48. The arrows within the box indicate the primers that have been used for a nested PCR scheme to amplify circIgf2r.
    Figure Legend Snippet: Circular RNA from the Igf2r locus, circIgf2r. ( A ) The gel image on top represents the results of the first PCR amplifying circIgf2r from a set of cDNA that have been derived from the total RNA of various mouse tissues and the image on bottom shows the expression profile of Igf2r . ( B ) Schematic representation of the exon structure of the mouse Igf2r locus. The black vertical lines indicate individual exons and the vertical lines within a parenthesis indicate the simplified version of the remaining exons, Exon 11 through 48. The arrows within the box indicate the primers that have been used for a nested PCR scheme to amplify circIgf2r.

    Techniques Used: Polymerase Chain Reaction, Derivative Assay, Expressing, Nested PCR

    Formation of circPeg3 in various mutant alleles. ( A ) The gel image represents the results of a nested PCR amplifying circPeg3 from a set of cDNA that have been derived from various mutant alleles. ( B ) Schematic representation of the mutant alleles used for the current study. The exons are indicated with black vertical lines, while the circE exon is indicated with a green vertical line. The 4-kb genomic region corresponding to the Peg3-DMR is deleted in the KO2 allele, which is indicated with a parenthesis. In the CoKO allele, a 7-kb expression cassette containing the β-galactosidase and neomycin resistance genes with Poly-A tails is inserted into the 5th intron, which is indicated with two open boxes. In the DelKO allele, the 6th exon is deleted as indicated with a parenthesis. In the Invert allele, the 4-kb Peg3-DMR is inverted relative to the orientation of the surrounding genomic regions.
    Figure Legend Snippet: Formation of circPeg3 in various mutant alleles. ( A ) The gel image represents the results of a nested PCR amplifying circPeg3 from a set of cDNA that have been derived from various mutant alleles. ( B ) Schematic representation of the mutant alleles used for the current study. The exons are indicated with black vertical lines, while the circE exon is indicated with a green vertical line. The 4-kb genomic region corresponding to the Peg3-DMR is deleted in the KO2 allele, which is indicated with a parenthesis. In the CoKO allele, a 7-kb expression cassette containing the β-galactosidase and neomycin resistance genes with Poly-A tails is inserted into the 5th intron, which is indicated with two open boxes. In the DelKO allele, the 6th exon is deleted as indicated with a parenthesis. In the Invert allele, the 4-kb Peg3-DMR is inverted relative to the orientation of the surrounding genomic regions.

    Techniques Used: Mutagenesis, Nested PCR, Derivative Assay, Expressing

    38) Product Images from "Developmental conservation of microRNA gene localization at the nuclear periphery"

    Article Title: Developmental conservation of microRNA gene localization at the nuclear periphery

