first-strand cdna Search Results


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  • 99
    New England Biolabs protoscript first strand cdna synthesis kit
    Protoscript First Strand Cdna Synthesis Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1533 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    First Strand cDNA Synthesis Kit 11801 025
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    Thermo Fisher first strand cdna
    First Strand Cdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 86991 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher revertaid first strand cdna synthesis kit
    Revertaid First Strand Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 40502 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher maxima first strand cdna synthesis kit
    Maxima First Strand Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 15392 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher revertaid h minus first strand cdna synthesis kit
    Revertaid H Minus First Strand Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9555 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare first strand cdna synthesis kit
    First Strand Cdna Synthesis Kit, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 5249 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad first strand cdna
    Alternative AID splicing in primary CLL cells and MEC1 cells. <t>RNA</t> and protein were extracted from primary CLL samples that were stimulated with IL-2, IL-4, and CpG (+) or left unstimulated (–) for 6 days. Results of PCR on <t>cDNA</t> using AID-specific and GAPDH-specific primers as loading control are shown (upper panel). Schematic presentations of the alternative splice variants are indicated right to each PCR band. PCR on TOPO-cloned splice variants as templates was performed as size controls and loaded in the first four lanes of the gel. The lower panel shows an immunoblot (IB) on cell lysates using AID- and tubulin-specific antibodies as loading control. Data are representative of two independent experiments.
    First Strand Cdna, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 6999 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa primescript first strand cdna synthesis kit
    Alternative AID splicing in primary CLL cells and MEC1 cells. <t>RNA</t> and protein were extracted from primary CLL samples that were stimulated with IL-2, IL-4, and CpG (+) or left unstimulated (–) for 6 days. Results of PCR on <t>cDNA</t> using AID-specific and GAPDH-specific primers as loading control are shown (upper panel). Schematic presentations of the alternative splice variants are indicated right to each PCR band. PCR on TOPO-cloned splice variants as templates was performed as size controls and loaded in the first four lanes of the gel. The lower panel shows an immunoblot (IB) on cell lysates using AID- and tubulin-specific antibodies as loading control. Data are representative of two independent experiments.
    Primescript First Strand Cdna Synthesis Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 2734 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Toyobo first strand cdna
    TDP-43 is downregulated via reduction in the amount of cytoplasmic TARDBP mRNA by nonsense-mediated mRNA decay. (A) Schematic representation of TARDBP mRNA and primers for <t>RT-PCR.</t> Black boxes represent the coding region. White boxes represent the non-coding region. Vertical red lines indicate the splicing sites. The diagonal dashed red lines indicate the splicing. Intron 6 has three donor sites (nucleotide positions 769, 833 and 842 in <t>cDNA).</t> The 833 and 842 sites are represented as a single line. Bold black vertical lines indicate the polyadenylation signals (pA) (Supplementary Table S1). Arrows indicate primers (Supplementary Table S2). (B) Western blot analysis of Flp-In T-REx™HEK293 cells with a TDP-43 complementary DNA with a myc sequence inserted into the FRT site with or without doxycycline (Dox) for 72 h using an anti-TDP-43 antibody. (C) RT-PCR analysis of endogenous TARDBP mRNA (amplified product between F2 and R2A) and endogenous TARDBP pre-mRNA (amplified product between F1 and R1). Samples treated with actinomycin D (ActD) (to halt transcription) were used as negative controls for amplification of endogenous TARDBP pre-mRNA. (D) Northern blot analysis of TARDBP mRNA. Poly (A) + RNA was extracted from the nuclear or cytoplasmic fraction in the absence or presence of Dox or cycloheximide (CHX) for 6 h. *Ectopic TARDBP mRNA. M indicates RNA size marker. Roman numerals correspond to those in (E). Hybridized with a probe for exons 2–4 (probe A); by a probe for intron 6 (probe B); and with a probe for intron 7 (probe C) (Supplementary Table S2). The lower panel hybridized with a probe for GAPDH. (E) Schematic representation of TARDBP mRNA isoforms and probes for northern blot analysis. Bold green lines represent probes. Vertical purple lines indicate the additional termination codons (ATCs). (F) Quantification of TARDBP mRNA isoforms by northern blot analysis using probe A. The roman numerals correspond to those in (E). Data represent the relative level of expression in lane 1. Orange bars correspond to samples without Dox. Blue bars correspond to samples with Dox. Data information: data are presented as mean ± SD ( n = 3) * P
    First Strand Cdna, supplied by Toyobo, used in various techniques. Bioz Stars score: 92/100, based on 4326 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa first strand cdna synthesis kit
