strand cdna synthesis Search Results


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  • 99
    New England Biolabs protoscript first strand cdna synthesis kit
    qPCR analyses of candidate circRNA expression. Cells were treated with DMSO (control) and cisplatin as explained in Materials and Methods. RNAse R-treated RNA samples were used to prepare cDNAs using <t>ProtoScript</t> ® first strand <t>cDNA</t> synthesis kit (NEB, United States) with random primers. GoTaq q-PCR Master Mix (Promega, United States) was used for qPCR analyses. Data collected in three biological replicates were normalized against DMSO control and GAPDH. ∗∗ and ∗∗∗ denote p ≤ 0.01 and p ≤ 0.001, respectively. (A) HeLa cells; (B) MCF and Jurkat cells.
    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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    Thermo Fisher revertaid first strand cdna synthesis kit
    IL-37 + T-cells play a protective role in DSS-induced chronic colitis in mice. ( A ) Relative weight curves of WT mice, DSS-induced WT mice, DSS-induced WT mice treated with PBS, and DSS-induced WT mice injected with IL-37 + T-cells in chronic colitis. The statistical significance between the relative weights of DSS-induced mice injected with IL-37 + T-cells versus DSS-induced mice treated with PBS at the end of the experiment is shown. ( B ) The disease activity index (DAI) was determined at day 63 of the study period, based on body weight loss (0, none; 1, 1–5%; 2, 5–10%; 3, 10–20%; 4, > 20%), stool consistency (0, normal; 2, loose stool; 4, diarrhea), and stool blood (0, negative; 2, fecal occult blood test positive; 4, gross bleeding). ( C ) Representative pictures of the colon from indicated treatment cohorts. ( D ) Macroscopic damage score of the colon in the four groups. ( E ) Representative haematoxylin and eosin (H E) staining of colon sections of mice. ( F ) Histological inflammation score. This score comprises the sum of architecture of the bowel, infiltration of neutrophils and mononuclear cells, gobleT-cell depletion, and epithelial cell erosion. Three sections per animal were evaluated. ( G ) Expressions of IFN-γ, IL-1β, TNF-α, and IL-10 mRNAs within colonic tissues in the four groups. Total RNA with the RNAiso Plusi Kit. cDNAs were synthesized by using the <t>RevertAid</t> First Strand <t>cDNA</t> Synthesis Kit according to the manufacturer’s instructions. qPCR amplification reactions were prepared with the SYBR Green PCR Kit and performed using the CFX96 Real-Time System. Data are means ± SEM ( n = 6). * p
    Revertaid First Strand Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 40345 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher first strand cdna synthesis
    Elevated gene 50 transcription is accompanied by hypomethylation of the gene 50 promoter at day 18. (A) Quantitative RT-PCR for total gene 50 (G50) (exon 2) transcripts on <t>cDNA</t> generated from total splenocyte <t>RNA.</t> Transcript levels are elevated in Cre-positive
    First Strand Cdna Synthesis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 40815 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher revertaid h minus first strand cdna synthesis kit
    Elevated gene 50 transcription is accompanied by hypomethylation of the gene 50 promoter at day 18. (A) Quantitative RT-PCR for total gene 50 (G50) (exon 2) transcripts on <t>cDNA</t> generated from total splenocyte <t>RNA.</t> Transcript levels are elevated in Cre-positive
    Revertaid H Minus First Strand Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 9555 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa primescript 1st strand cdna synthesis kit
    Elevated gene 50 transcription is accompanied by hypomethylation of the gene 50 promoter at day 18. (A) Quantitative RT-PCR for total gene 50 (G50) (exon 2) transcripts on <t>cDNA</t> generated from total splenocyte <t>RNA.</t> Transcript levels are elevated in Cre-positive
    Primescript 1st Strand Cdna Synthesis Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 7752 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare first strand cdna synthesis kit
    Northern blot analysis of rasGEFM expression in parental and mutant strains . ( A ) Total <t>RNA</t> was extracted from AX2 cells developed in suspension for 0 to 9 hours or on filters. In the latter case the cells were harvested at mound (12 hours), first finger (16 hours) and preculminant (20 hours) stages. The membrane was hybridized to a radiolabelled rasGEFM specific probe (probe a ) corresponding to bp 760–1518 of the <t>cDNA</t> clone and to the actin gene used as a loading control. ( B ) Total RNA was extracted from different developmental Dictyostelium mutants starved in shaking suspension up to 6 hours. LW6 (G protein β subunit minus), HSB1 (PIA ts -mutant, defective in the G-protein adenylyl cyclase activation) and HSB50 (mutant blocked at mound stage).