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0223759

    Allelic expression profile analysis of microRNA gene loci reveals their preferential monoallelic expression in the murine immune system. A) Representative images of RNA-DNA FISH experiments for microRNA genes in thymocytes, CD4 + , TH1, TH2 cells. microRNA gene locus DNA (red) and biotinylated cDNA probes (green) were used to detect the nascent microRNA transcript. The number of nuclei measured in the RNA-DNA FISH experiments were: n = 1950 thymocytes, n = 2348 CD4 + , n = 3417 TH1, n = 3057 TH2. Scale bar 2μm. B) The mono- and bi-allelic expression profile of the portrayed microRNAs in the graph corresponds to the percentage of cells expressing either one or both alleles, before and after differentiation of the cells into the TH1 and TH2 cell lineages. The number of nuclei measured are the same as in Fig 1A. C) Representative confocal microscopy images of RNA-DNA FISH experiments showing the allelic expression profile of microRNA genes in thioglycollate elicited peritoneal macrophages (TEPMs). Bar graphs indicate the percentage of cells with either mono- or bi-allelic expression of the nascent pri-miRNA transcript before and after LPS stimulation of macrophages. A total number of 3558 and 2606 nuclei were measured in naive (-LPS) and LPS activated (+LPS) TEPMs respectively. Scale bar 2μm. D) Confocal microscopy single z-stack images indicating the colocalization of nascent pri-miR-155 transcript (green) along with its corresponding gene locus (red), in 2h LPS-stimulated BMDMs (blue-DAPI, for DNA counterstain). Bar graph portraying the mono- and bi-allelic expression pattern of miR-146a , miR-155 and miR-let7e in naive and LPS-stimulated BMDMs. The total number of nuclei measured for each microRNA locus were n = 691 for miR-146a , n = 931 for miR-155 and n = 168 for miR-let7e . Scale bar 2 μm. E) Representative confocal microscopy images of RNA-DNA FISH experiments for microRNA gene loci (DNA- miR-155 red) with the respective nascent pri-microRNA transcript (green) in thymocytes (DNA counterstained with DAPI—blue). The upper panel depicts DNA, but not RNA signals in RNase A treated cells. The second and third panel depict mono- and biallelically expressing cells. The fourth panel presents RNA-DNA hybridization with the TOPO-TA cloning vector used for RNA FISH probe synthesis. Scale bar 2 μm.
    Figure Legend Snippet: Allelic expression profile analysis of microRNA gene loci reveals their preferential monoallelic expression in the murine immune system. A) Representative images of RNA-DNA FISH experiments for microRNA genes in thymocytes, CD4 + , TH1, TH2 cells. microRNA gene locus DNA (red) and biotinylated cDNA probes (green) were used to detect the nascent microRNA transcript. The number of nuclei measured in the RNA-DNA FISH experiments were: n = 1950 thymocytes, n = 2348 CD4 + , n = 3417 TH1, n = 3057 TH2. Scale bar 2μm. B) The mono- and bi-allelic expression profile of the portrayed microRNAs in the graph corresponds to the percentage of cells expressing either one or both alleles, before and after differentiation of the cells into the TH1 and TH2 cell lineages. The number of nuclei measured are the same as in Fig 1A. C) Representative confocal microscopy images of RNA-DNA FISH experiments showing the allelic expression profile of microRNA genes in thioglycollate elicited peritoneal macrophages (TEPMs). Bar graphs indicate the percentage of cells with either mono- or bi-allelic expression of the nascent pri-miRNA transcript before and after LPS stimulation of macrophages. A total number of 3558 and 2606 nuclei were measured in naive (-LPS) and LPS activated (+LPS) TEPMs respectively. Scale bar 2μm. D) Confocal microscopy single z-stack images indicating the colocalization of nascent pri-miR-155 transcript (green) along with its corresponding gene locus (red), in 2h LPS-stimulated BMDMs (blue-DAPI, for DNA counterstain). Bar graph portraying the mono- and bi-allelic expression pattern of miR-146a , miR-155 and miR-let7e in naive and LPS-stimulated BMDMs. The total number of nuclei measured for each microRNA locus were n = 691 for miR-146a , n = 931 for miR-155 and n = 168 for miR-let7e . Scale bar 2 μm. E) Representative confocal microscopy images of RNA-DNA FISH experiments for microRNA gene loci (DNA- miR-155 red) with the respective nascent pri-microRNA transcript (green) in thymocytes (DNA counterstained with DAPI—blue). The upper panel depicts DNA, but not RNA signals in RNase A treated cells. The second and third panel depict mono- and biallelically expressing cells. The fourth panel presents RNA-DNA hybridization with the TOPO-TA cloning vector used for RNA FISH probe synthesis. Scale bar 2 μm.

    Techniques Used: Expressing, Fluorescence In Situ Hybridization, Confocal Microscopy, DNA Hybridization, TA Cloning, Plasmid Preparation