    TDP-43 is downregulated via reduction in the amount of cytoplasmic TARDBP mRNA by nonsense-mediated mRNA decay. (A) Schematic representation of TARDBP mRNA and primers for <t>RT-PCR.</t> Black boxes represent the coding region. White boxes represent the non-coding region. Vertical red lines indicate the splicing sites. The diagonal dashed red lines indicate the splicing. Intron 6 has three donor sites (nucleotide positions 769, 833 and 842 in <t>cDNA).</t> The 833 and 842 sites are represented as a single line. Bold black vertical lines indicate the polyadenylation signals (pA) (Supplementary Table S1). Arrows indicate primers (Supplementary Table S2). (B) Western blot analysis of Flp-In T-REx™HEK293 cells with a TDP-43 complementary DNA with a myc sequence inserted into the FRT site with or without doxycycline (Dox) for 72 h using an anti-TDP-43 antibody. (C) RT-PCR analysis of endogenous TARDBP mRNA (amplified product between F2 and R2A) and endogenous TARDBP pre-mRNA (amplified product between F1 and R1). Samples treated with actinomycin D (ActD) (to halt transcription) were used as negative controls for amplification of endogenous TARDBP pre-mRNA. (D) Northern blot analysis of TARDBP mRNA. Poly (A) + RNA was extracted from the nuclear or cytoplasmic fraction in the absence or presence of Dox or cycloheximide (CHX) for 6 h. *Ectopic TARDBP mRNA. M indicates RNA size marker. Roman numerals correspond to those in (E). Hybridized with a probe for exons 2–4 (probe A); by a probe for intron 6 (probe B); and with a probe for intron 7 (probe C) (Supplementary Table S2). The lower panel hybridized with a probe for GAPDH. (E) Schematic representation of TARDBP mRNA isoforms and probes for northern blot analysis. Bold green lines represent probes. Vertical purple lines indicate the additional termination codons (ATCs). (F) Quantification of TARDBP mRNA isoforms by northern blot analysis using probe A. The roman numerals correspond to those in (E). Data represent the relative level of expression in lane 1. Orange bars correspond to samples without Dox. Blue bars correspond to samples with Dox. Data information: data are presented as mean ± SD ( n = 3) * P
    First Strand Cdna Synthesis Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 2195 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher maxima h minus first strand cdna synthesis kit
    TDP-43 is downregulated via reduction in the amount of cytoplasmic TARDBP mRNA by nonsense-mediated mRNA decay. (A) Schematic representation of TARDBP mRNA and primers for <t>RT-PCR.</t> Black boxes represent the coding region. White boxes represent the non-coding region. Vertical red lines indicate the splicing sites. The diagonal dashed red lines indicate the splicing. Intron 6 has three donor sites (nucleotide positions 769, 833 and 842 in <t>cDNA).</t> The 833 and 842 sites are represented as a single line. Bold black vertical lines indicate the polyadenylation signals (pA) (Supplementary Table S1). Arrows indicate primers (Supplementary Table S2). (B) Western blot analysis of Flp-In T-REx™HEK293 cells with a TDP-43 complementary DNA with a myc sequence inserted into the FRT site with or without doxycycline (Dox) for 72 h using an anti-TDP-43 antibody. (C) RT-PCR analysis of endogenous TARDBP mRNA (amplified product between F2 and R2A) and endogenous TARDBP pre-mRNA (amplified product between F1 and R1). Samples treated with actinomycin D (ActD) (to halt transcription) were used as negative controls for amplification of endogenous TARDBP pre-mRNA. (D) Northern blot analysis of TARDBP mRNA. Poly (A) + RNA was extracted from the nuclear or cytoplasmic fraction in the absence or presence of Dox or cycloheximide (CHX) for 6 h. *Ectopic TARDBP mRNA. M indicates RNA size marker. Roman numerals correspond to those in (E). Hybridized with a probe for exons 2–4 (probe A); by a probe for intron 6 (probe B); and with a probe for intron 7 (probe C) (Supplementary Table S2). The lower panel hybridized with a probe for GAPDH. (E) Schematic representation of TARDBP mRNA isoforms and probes for northern blot analysis. Bold green lines represent probes. Vertical purple lines indicate the additional termination codons (ATCs). (F) Quantification of TARDBP mRNA isoforms by northern blot analysis using probe A. The roman numerals correspond to those in (E). Data represent the relative level of expression in lane 1. Orange bars correspond to samples without Dox. Blue bars correspond to samples with Dox. Data information: data are presented as mean ± SD ( n = 3) * P
    Maxima H Minus First Strand Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1493 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs protoscript ii first strand cdna synthesis kit
    TDP-43 is downregulated via reduction in the amount of cytoplasmic TARDBP mRNA by nonsense-mediated mRNA decay. (A) Schematic representation of TARDBP mRNA and primers for <t>RT-PCR.</t> Black boxes represent the coding region. White boxes represent the non-coding region. Vertical red lines indicate the splicing sites. The diagonal dashed red lines indicate the splicing. Intron 6 has three donor sites (nucleotide positions 769, 833 and 842 in <t>cDNA).</t> The 833 and 842 sites are represented as a single line. Bold black vertical lines indicate the polyadenylation signals (pA) (Supplementary Table S1). Arrows indicate primers (Supplementary Table S2). (B) Western blot analysis of Flp-In T-REx™HEK293 cells with a TDP-43 complementary DNA with a myc sequence inserted into the FRT site with or without doxycycline (Dox) for 72 h using an anti-TDP-43 antibody. (C) RT-PCR analysis of endogenous TARDBP mRNA (amplified product between F2 and R2A) and endogenous TARDBP pre-mRNA (amplified product between F1 and R1). Samples treated with actinomycin D (ActD) (to halt transcription) were used as negative controls for amplification of endogenous TARDBP pre-mRNA. (D) Northern blot analysis of TARDBP mRNA. Poly (A) + RNA was extracted from the nuclear or cytoplasmic fraction in the absence or presence of Dox or cycloheximide (CHX) for 6 h. *Ectopic TARDBP mRNA. M indicates RNA size marker. Roman numerals correspond to those in (E). Hybridized with a probe for exons 2–4 (probe A); by a probe for intron 6 (probe B); and with a probe for intron 7 (probe C) (Supplementary Table S2). The lower panel hybridized with a probe for GAPDH. (E) Schematic representation of TARDBP mRNA isoforms and probes for northern blot analysis. Bold green lines represent probes. Vertical purple lines indicate the additional termination codons (ATCs). (F) Quantification of TARDBP mRNA isoforms by northern blot analysis using probe A. The roman numerals correspond to those in (E). Data represent the relative level of expression in lane 1. Orange bars correspond to samples without Dox. Blue bars correspond to samples with Dox. Data information: data are presented as mean ± SD ( n = 3) * P