    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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    Thermo Fisher superscript double stranded cdna synthesis kit
    Overview of Cufflinks. The algorithm takes as input <t>cDNA</t> fragment sequences that have been ( a ) aligned to the genome by software capable of producing spliced alignments, such as TopHat. With paired-end RNA-Seq, Cufflinks treats each pair of fragment reads as a single alignment. The algorithm assembles overlapping ‘bundles’ of fragment alignments ( b-c ) separately, which reduces running time and memory use because each bundle typically contains the fragments from no more than a few genes. Cufflinks then estimates the abundances of the assembled transcripts ( d-e ). ( b ) The first step in fragment assembly is to identify pairs of ‘incompatible’ fragments that must have originated from distinct spliced <t>mRNA</t> isoforms. Fragments are connected in an ‘overlap graph’ when they are compatible and their alignments overlap in the genome. Each fragment has one node in the graph, and an edge, directed from left to right along the genome, is placed between each pair of compatible fragments. In this example, the yellow, blue, and red fragments must have originated from separate isoforms, but any other fragment could have come from the same transcript as one of these three. ( c ) Assembling isoforms from the overlap graph. Paths through the graph correspond to sets of mutually compatible fragments that could be merged into complete isoforms. The overlap graph here can be minimally ‘covered’ by three paths, each representing a different isoform. Dilworth's Theorem states that the number of mutually incompatible reads is the same as the minimum number of transcripts needed to “explain” all the fragments. Cufflinks implements a proof of Dilworth's Theorem that produces a minimal set of paths that cover all the fragments in the overlap graph by finding the largest set of reads with the property that no two could have originated from the same isoform. ( d ) Estimating transcript abundance. Fragments are matched (denoted here using color) to the transcripts from which they could have originated. The violet fragment could have originated from the blue or red isoform. Gray fragments could have come from any of the three shown. Cufflinks estimates transcript abundances using a statistical model in which the probability of observing each fragment is a linear function of the abundances of the transcripts from which it could have originated. Because only the ends of each fragment are sequenced, the length of each may be unknown. Assigning a fragment to different isoforms often implies a different length for it. Cufflinks can incorporate the distribution of fragment lengths to help assign fragments to isoforms. For example, the violet fragment would be much longer, and very improbable according to Cufflinks' model, if it were to come from the red isoform instead of the blue isoform. ( e ) The program then numerically maximizes a function that assigns a likelihood to all possible sets of relative abundances of the yellow, red and blue isoforms (γ 1 ,γ 2 ,γ 3 ), producing the abundances that best explain the observed fragments, shown as a pie chart.
    Superscript Double Stranded Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6021 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa primescript first strand cdna synthesis kit
    Basic characteristics of pJAB1. ( a ) The <t>PCR</t> result of the pJAB1 gene. M, 5000 bp DNA marker; 1, the pJAB1 gene; 2, blank control. ( b ) The exon–intron structure. Eight exons were found in the <t>cDNA</t> sequence. ( c ) The secondary structure showing chromo, MPN and DEAD domains in the pJAB1 protein. ( d ) Comparison of the nucleotide sequences of pJAB1 and hJAB1. Short bars represent the identical nucleotides among the two sequences. Different regions were indicated by lowercase letters
    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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    93
    Thermo Fisher revertaidtm first strand cdna synthesis kit
    Basic characteristics of pJAB1. ( a ) The <t>PCR</t> result of the pJAB1 gene. M, 5000 bp DNA marker; 1, the pJAB1 gene; 2, blank control. ( b ) The exon–intron structure. Eight exons were found in the <t>cDNA</t> sequence. ( c ) The secondary structure showing chromo, MPN and DEAD domains in the pJAB1 protein. ( d ) Comparison of the nucleotide sequences of pJAB1 and hJAB1. Short bars represent the identical nucleotides among the two sequences. Different regions were indicated by lowercase letters
    Revertaidtm First Strand Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 2571 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
    Basic characteristics of pJAB1. ( a ) The <t>PCR</t> result of the pJAB1 gene. M, 5000 bp DNA marker; 1, the pJAB1 gene; 2, blank control. ( b ) The exon–intron structure. Eight exons were found in the <t>cDNA</t> sequence. ( c ) The secondary structure showing chromo, MPN and DEAD domains in the pJAB1 protein. ( d ) Comparison of the nucleotide sequences of pJAB1 and hJAB1. Short bars represent the identical nucleotides among the two sequences. Different regions were indicated by lowercase letters