    Perinuclear localization of microRNA gene loci in Bach1 -/- thymocytes and BMDMs. (A) miR-155 gene alleles distribution in wild type (wt) and Bach1 -/- thymocytes and BMDMs. (B) Cumulative frequency graphs and KS-test for the comparison of miR-155 gene alleles distribution in wt versus Bach1 -/- thymocytes and BMDMs before and after LPS stimulation for the indicated time points. (C) miR-146α gene alleles distribution in wt and Bach1 -/- thymocytes and BMDMs. (D) Cumulative frequency graphs of miR-146α gene alleles NDs in wt and Bach1 -/- thymocytes and BMDMs before and after LPS stimulation for the indicated time points. KS-test analysis results are portrayed on the table. (E) Allelic expression profile of pri-miRNA-155 and pri-miRNA-146α in naive (0h) and LPS stimulated (2h, 6h) BMDMs, following RNA-DNA FISH.
    Figure Legend Snippet: Perinuclear localization of microRNA gene loci in Bach1 -/- thymocytes and BMDMs. (A) miR-155 gene alleles distribution in wild type (wt) and Bach1 -/- thymocytes and BMDMs. (B) Cumulative frequency graphs and KS-test for the comparison of miR-155 gene alleles distribution in wt versus Bach1 -/- thymocytes and BMDMs before and after LPS stimulation for the indicated time points. (C) miR-146α gene alleles distribution in wt and Bach1 -/- thymocytes and BMDMs. (D) Cumulative frequency graphs of miR-146α gene alleles NDs in wt and Bach1 -/- thymocytes and BMDMs before and after LPS stimulation for the indicated time points. KS-test analysis results are portrayed on the table. (E) Allelic expression profile of pri-miRNA-155 and pri-miRNA-146α in naive (0h) and LPS stimulated (2h, 6h) BMDMs, following RNA-DNA FISH.

    Techniques Used: Expressing, Fluorescence In Situ Hybridization

    39) Product Images from "mi RNA profiling of human naive CD4 T cells links miR‐34c‐5p to cell activation and HIV replication"

    Article Title: mi RNA profiling of human naive CD4 T cells links miR‐34c‐5p to cell activation and HIV replication

    Journal: The EMBO Journal

    doi: 10.15252/embj.201694335

    miR‐34c‐5p targets in the HIV interactome Interaction network between genes regulated by miR‐34c‐5p and HIV‐1 proteins. Figure depicts the subset of DE genes identified in J34c cells that have reported interactions with HIV‐1 proteins in the HIV interactome database (Ako‐Adjei et al ). Western blot of PCAF/KAT2B and histone H3 in JØ or J34c (top), confirming decrease in protein levels. Bottom: AntagomiR to miR‐34c‐5p results in increased KAT2B mRNA levels compared to control‐treated cells. Plot presents relative expression values for mRNA in three biological replicates; bars represent mean and SEM. Differences were found to be significant with P
    Figure Legend Snippet: miR‐34c‐5p targets in the HIV interactome Interaction network between genes regulated by miR‐34c‐5p and HIV‐1 proteins. Figure depicts the subset of DE genes identified in J34c cells that have reported interactions with HIV‐1 proteins in the HIV interactome database (Ako‐Adjei et al ). Western blot of PCAF/KAT2B and histone H3 in JØ or J34c (top), confirming decrease in protein levels. Bottom: AntagomiR to miR‐34c‐5p results in increased KAT2B mRNA levels compared to control‐treated cells. Plot presents relative expression values for mRNA in three biological replicates; bars represent mean and SEM. Differences were found to be significant with P

    Techniques Used: Western Blot, Expressing

    Functional enrichment and network analysis of the miR‐34c‐5p‐dependent transcriptome in Jurkat T cells Distribution of enriched GO terms (Biological Processes) in J34c DE genes grouped into nine functional categories. Bar chart represents the number of expected and observed hits for GO terms associated with “Immune response”. Gene interaction network for J34c DE genes (filled nodes) and additional highly connected genes (transparent nodes). Node size reflects the number of network connections.
    Figure Legend Snippet: Functional enrichment and network analysis of the miR‐34c‐5p‐dependent transcriptome in Jurkat T cells Distribution of enriched GO terms (Biological Processes) in J34c DE genes grouped into nine functional categories. Bar chart represents the number of expected and observed hits for GO terms associated with “Immune response”. Gene interaction network for J34c DE genes (filled nodes) and additional highly connected genes (transparent nodes). Node size reflects the number of network connections.