    Protoscript Ii First Strand Cdna Synthesis Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1292 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Toyobo first strand cdna synthesis kit
    The expression level of cir-ITCH is closely related to lung cancer. (a) cir-ITCH was amplified by RT-PCR with divergent primers in <t>cDNA</t> but was not amplified in genomic DNA (gDNA). GAPDH, linear control. (b) qRT-PCR based on TaqMan probe was used to analyze the expression level of cir-ITCH in lung cancer tissues and paired noncancerous tissues. GAPDH was used as endogenous control. (c) The linear correlations between the cir-ITCH expression levels and linear ITCH were tested by Spearman analysis. The relative expression value was normalized by GAPDH expression level. (d) Random primers and oligo dT primers were used, respectively, in the reverse transcription experiments. The predicted circular <t>RNA</t> is absent in poly(A)-enriched samples. (e) The predicted circular RNA is resistant to RNase R treatment. Data are presented as mean ± SEM from three independent experiments. ∗ p
    First Strand Cdna Synthesis Kit, supplied by Toyobo, used in various techniques. Bioz Stars score: 92/100, based on 1692 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche transcriptor first strand cdna synthesis kit
    The expression level of cir-ITCH is closely related to lung cancer. (a) cir-ITCH was amplified by RT-PCR with divergent primers in <t>cDNA</t> but was not amplified in genomic DNA (gDNA). GAPDH, linear control. (b) qRT-PCR based on TaqMan probe was used to analyze the expression level of cir-ITCH in lung cancer tissues and paired noncancerous tissues. GAPDH was used as endogenous control. (c) The linear correlations between the cir-ITCH expression levels and linear ITCH were tested by Spearman analysis. The relative expression value was normalized by GAPDH expression level. (d) Random primers and oligo dT primers were used, respectively, in the reverse transcription experiments. The predicted circular <t>RNA</t> is absent in poly(A)-enriched samples. (e) The predicted circular RNA is resistant to RNase R treatment. Data are presented as mean ± SEM from three independent experiments. ∗ p
    Transcriptor First Strand Cdna Synthesis Kit, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 23862 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cdna synthesis
    The expression level of cir-ITCH is closely related to lung cancer. (a) cir-ITCH was amplified by RT-PCR with divergent primers in <t>cDNA</t> but was not amplified in genomic DNA (gDNA). GAPDH, linear control. (b) qRT-PCR based on TaqMan probe was used to analyze the expression level of cir-ITCH in lung cancer tissues and paired noncancerous tissues. GAPDH was used as endogenous control. (c) The linear correlations between the cir-ITCH expression levels and linear ITCH were tested by Spearman analysis. The relative expression value was normalized by GAPDH expression level. (d) Random primers and oligo dT primers were used, respectively, in the reverse transcription experiments. The predicted circular <t>RNA</t> is absent in poly(A)-enriched samples. (e) The predicted circular RNA is resistant to RNase R treatment. Data are presented as mean ± SEM from three independent experiments. ∗ p
    Cdna Synthesis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 106131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad first strand cdna synthesis
    C. neoformans CDA1 is transcriptionally upregulated during host infection. Mice were inoculated with 10 7 CFU of KN99 cells. At day 7 PI, the lungs were excised and homogenized, and fungal cells were harvested and used for the isolation of total <t>RNA.</t> Total RNA (0.5 µg) was used for the synthesis of <t>cDNA,</t> which was subsequently subjected to quantitative real-time PCR using CDA1 -specific primers. C. neoformans 18S rRNA transcript levels were used as a reference gene. Transcript levels of the respective genes in the cells used as inoculum served as control. Data are the averages for two independent experiments each with three animals per group. Fold expression was calculated for each gene comparing the extent of its upregulation in the lung samples to YPD-grown samples. Significant differences in the expression levels between genes were compared by ordinary one-way ANOVA, followed by Dunnett’s multiple-comparison test. (****, P
    First Strand Cdna Synthesis, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 2433 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    First Strand cDNA Human Brain 30 rxns
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    miRNAs and other noncoding RNAs are polyadenylated by poly A polymerase and subsequently converted into cDNA by reverse transcriptase with oligodT priming miRNA s are converted into cDNA by reverse
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    Image Search Results