    Techniques Used: Functional Assay

    Impact of miR‐34c‐5p expression on HIV infection and replication Cell‐associated viral DNA levels (left), tat mRNA levels (center), and RT activity levels (right) in cell culture supernatants in infected J34c cells versus infected JØ cells at 24 and 48 h post‐infection. * P ≤ 0.05 (paired two‐tailed t ‐test). Representative flow cytometry plot of intracellular HIV‐1 Gag protein staining (KC57) versus side scatter (SSC) in JØ and J34c cells, mock infected or infected with HIV‐1 NL4‐3 , after 48 h of culture and corresponding fold change (J34c/JØ) plot in four biological replicate assays. Differences to control were found to be significant with a paired two‐tailed t ‐test ( P ≤ 0.05). Fold change levels for miR‐34c‐5p, cell‐associated viral DNA level, tat mRNA, and RT activity levels in J34c cells treated with miR‐34c‐5p antagomiR versus control antagomiR, 48 h post‐infection. Differences to control were found to be significant with a paired two‐tailed t ‐test ( P ≤ 0.05). Data information: All plots show fold change levels observed in a total of three biological replicate experiments performed (four for panel B). Lines depict the corresponding mean and SEM values.
    Figure Legend Snippet: Impact of miR‐34c‐5p expression on HIV infection and replication Cell‐associated viral DNA levels (left), tat mRNA levels (center), and RT activity levels (right) in cell culture supernatants in infected J34c cells versus infected JØ cells at 24 and 48 h post‐infection. * P ≤ 0.05 (paired two‐tailed t ‐test). Representative flow cytometry plot of intracellular HIV‐1 Gag protein staining (KC57) versus side scatter (SSC) in JØ and J34c cells, mock infected or infected with HIV‐1 NL4‐3 , after 48 h of culture and corresponding fold change (J34c/JØ) plot in four biological replicate assays. Differences to control were found to be significant with a paired two‐tailed t ‐test ( P ≤ 0.05). Fold change levels for miR‐34c‐5p, cell‐associated viral DNA level, tat mRNA, and RT activity levels in J34c cells treated with miR‐34c‐5p antagomiR versus control antagomiR, 48 h post‐infection. Differences to control were found to be significant with a paired two‐tailed t ‐test ( P ≤ 0.05). Data information: All plots show fold change levels observed in a total of three biological replicate experiments performed (four for panel B). Lines depict the corresponding mean and SEM values.

    Techniques Used: Expressing, Infection, Activity Assay, Cell Culture, Two Tailed Test, Flow Cytometry, Cytometry, Staining

    miR‐34c‐5p and PCAF/KAT2B expression in naive CD4 T cells Quantification of PCAF/KAT2B and miR‐34c‐5p expression levels in naive CD4 T cells isolated from two healthy donors by qRT–PCR in response to TCR stimulation. Same as in (A), except in HIV‐1‐infected versus non‐infected stimulated cells after 72 h TCR stimulation. Data information: Each symbol represents one individual. Relative expression corresponds to 2 −ΔCt values, normalized to RNU48.
    Figure Legend Snippet: miR‐34c‐5p and PCAF/KAT2B expression in naive CD4 T cells Quantification of PCAF/KAT2B and miR‐34c‐5p expression levels in naive CD4 T cells isolated from two healthy donors by qRT–PCR in response to TCR stimulation. Same as in (A), except in HIV‐1‐infected versus non‐infected stimulated cells after 72 h TCR stimulation. Data information: Each symbol represents one individual. Relative expression corresponds to 2 −ΔCt values, normalized to RNU48.

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Infection

    miR‐34c‐5p is up‐regulated in naive CD4 T cells in response to TCR‐mediated stimulation Average miR expression levels in naive CD4 T cells (purity > 97%) before and after 72 h TCR stimulation. Lines indicate changes of ± 1 log 2 fold between samples ( n ). miRs with significant changes are highlighted (black) and with insets presenting mean read counts in unstimulated samples (mean), log 2 fold change values in stimulated samples (log FC), and corresponding adjusted P ‐value ( P ; Fisher's test implemented in Deseq). The two most highly expressed miRs (mir‐21‐5p and miR‐146b‐5p) are also indicated. Relative miR‐34c‐5p and miR‐155‐5p expression levels (2 −ΔCt normalized to RNU48) in unstimulated and stimulated naive CD4 T cells of individual samples pooled to generate the sequencing libraries. Mean ± SEM and P ‐value for differences in miR‐155‐5p expression (paired two‐tailed t ‐test) are shown. Comparison could not be performed for miR‐34c‐5p as it is undetermined in unstimulated samples. Time‐course quantification of miR‐34c‐5p expression (top panel, 2 −ΔCt values), cell proliferation (middle panel), and cell activation (bottom panel) markers by flow cytometry, in three different donors.
    Figure Legend Snippet: miR‐34c‐5p is up‐regulated in naive CD4 T cells in response to TCR‐mediated stimulation Average miR expression levels in naive CD4 T cells (purity > 97%) before and after 72 h TCR stimulation. Lines indicate changes of ± 1 log 2 fold between samples ( n ). miRs with significant changes are highlighted (black) and with insets presenting mean read counts in unstimulated samples (mean), log 2 fold change values in stimulated samples (log FC), and corresponding adjusted P ‐value ( P ; Fisher's test implemented in Deseq). The two most highly expressed miRs (mir‐21‐5p and miR‐146b‐5p) are also indicated. Relative miR‐34c‐5p and miR‐155‐5p expression levels (2 −ΔCt normalized to RNU48) in unstimulated and stimulated naive CD4 T cells of individual samples pooled to generate the sequencing libraries. Mean ± SEM and P ‐value for differences in miR‐155‐5p expression (paired two‐tailed t ‐test) are shown. Comparison could not be performed for miR‐34c‐5p as it is undetermined in unstimulated samples. Time‐course quantification of miR‐34c‐5p expression (top panel, 2 −ΔCt values), cell proliferation (middle panel), and cell activation (bottom panel) markers by flow cytometry, in three different donors.