    Alternative AID splicing in primary CLL cells and MEC1 cells. RNA and protein were extracted from primary CLL samples that were stimulated with IL-2, IL-4, and CpG (+) or left unstimulated (–) for 6 days. Results of PCR on cDNA using AID-specific and GAPDH-specific primers as loading control are shown (upper panel). Schematic presentations of the alternative splice variants are indicated right to each PCR band. PCR on TOPO-cloned splice variants as templates was performed as size controls and loaded in the first four lanes of the gel. The lower panel shows an immunoblot (IB) on cell lysates using AID- and tubulin-specific antibodies as loading control. Data are representative of two independent experiments.

    Journal: European Journal of Immunology

    Article Title: Alternative splice variants of AID are not stoichiometrically present at the protein level in chronic lymphocytic leukemia

    doi: 10.1002/eji.201343853

    Figure Lengend Snippet: Alternative AID splicing in primary CLL cells and MEC1 cells. RNA and protein were extracted from primary CLL samples that were stimulated with IL-2, IL-4, and CpG (+) or left unstimulated (–) for 6 days. Results of PCR on cDNA using AID-specific and GAPDH-specific primers as loading control are shown (upper panel). Schematic presentations of the alternative splice variants are indicated right to each PCR band. PCR on TOPO-cloned splice variants as templates was performed as size controls and loaded in the first four lanes of the gel. The lower panel shows an immunoblot (IB) on cell lysates using AID- and tubulin-specific antibodies as loading control. Data are representative of two independent experiments.

    Article Snippet: RT-PCR and immunoblot For RT-PCR, RNA was isolated from cells (High Pure RNA Isolation Kit, Roche) and first strand cDNA was generated (iScrpt, BioRad).

    Techniques: Polymerase Chain Reaction, Clone Assay

    Quantification of AID splice transcripts. (A) RPA was performed on RNA from MEC1 cells, MEC1 AID-SRC cells, and stimulated CLL samples (ID #25 and #151), using an antisense RNA probe spanning intron 3, exon 4/5 junction, and the 3′UTR of endogenous AID. White triangles represent splice variants of endogenous AID (AIDendo), and filled triangles indicate splice variants descending from AID-SRC (AIDtrans). Nonspecific bands that also appear using yeast RNA as control (no target) are indicated with asterisks. Protected fragments are shown to the right of the autoradiograph. Data are representative of two independent experiments. (B) Quantification of PCR-amplified splice variants by amplicon sequencing. AID transcripts were PCR amplified from cDNA of the respective samples (stimulated CLL sample ID #31, MEC1, and MEC1 AID-SRC ). The PCR products were sequenced and aligned to the reference sequences of splice variants. Based on the relative mapping frequencies, the relative quantities of the PCR-amplified splice variants were calculated. The coverage of uniquely mapped reads (read count) is indicated below each sample. (C) Relative quantification of splice variants was performed using SYBR green qRT-PCR on cDNA from MEC1 cells, MEC1 AID-SRC cells, and stimulated CLL samples (ID #13, #31, and #279). Data show mean + SD of duplicate samples of one experiment.

    Journal: European Journal of Immunology

    Article Title: Alternative splice variants of AID are not stoichiometrically present at the protein level in chronic lymphocytic leukemia

    doi: 10.1002/eji.201343853

    Figure Lengend Snippet: Quantification of AID splice transcripts. (A) RPA was performed on RNA from MEC1 cells, MEC1 AID-SRC cells, and stimulated CLL samples (ID #25 and #151), using an antisense RNA probe spanning intron 3, exon 4/5 junction, and the 3′UTR of endogenous AID. White triangles represent splice variants of endogenous AID (AIDendo), and filled triangles indicate splice variants descending from AID-SRC (AIDtrans). Nonspecific bands that also appear using yeast RNA as control (no target) are indicated with asterisks. Protected fragments are shown to the right of the autoradiograph. Data are representative of two independent experiments. (B) Quantification of PCR-amplified splice variants by amplicon sequencing. AID transcripts were PCR amplified from cDNA of the respective samples (stimulated CLL sample ID #31, MEC1, and MEC1 AID-SRC ). The PCR products were sequenced and aligned to the reference sequences of splice variants. Based on the relative mapping frequencies, the relative quantities of the PCR-amplified splice variants were calculated. The coverage of uniquely mapped reads (read count) is indicated below each sample. (C) Relative quantification of splice variants was performed using SYBR green qRT-PCR on cDNA from MEC1 cells, MEC1 AID-SRC cells, and stimulated CLL samples (ID #13, #31, and #279). Data show mean + SD of duplicate samples of one experiment.

    Article Snippet: RT-PCR and immunoblot For RT-PCR, RNA was isolated from cells (High Pure RNA Isolation Kit, Roche) and first strand cDNA was generated (iScrpt, BioRad).

    Techniques: Recombinase Polymerase Amplification, Autoradiography, Polymerase Chain Reaction, Amplification, Sequencing, SYBR Green Assay, Quantitative RT-PCR