    Techniques Used: Expressing, Sequencing, Two Tailed Test, Activation Assay, Flow Cytometry, Cytometry

    Impact of miR‐34c‐5p overexpression on Jurkat T cells Hierarchical clustering of the 82 differentially expressed (DE) genes identified in Jurkat cell lines overexpressing miR‐34c‐5p (J34c‐2 and J34c‐5) versus control (JØ‐1, 2 and 3 clones). Color scale represents mean centered log 2 fold expression level across samples. Pie charts represent the relative proportion of predicted miR‐34c‐5p binding sites by the Miranda or PITA algorithms (or both) in down‐regulated (top) and up‐regulated (bottom) gene sets. qRT–PCR validation of selected genes. Plot represents the average percentage of target gene mRNA (normalized to GAPDH) in J34c versus JØ cells (all differences were found to be significant with a paired two‐tailed t ‐test, P
    Figure Legend Snippet: Impact of miR‐34c‐5p overexpression on Jurkat T cells Hierarchical clustering of the 82 differentially expressed (DE) genes identified in Jurkat cell lines overexpressing miR‐34c‐5p (J34c‐2 and J34c‐5) versus control (JØ‐1, 2 and 3 clones). Color scale represents mean centered log 2 fold expression level across samples. Pie charts represent the relative proportion of predicted miR‐34c‐5p binding sites by the Miranda or PITA algorithms (or both) in down‐regulated (top) and up‐regulated (bottom) gene sets. qRT–PCR validation of selected genes. Plot represents the average percentage of target gene mRNA (normalized to GAPDH) in J34c versus JØ cells (all differences were found to be significant with a paired two‐tailed t ‐test, P

    Techniques Used: Over Expression, Expressing, Binding Assay, Quantitative RT-PCR, Two Tailed Test

    40) Product Images from "Inhibition of mitochondrial respiration under hypoxia and increased antioxidant activity after reoxygenation of Tribolium castaneum"

    Article Title: Inhibition of mitochondrial respiration under hypoxia and increased antioxidant activity after reoxygenation of Tribolium castaneum

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0199056

    qRT-PCR analysis of selected transcripts to confirm expression profiles identified by RNA-seq. Tc ELOVL, elongation of very long chain fatty acids protein 7; Tc MRP, ATP-binding cassette subfamily C (CFTR/MRP) member 4; Tc HSP70, heat shock 70kDa protein; Tc MAPK, MAP kinase; Tc DUSP, Dual specificity MAP kinase phosphatase; Tc SD, superoxide dismutase, Cu-Zn family; Tc AG, alpha-glucosidase; Tc DACHS, DACHS, Hippo signling pathway; Tc FJBP Four-jointed box protein 1 (FJBP). Value represents mean ± SE of three independent PCR amplifications and quantifications.
    Figure Legend Snippet: qRT-PCR analysis of selected transcripts to confirm expression profiles identified by RNA-seq. Tc ELOVL, elongation of very long chain fatty acids protein 7; Tc MRP, ATP-binding cassette subfamily C (CFTR/MRP) member 4; Tc HSP70, heat shock 70kDa protein; Tc MAPK, MAP kinase; Tc DUSP, Dual specificity MAP kinase phosphatase; Tc SD, superoxide dismutase, Cu-Zn family; Tc AG, alpha-glucosidase; Tc DACHS, DACHS, Hippo signling pathway; Tc FJBP Four-jointed box protein 1 (FJBP). Value represents mean ± SE of three independent PCR amplifications and quantifications.