    TDP-43 is downregulated via reduction in the amount of cytoplasmic TARDBP mRNA by nonsense-mediated mRNA decay. (A) Schematic representation of TARDBP mRNA and primers for RT-PCR. Black boxes represent the coding region. White boxes represent the non-coding region. Vertical red lines indicate the splicing sites. The diagonal dashed red lines indicate the splicing. Intron 6 has three donor sites (nucleotide positions 769, 833 and 842 in cDNA). The 833 and 842 sites are represented as a single line. Bold black vertical lines indicate the polyadenylation signals (pA) (Supplementary Table S1). Arrows indicate primers (Supplementary Table S2). (B) Western blot analysis of Flp-In T-REx™HEK293 cells with a TDP-43 complementary DNA with a myc sequence inserted into the FRT site with or without doxycycline (Dox) for 72 h using an anti-TDP-43 antibody. (C) RT-PCR analysis of endogenous TARDBP mRNA (amplified product between F2 and R2A) and endogenous TARDBP pre-mRNA (amplified product between F1 and R1). Samples treated with actinomycin D (ActD) (to halt transcription) were used as negative controls for amplification of endogenous TARDBP pre-mRNA. (D) Northern blot analysis of TARDBP mRNA. Poly (A) + RNA was extracted from the nuclear or cytoplasmic fraction in the absence or presence of Dox or cycloheximide (CHX) for 6 h. *Ectopic TARDBP mRNA. M indicates RNA size marker. Roman numerals correspond to those in (E). Hybridized with a probe for exons 2–4 (probe A); by a probe for intron 6 (probe B); and with a probe for intron 7 (probe C) (Supplementary Table S2). The lower panel hybridized with a probe for GAPDH. (E) Schematic representation of TARDBP mRNA isoforms and probes for northern blot analysis. Bold green lines represent probes. Vertical purple lines indicate the additional termination codons (ATCs). (F) Quantification of TARDBP mRNA isoforms by northern blot analysis using probe A. The roman numerals correspond to those in (E). Data represent the relative level of expression in lane 1. Orange bars correspond to samples without Dox. Blue bars correspond to samples with Dox. Data information: data are presented as mean ± SD ( n = 3) * P

    Journal: Nucleic Acids Research

    Article Title: Increased cytoplasmic TARDBP mRNA in affected spinal motor neurons in ALS caused by abnormal autoregulation of TDP-43

    doi: 10.1093/nar/gkw499

    Figure Lengend Snippet: TDP-43 is downregulated via reduction in the amount of cytoplasmic TARDBP mRNA by nonsense-mediated mRNA decay. (A) Schematic representation of TARDBP mRNA and primers for RT-PCR. Black boxes represent the coding region. White boxes represent the non-coding region. Vertical red lines indicate the splicing sites. The diagonal dashed red lines indicate the splicing. Intron 6 has three donor sites (nucleotide positions 769, 833 and 842 in cDNA). The 833 and 842 sites are represented as a single line. Bold black vertical lines indicate the polyadenylation signals (pA) (Supplementary Table S1). Arrows indicate primers (Supplementary Table S2). (B) Western blot analysis of Flp-In T-REx™HEK293 cells with a TDP-43 complementary DNA with a myc sequence inserted into the FRT site with or without doxycycline (Dox) for 72 h using an anti-TDP-43 antibody. (C) RT-PCR analysis of endogenous TARDBP mRNA (amplified product between F2 and R2A) and endogenous TARDBP pre-mRNA (amplified product between F1 and R1). Samples treated with actinomycin D (ActD) (to halt transcription) were used as negative controls for amplification of endogenous TARDBP pre-mRNA. (D) Northern blot analysis of TARDBP mRNA. Poly (A) + RNA was extracted from the nuclear or cytoplasmic fraction in the absence or presence of Dox or cycloheximide (CHX) for 6 h. *Ectopic TARDBP mRNA. M indicates RNA size marker. Roman numerals correspond to those in (E). Hybridized with a probe for exons 2–4 (probe A); by a probe for intron 6 (probe B); and with a probe for intron 7 (probe C) (Supplementary Table S2). The lower panel hybridized with a probe for GAPDH. (E) Schematic representation of TARDBP mRNA isoforms and probes for northern blot analysis. Bold green lines represent probes. Vertical purple lines indicate the additional termination codons (ATCs). (F) Quantification of TARDBP mRNA isoforms by northern blot analysis using probe A. The roman numerals correspond to those in (E). Data represent the relative level of expression in lane 1. Orange bars correspond to samples without Dox. Blue bars correspond to samples with Dox. Data information: data are presented as mean ± SD ( n = 3) * P

    Article Snippet: Quantitative real-time PCR For cultured cells, first-strand cDNA was synthesized from total RNA by ReverTra Ace (TOYOBO) with random primers and an oligo(dT) primer (for standard qRT-PCR), or with P7-t25-vn primer (for 3′-end qRT-PCR) ( ).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Sequencing, Amplification, Northern Blot, Marker, Expressing

    The significance of splicing intron 6. ( A ) Schematic representation of the EGFP-SMN-TDP-43 fused minigene and the location of the primers (primer sequences are listed in Supplementary Table S2). In m-intron 6, we substituted nucleotides at positions 769–770, 833–834 and 842–843 with guanine and at positions 1782–1783 with cytosine. Green box: EGFP; gray box: exons of the SMN gene; black box: coding region of exon 6; white box: non - coding region of exon 6; vertical red lines: splicing sites; diagonal lines: splicing between exons; arrows: primers; diagonal dashed lines within arrows: spanning exons; pA: polyadenylation signal. ( B ) Northern blot analysis of poly(A) + RNA derived from EGFP-fused minigene co-transfected with empty vector or TDP-43 cDNA in HEK293T cells (left upper panel). M indicates RNA size marker. The middle panel hybridized with a probe for GAPDH. The lower panel represents the TARDBP mRNA amplified by the primers between exon 5 (ex5) and exon 6 (ex6). The right panel represents the schematic representation of fusion minigene poly(A) + RNA isoforms and the probe for northern blot analysis. Gray boxes represent exon 7 of the SMN gene. The white boxes represent exon 6 of the human TARDBP gene, and the green line represents a probe. The diagonal dashed red lines indicate the splicing between exons in exon 6. pA indicates polyadenylation signal. ( C ) Quantitative analysis of EGFP-SMN-TDP-43 fusion poly(A) + RNA isoform I in (B). The levels of EGFP-SMN-TDP-43 fusion poly(A) + RNA isoform I were normalized to the levels of GAPDH, and then they were expressed as fold change relative to control (empty vector)(log 2 ) (mean ± SEM, n = 3) (black triangle: empty vector; white box: TDP-43 expression vector). * P

    Journal: Nucleic Acids Research

    Article Title: Increased cytoplasmic TARDBP mRNA in affected spinal motor neurons in ALS caused by abnormal autoregulation of TDP-43

    doi: 10.1093/nar/gkw499

    Figure Lengend Snippet: The significance of splicing intron 6. ( A ) Schematic representation of the EGFP-SMN-TDP-43 fused minigene and the location of the primers (primer sequences are listed in Supplementary Table S2). In m-intron 6, we substituted nucleotides at positions 769–770, 833–834 and 842–843 with guanine and at positions 1782–1783 with cytosine. Green box: EGFP; gray box: exons of the SMN gene; black box: coding region of exon 6; white box: non - coding region of exon 6; vertical red lines: splicing sites; diagonal lines: splicing between exons; arrows: primers; diagonal dashed lines within arrows: spanning exons; pA: polyadenylation signal. ( B ) Northern blot analysis of poly(A) + RNA derived from EGFP-fused minigene co-transfected with empty vector or TDP-43 cDNA in HEK293T cells (left upper panel). M indicates RNA size marker. The middle panel hybridized with a probe for GAPDH. The lower panel represents the TARDBP mRNA amplified by the primers between exon 5 (ex5) and exon 6 (ex6). The right panel represents the schematic representation of fusion minigene poly(A) + RNA isoforms and the probe for northern blot analysis. Gray boxes represent exon 7 of the SMN gene. The white boxes represent exon 6 of the human TARDBP gene, and the green line represents a probe. The diagonal dashed red lines indicate the splicing between exons in exon 6. pA indicates polyadenylation signal. ( C ) Quantitative analysis of EGFP-SMN-TDP-43 fusion poly(A) + RNA isoform I in (B). The levels of EGFP-SMN-TDP-43 fusion poly(A) + RNA isoform I were normalized to the levels of GAPDH, and then they were expressed as fold change relative to control (empty vector)(log 2 ) (mean ± SEM, n = 3) (black triangle: empty vector; white box: TDP-43 expression vector). * P

    Article Snippet: Quantitative real-time PCR For cultured cells, first-strand cDNA was synthesized from total RNA by ReverTra Ace (TOYOBO) with random primers and an oligo(dT) primer (for standard qRT-PCR), or with P7-t25-vn primer (for 3′-end qRT-PCR) ( ).

    Techniques: Northern Blot, Derivative Assay, Transfection, Plasmid Preparation, Marker, Amplification, Expressing

    TDP-43 regulates the use of polyadenylation signals. ( A ) Schematic representation of cDNA derived from the minigene construct (Figure 3A ) and the location of the primers. Gray boxes represent exons of the SMN gene. The arrows represent the primers. For analysis of the mRNA using pA1, we used P7-t25-vn primer first-strand cDNA synthesis (Supplementary Table S2) ( 21 ). ( B ) qRT-PCR analysis of fusion mRNA using each polyadenylation site with the minigene (Figure 3A ) and TDP-43 plasmid (left panel) or TDP-43 siRNA (right panel) in HEK293T cells. Western blot analysis of TDP-43 expression (upper panel). Levels of each pA minigene isoform were measured by qRT-PCR and the relative values of whole unspliced isoforms were determined ( n = 3) (lower panel). EV: empty vector; siCtrl: control siRNA. The probes are listed in Supplementary Table S2. ( C ) Schematic representation of the minigene with a mutation in the intron 7 donor site and the location of the primers for amplification. Gray boxes represent exons of the SMN gene. The arrows represent the primers (Supplementary Table S2). ( D ) qRT-PCR analysis of fusion mRNA from the minigene with a mutation at the intron 7 donor site (C) and TDP-43 plasmid (left panel) or TDP-43 siRNA (right panel) in HEK293T cells. Western blot analysis of TDP-43 expression (upper panel). Levels of isoforms containing sequences beyond pA1 were measured by qRT-PCR and the relative values of whole unspliced isoforms were determined (lower panel). TDP-43 EV: empty vector; siCtrl: control siRNA. The probes are listed in Supplementary Table S2. Data are expressed as fold-change relative to control (log 2 ) and presented as mean ± SEM. Asterisks indicate statistically significant ( P