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

    Gene expression pattern of glycolytic (A) and Krebs (B) cycle enzymes of Tribolium castaneum larvae in response to hypoxia. Total RNA was isolated from the larvae after 12 hours’ hypoxia treatment. qRT-PCR was used to illustrate gene expression. Tc HK, hexokinase; Tc PGI, phosphoglucose isomerase; Tc PFKFB, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; Tc FBP, fructose 1,6-bisphosphate aldolase; Tc TPI, triosephosphate isomerase; Tc GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Tc PGK, phosphoglycerate kinase; Tc PGM, phosphoglycerate mutase; Tc ENO, Enolase; Tc PK, Pyruvate kinase; Tc LDH, L-lactate dehydrogenase; Tc PDC, pyruvate dehydrogenase; Tc ACO, aconitate hydratase, mitochondria; Tc IDH, isocitrate dehydrogenase; Tc SCSb, succinyl-CoA synthetase beta chain; Tc SDH, succinate dehydrogenase; Tc FH, fumarate hydratase; Tc MDH, malate dehydrogenase. Red color represents upregulate, green color represents downregulate and black color represents no change.
    Figure Legend Snippet: Gene expression pattern of glycolytic (A) and Krebs (B) cycle enzymes of Tribolium castaneum larvae in response to hypoxia. Total RNA was isolated from the larvae after 12 hours’ hypoxia treatment. qRT-PCR was used to illustrate gene expression. Tc HK, hexokinase; Tc PGI, phosphoglucose isomerase; Tc PFKFB, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; Tc FBP, fructose 1,6-bisphosphate aldolase; Tc TPI, triosephosphate isomerase; Tc GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Tc PGK, phosphoglycerate kinase; Tc PGM, phosphoglycerate mutase; Tc ENO, Enolase; Tc PK, Pyruvate kinase; Tc LDH, L-lactate dehydrogenase; Tc PDC, pyruvate dehydrogenase; Tc ACO, aconitate hydratase, mitochondria; Tc IDH, isocitrate dehydrogenase; Tc SCSb, succinyl-CoA synthetase beta chain; Tc SDH, succinate dehydrogenase; Tc FH, fumarate hydratase; Tc MDH, malate dehydrogenase. Red color represents upregulate, green color represents downregulate and black color represents no change.

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR

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    Quantitative RT-PCR:

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    SYBR Green Assay:

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    Incubation:

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    New England Biolabs mmlv rt
    Reverse transcriptase assays. A. Fluorogenic assay. RT activity was measured by detection of RNA:DNA heteroduplex by fluorescence of EvaGreen binding. Oligo dT primed poly A was incubated at 37°C and 65°C in the presence of indicated Pol enzymes in manufacturer recommended buffers and dTTP. Fluorescence measurements were obtained every 6 seconds for 10 minutes. The initial slopes from a plot of RFU vs. time in seconds were determined by linear least square regression from 30 to 150 seconds at 37°C and from 30 to 90 seconds at 65°C. Error bars are standard error of regression slope. B. RT primer extension assay. HEX-labeled dT 20 primed poly A was incubated 10 minutes at 37°C and then 10 minutes at 65°C in the presence of indicated Pol enzymes and dTTP in manufacturer recommended buffers. Primer extension products were resolved by 10% denaturing PAGE and imaged on a Molecular Imager FX (Bio-Rad). Left facing triangle indicates migration of unextended dT20 primer and asterisk indicates bromophenol blue dye front. C. RT MS2-specific primer extension. 5′-labeled primer was annealed to MS2 RNA and incubated 10 minutes at 37°C and then 30 minutes at 65°C in the presence of indicated Pol enzymes with dNTPS (N = A,C,G,T) in manufacturer recommended buffers. Primer extension products were resolved by 5% denaturing PAGE. Lane 1 No <t>RNA+MMLV</t> RT; Lane 2: MS2 RNA No RT; Lane 3 MS2 RNA+MMLV RT, Lane 3 MS2 <t>RNA+3173</t> Pol. Molecular weight in bases indicted. Red Arrow: ∼650 base MMLV extension product. Blue Arrow: ∼715 base PyroScript extension product. Green arrow: Non-templated MMLV reaction product.
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    Reverse transcriptase assays. A. Fluorogenic assay. RT activity was measured by detection of RNA:DNA heteroduplex by fluorescence of EvaGreen binding. Oligo dT primed poly A was incubated at 37°C and 65°C in the presence of indicated Pol enzymes in manufacturer recommended buffers and dTTP. Fluorescence measurements were obtained every 6 seconds for 10 minutes. The initial slopes from a plot of RFU vs. time in seconds were determined by linear least square regression from 30 to 150 seconds at 37°C and from 30 to 90 seconds at 65°C. Error bars are standard error of regression slope. B. RT primer extension assay. HEX-labeled dT 20 primed poly A was incubated 10 minutes at 37°C and then 10 minutes at 65°C in the presence of indicated Pol enzymes and dTTP in manufacturer recommended buffers. Primer extension products were resolved by 10% denaturing PAGE and imaged on a Molecular Imager FX (Bio-Rad). Left facing triangle indicates migration of unextended dT20 primer and asterisk indicates bromophenol blue dye front. C. RT MS2-specific primer extension. 5′-labeled primer was annealed to MS2 RNA and incubated 10 minutes at 37°C and then 30 minutes at 65°C in the presence of indicated Pol enzymes with dNTPS (N = A,C,G,T) in manufacturer recommended buffers. Primer extension products were resolved by 5% denaturing PAGE. Lane 1 No RNA+MMLV RT; Lane 2: MS2 RNA No RT; Lane 3 MS2 RNA+MMLV RT, Lane 3 MS2 RNA+3173 Pol. Molecular weight in bases indicted. Red Arrow: ∼650 base MMLV extension product. Blue Arrow: ∼715 base PyroScript extension product. Green arrow: Non-templated MMLV reaction product.

    Journal: PLoS ONE

    Article Title: Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme

    doi: 10.1371/journal.pone.0038371

    Figure Lengend Snippet: Reverse transcriptase assays. A. Fluorogenic assay. RT activity was measured by detection of RNA:DNA heteroduplex by fluorescence of EvaGreen binding. Oligo dT primed poly A was incubated at 37°C and 65°C in the presence of indicated Pol enzymes in manufacturer recommended buffers and dTTP. Fluorescence measurements were obtained every 6 seconds for 10 minutes. The initial slopes from a plot of RFU vs. time in seconds were determined by linear least square regression from 30 to 150 seconds at 37°C and from 30 to 90 seconds at 65°C. Error bars are standard error of regression slope. B. RT primer extension assay. HEX-labeled dT 20 primed poly A was incubated 10 minutes at 37°C and then 10 minutes at 65°C in the presence of indicated Pol enzymes and dTTP in manufacturer recommended buffers. Primer extension products were resolved by 10% denaturing PAGE and imaged on a Molecular Imager FX (Bio-Rad). Left facing triangle indicates migration of unextended dT20 primer and asterisk indicates bromophenol blue dye front. C. RT MS2-specific primer extension. 5′-labeled primer was annealed to MS2 RNA and incubated 10 minutes at 37°C and then 30 minutes at 65°C in the presence of indicated Pol enzymes with dNTPS (N = A,C,G,T) in manufacturer recommended buffers. Primer extension products were resolved by 5% denaturing PAGE. Lane 1 No RNA+MMLV RT; Lane 2: MS2 RNA No RT; Lane 3 MS2 RNA+MMLV RT, Lane 3 MS2 RNA+3173 Pol. Molecular weight in bases indicted. Red Arrow: ∼650 base MMLV extension product. Blue Arrow: ∼715 base PyroScript extension product. Green arrow: Non-templated MMLV reaction product.

    Article Snippet: Two-step RT-PCR reactions were performed using either 5 units 3173 Pol, exonuclease negative mutant or 200 Units MMLV RT (NEB).