    Journal: Nucleic Acids Research

    Article Title: Increased cytoplasmic TARDBP mRNA in affected spinal motor neurons in ALS caused by abnormal autoregulation of TDP-43

    doi: 10.1093/nar/gkw499

    Figure Lengend Snippet: TDP-43 regulates the use of polyadenylation signals. ( A ) Schematic representation of cDNA derived from the minigene construct (Figure 3A ) and the location of the primers. Gray boxes represent exons of the SMN gene. The arrows represent the primers. For analysis of the mRNA using pA1, we used P7-t25-vn primer first-strand cDNA synthesis (Supplementary Table S2) ( 21 ). ( B ) qRT-PCR analysis of fusion mRNA using each polyadenylation site with the minigene (Figure 3A ) and TDP-43 plasmid (left panel) or TDP-43 siRNA (right panel) in HEK293T cells. Western blot analysis of TDP-43 expression (upper panel). Levels of each pA minigene isoform were measured by qRT-PCR and the relative values of whole unspliced isoforms were determined ( n = 3) (lower panel). EV: empty vector; siCtrl: control siRNA. The probes are listed in Supplementary Table S2. ( C ) Schematic representation of the minigene with a mutation in the intron 7 donor site and the location of the primers for amplification. Gray boxes represent exons of the SMN gene. The arrows represent the primers (Supplementary Table S2). ( D ) qRT-PCR analysis of fusion mRNA from the minigene with a mutation at the intron 7 donor site (C) and TDP-43 plasmid (left panel) or TDP-43 siRNA (right panel) in HEK293T cells. Western blot analysis of TDP-43 expression (upper panel). Levels of isoforms containing sequences beyond pA1 were measured by qRT-PCR and the relative values of whole unspliced isoforms were determined (lower panel). TDP-43 EV: empty vector; siCtrl: control siRNA. The probes are listed in Supplementary Table S2. Data are expressed as fold-change relative to control (log 2 ) and presented as mean ± SEM. Asterisks indicate statistically significant ( P

    Article Snippet: Quantitative real-time PCR For cultured cells, first-strand cDNA was synthesized from total RNA by ReverTra Ace (TOYOBO) with random primers and an oligo(dT) primer (for standard qRT-PCR), or with P7-t25-vn primer (for 3′-end qRT-PCR) ( ).

    Techniques: Derivative Assay, Construct, Quantitative RT-PCR, Plasmid Preparation, Western Blot, Expressing, Mutagenesis, Amplification

    The depletion of TDP-43 inhibits splicing in exon 6 in the TARDBP gene. ( A ) Schematic representation of a minigene containing exon 6 of the human TARDBP gene. Gray boxes represent exons of the SMN gene. White boxes represent exon 6 of the human TARDBP gene. Vertical red lines indicate the splicing sites. Diagonal dashed red lines indicate the splicing between exons. Lines represent introns. Bold black vertical lines indicate the polyadenylation signals (pA). ( B ) Western blot analysis for TDP-43 in HEK293T cells co-transfected with control siRNA (siCtrl), TDP-43 siRNA (siTDP-43) or TDP-43 expression vector (probes are listed in Supplementary Table S2). ( C ) Northern blot analysis of poly(A) + RNA derived from the minigene. Poly(A) + RNA was extracted from the nuclear or cytoplasmic fraction from HEK293T cells transfected with the minigene (A). The results for cells co-transfected with TDP-43 cDNA plasmid or vector (left panel). The results for cells co-transfected with TDP-43 siRNA or control siRNA (right panel). *Ectopic TARDBP mRNA. M indicates RNA size marker. ( D ) Schematic representation of fusion minigene poly(A) + RNA isoforms and the probe for northern blot analysis. Gray boxes represent exon 7 of the SMN gene. White boxes represent exon 6 of the human TARDBP gene. Green line represents a probe. The diagonal dashed red lines indicate the splicing between exons in exon 6. pA indicates polyadenylation signal. ( E ) Quantification of minigene mRNA isoforms co-transfected with TDP-43 expression vector by northern blot analysis. ( F ) Quantification of minigene mRNA isoforms co-transfected with TDP-43 siRNA by northern blot analysis. Data information: the roman numerals in each graph correspond to those in (D). Data (mean ± SD, n = 3) represent values relative to the value in lane 1. * P

    Journal: Nucleic Acids Research

    Article Title: Increased cytoplasmic TARDBP mRNA in affected spinal motor neurons in ALS caused by abnormal autoregulation of TDP-43

    doi: 10.1093/nar/gkw499

    Figure Lengend Snippet: The depletion of TDP-43 inhibits splicing in exon 6 in the TARDBP gene. ( A ) Schematic representation of a minigene containing exon 6 of the human TARDBP gene. Gray boxes represent exons of the SMN gene. White boxes represent exon 6 of the human TARDBP gene. Vertical red lines indicate the splicing sites. Diagonal dashed red lines indicate the splicing between exons. Lines represent introns. Bold black vertical lines indicate the polyadenylation signals (pA). ( B ) Western blot analysis for TDP-43 in HEK293T cells co-transfected with control siRNA (siCtrl), TDP-43 siRNA (siTDP-43) or TDP-43 expression vector (probes are listed in Supplementary Table S2). ( C ) Northern blot analysis of poly(A) + RNA derived from the minigene. Poly(A) + RNA was extracted from the nuclear or cytoplasmic fraction from HEK293T cells transfected with the minigene (A). The results for cells co-transfected with TDP-43 cDNA plasmid or vector (left panel). The results for cells co-transfected with TDP-43 siRNA or control siRNA (right panel). *Ectopic TARDBP mRNA. M indicates RNA size marker. ( D ) Schematic representation of fusion minigene poly(A) + RNA isoforms and the probe for northern blot analysis. Gray boxes represent exon 7 of the SMN gene. White boxes represent exon 6 of the human TARDBP gene. Green line represents a probe. The diagonal dashed red lines indicate the splicing between exons in exon 6. pA indicates polyadenylation signal. ( E ) Quantification of minigene mRNA isoforms co-transfected with TDP-43 expression vector by northern blot analysis. ( F ) Quantification of minigene mRNA isoforms co-transfected with TDP-43 siRNA by northern blot analysis. Data information: the roman numerals in each graph correspond to those in (D). Data (mean ± SD, n = 3) represent values relative to the value in lane 1. * P