    Techniques: Activity Assay, Fluorescence, Binding Assay, Incubation, Primer Extension Assay, Labeling, Polyacrylamide Gel Electrophoresis, Migration, Molecular Weight

    RT-PCR detection of human transcript RNAs. Beta-actin, beta2-microglobulin and cyclophilin target sequences of the indicated sizes were amplified from human liver total RNA using the primers described in Table 1 . Shown are products of two step reactions where either MMLV RT or 3173 Pol were used for first strand cDNA synthesis, as indicated. Taq Pol was used for PCR. Products were resolved on a 1% agarose gel.

    Journal: PLoS ONE

    Article Title: Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme

    doi: 10.1371/journal.pone.0038371

    Figure Lengend Snippet: RT-PCR detection of human transcript RNAs. Beta-actin, beta2-microglobulin and cyclophilin target sequences of the indicated sizes were amplified from human liver total RNA using the primers described in Table 1 . Shown are products of two step reactions where either MMLV RT or 3173 Pol were used for first strand cDNA synthesis, as indicated. Taq Pol was used for PCR. Products were resolved on a 1% agarose gel.

    Article Snippet: Two-step RT-PCR reactions were performed using either 5 units 3173 Pol, exonuclease negative mutant or 200 Units MMLV RT (NEB).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Two step RT-PCR comparing 3173 Pol and MMLV RT. A. MS2 viral RNA and B., C. total human liver RNA were reverse transcribed using either 3173 Pol or MMLV RT and then PCR amplified using Taq Polymerase. Target amplicons : A. MS2 RNA phage 77 bp amplicon, 2% gel, B. Human beta-actin 144 bp amplicon, 2% gel, C. Human beta-actin 821 bp amplicon, 1% gel. Lanes : 1 , 11 : oligo dT primer; 2–4 , 12–14 : Gene specific primers; 5 , 15 : random hexamers; 6 , 16 : random nonamers; 7 , 17 : No primer plus RT; 8 : No RT enzyme; 9 : PCR No Target Control; 10 : Molecular Weight Marker (MW), 100 bp (50 bp lowest) for Panels A, B and 1000 bp (300, 500, 700 lowest) for Panel C. Correct PCR product size indicated by black triangle.

    Journal: PLoS ONE

    Article Title: Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme

    doi: 10.1371/journal.pone.0038371

    Figure Lengend Snippet: Two step RT-PCR comparing 3173 Pol and MMLV RT. A. MS2 viral RNA and B., C. total human liver RNA were reverse transcribed using either 3173 Pol or MMLV RT and then PCR amplified using Taq Polymerase. Target amplicons : A. MS2 RNA phage 77 bp amplicon, 2% gel, B. Human beta-actin 144 bp amplicon, 2% gel, C. Human beta-actin 821 bp amplicon, 1% gel. Lanes : 1 , 11 : oligo dT primer; 2–4 , 12–14 : Gene specific primers; 5 , 15 : random hexamers; 6 , 16 : random nonamers; 7 , 17 : No primer plus RT; 8 : No RT enzyme; 9 : PCR No Target Control; 10 : Molecular Weight Marker (MW), 100 bp (50 bp lowest) for Panels A, B and 1000 bp (300, 500, 700 lowest) for Panel C. Correct PCR product size indicated by black triangle.

    Article Snippet: Two-step RT-PCR reactions were performed using either 5 units 3173 Pol, exonuclease negative mutant or 200 Units MMLV RT (NEB).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Molecular Weight, Marker

    Reverse transcription using AHP dUTP. ( A ) RNA template T5 with primer P3. ( B and C ) Twenty percent denaturing PAGE analysis of reactions using AMV and M-MuLV (RNase H-) reverse transcriptases at 42°C for 15 h.

    Journal: Nucleic Acids Research

    Article Title: Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes

    doi: 10.1093/nar/gkw028

    Figure Lengend Snippet: Reverse transcription using AHP dUTP. ( A ) RNA template T5 with primer P3. ( B and C ) Twenty percent denaturing PAGE analysis of reactions using AMV and M-MuLV (RNase H-) reverse transcriptases at 42°C for 15 h.

    Article Snippet: Klenow large fragment, Therminator™ II, M-MuLV (RNase H− ) reverse transcriptase, AMV reverse transcriptase, RNase inhibitor and λ-exonuclease were purchased from New England Biolabs.

    Techniques: Polyacrylamide Gel Electrophoresis