    Article Snippet: Quantitative real-time PCR For cultured cells, first-strand cDNA was synthesized from total RNA by ReverTra Ace (TOYOBO) with random primers and an oligo(dT) primer (for standard qRT-PCR), or with P7-t25-vn primer (for 3′-end qRT-PCR) ( ).

    Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, Northern Blot, Derivative Assay, Marker

    The expression level of cir-ITCH is closely related to lung cancer. (a) cir-ITCH was amplified by RT-PCR with divergent primers in cDNA but was not amplified in genomic DNA (gDNA). GAPDH, linear control. (b) qRT-PCR based on TaqMan probe was used to analyze the expression level of cir-ITCH in lung cancer tissues and paired noncancerous tissues. GAPDH was used as endogenous control. (c) The linear correlations between the cir-ITCH expression levels and linear ITCH were tested by Spearman analysis. The relative expression value was normalized by GAPDH expression level. (d) Random primers and oligo dT primers were used, respectively, in the reverse transcription experiments. The predicted circular RNA is absent in poly(A)-enriched samples. (e) The predicted circular RNA is resistant to RNase R treatment. Data are presented as mean ± SEM from three independent experiments. ∗ p

    Journal: BioMed Research International

    Article Title: Circular RNA-ITCH Suppresses Lung Cancer Proliferation via Inhibiting the Wnt/β-Catenin Pathway

    doi: 10.1155/2016/1579490

    Figure Lengend Snippet: The expression level of cir-ITCH is closely related to lung cancer. (a) cir-ITCH was amplified by RT-PCR with divergent primers in cDNA but was not amplified in genomic DNA (gDNA). GAPDH, linear control. (b) qRT-PCR based on TaqMan probe was used to analyze the expression level of cir-ITCH in lung cancer tissues and paired noncancerous tissues. GAPDH was used as endogenous control. (c) The linear correlations between the cir-ITCH expression levels and linear ITCH were tested by Spearman analysis. The relative expression value was normalized by GAPDH expression level. (d) Random primers and oligo dT primers were used, respectively, in the reverse transcription experiments. The predicted circular RNA is absent in poly(A)-enriched samples. (e) The predicted circular RNA is resistant to RNase R treatment. Data are presented as mean ± SEM from three independent experiments. ∗ p

    Article Snippet: RNA was reversely transcribed into cDNA using First Strand cDNA Synthesis Kit (Toyobo).

    Techniques: Expressing, Amplification, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

    C. neoformans CDA1 is transcriptionally upregulated during host infection. Mice were inoculated with 10 7 CFU of KN99 cells. At day 7 PI, the lungs were excised and homogenized, and fungal cells were harvested and used for the isolation of total RNA. Total RNA (0.5 µg) was used for the synthesis of cDNA, which was subsequently subjected to quantitative real-time PCR using CDA1 -specific primers. C. neoformans 18S rRNA transcript levels were used as a reference gene. Transcript levels of the respective genes in the cells used as inoculum served as control. Data are the averages for two independent experiments each with three animals per group. Fold expression was calculated for each gene comparing the extent of its upregulation in the lung samples to YPD-grown samples. Significant differences in the expression levels between genes were compared by ordinary one-way ANOVA, followed by Dunnett’s multiple-comparison test. (****, P

    Journal: mBio

    Article Title: Cryptococcus neoformans Cda1 and Its Chitin Deacetylase Activity Are Required for Fungal Pathogenesis

    doi: 10.1128/mBio.02087-18

    Figure Lengend Snippet: C. neoformans CDA1 is transcriptionally upregulated during host infection. Mice were inoculated with 10 7 CFU of KN99 cells. At day 7 PI, the lungs were excised and homogenized, and fungal cells were harvested and used for the isolation of total RNA. Total RNA (0.5 µg) was used for the synthesis of cDNA, which was subsequently subjected to quantitative real-time PCR using CDA1 -specific primers. C. neoformans 18S rRNA transcript levels were used as a reference gene. Transcript levels of the respective genes in the cells used as inoculum served as control. Data are the averages for two independent experiments each with three animals per group. Fold expression was calculated for each gene comparing the extent of its upregulation in the lung samples to YPD-grown samples. Significant differences in the expression levels between genes were compared by ordinary one-way ANOVA, followed by Dunnett’s multiple-comparison test. (****, P

    Article Snippet: Total RNA (0.5 µg) was used for first-strand cDNA synthesis using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) per the manufacturer’s instructions.

    Techniques: Infection, Mouse Assay, Isolation, Real-time Polymerase Chain Reaction, Expressing