    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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    92
    Thermo Fisher maxima h minus first strand cdna synthesis kit
    Footprint of a putative regulatory protein bound to the 5’ UTR of psbA : A , Predicted mRNA secondary structure of the psbA translation initiation region in low light (LL) using normalized DMS reactivities ( Figure 1A ) as constrains. The white box marks the position of the primer used to amplify the <t>cDNA.</t> For this region no DMS reactivities could be obtained. The green box marks the footprint of a <t>RNA</t> binding protein ( McDermott et al., 2019 ) (Supplemental Figure S11A-D), the grey boxes indicate the Shine-Dalgarno sequence (AGGA) and the start codon (AUG). For each nucleotide, the normalized DMS reactivity is shown in a color code. The kcal mol −1 value for the strength of the RNA structure is given. B , Predicted mRNA secondary structure in high light (HL) using normalized DMS reactivities ( Figure 1A ) and the protein binding site (forced to be single-stranded) as constrains. For the structure predictions for in vitro -folded RNA see Supplemental Figure S11G. C , Normalized DMS reactivities of the nucleotides predicted to form base pairs in low light ( A ) between the region of the footprint (between nucleotides 35-48) and the region including the start codon and the Shine-Dalgarno sequence (SD) (between nucleotides 69-86). The average normalized DMS reactivities are shown separately for both regions. Nucleotides in these regions predicted not to be paired are excluded. DMS reactivities at the SD side significantly increase in high light indicating a shift to single-stranded RNA. This suggests that in low light a stem loop structure is formed ( A ), whereas in high light a protein is bound to the psbA translation initiation region making the SD and the start codon accessible ( B ). Asterisks indicate statistically significant changes compared to LL ( P -values calculated with the Wilcoxon rank sum test; *** = P
    Maxima H Minus First Strand Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 10362 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Toyobo first strand cdna synthesis kit
    Reverse transcriptase-PCR analysis demonstrates a polycistronic transcript of mtsABC mRNA . Total <t>RNA</t> from S. iniae HD-1 was reverse transcribed into <t>cDNA,</t> and PCR was performed with ORF-specific primers. Each box contains products with the same primer pairs. For PCR, S. iniae HD-1 genomic DNA was used as the template (on the left), and for reverse transcriptase-PCR, S. iniae HD-1 RNA was used as the template (on the right).
    First Strand Cdna Synthesis Kit, supplied by Toyobo, used in various techniques. Bioz Stars score: 94/100, based on 1692 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad first strand cdna synthesis
    Identification of the transcriptional start of ndvB by 5′ RACE. (A) <t>RNA</t> was extracted from PA14 or PA14/pSC01, and first-strand <t>cDNA</t> synthesis was performed with a gfp -specific primer. First-strand cDNA was incubated with dATP without or with TdT (−TdT and +TdT, respectively). The dA-tailed cDNA was subsequently amplified by two rounds of PCR as described in Materials and Methods. The 5′ RACE products were analyzed by electrophoresis on a 1.2% agarose gel. Two 5′ RACE products (major and minor) were obtained. Note that 5′ RACE products were observed only for the reaction that used RNA from PA14/pSC01 and that had been incubated with TdT, demonstrating the specificity of the gfp ). Sequence identity is shown by asterisks. The GTCGACTAGTAC(T) 17 sequence is derived from the 3′ RACE adapter primer. The −10 RpoS element is shown in blue, the TSS is shown in yellow with a +1, and the gfp coding sequence is in green. (C) Two pUC18 plasmids carrying the minor 5′ RACE product (RACE4 and -5) were analyzed as described for the major product.
    First Strand Cdna Synthesis, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 2433 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    tiangen biotech co mircute mirna first strand cdna synthesis kit
    Identification of the transcriptional start of ndvB by 5′ RACE. (A) <t>RNA</t> was extracted from PA14 or PA14/pSC01, and first-strand <t>cDNA</t> synthesis was performed with a gfp -specific primer. First-strand cDNA was incubated with dATP without or with TdT (−TdT and +TdT, respectively). The dA-tailed cDNA was subsequently amplified by two rounds of PCR as described in Materials and Methods. The 5′ RACE products were analyzed by electrophoresis on a 1.2% agarose gel. Two 5′ RACE products (major and minor) were obtained. Note that 5′ RACE products were observed only for the reaction that used RNA from PA14/pSC01 and that had been incubated with TdT, demonstrating the specificity of the gfp ). Sequence identity is shown by asterisks. The GTCGACTAGTAC(T) 17 sequence is derived from the 3′ RACE adapter primer. The −10 RpoS element is shown in blue, the TSS is shown in yellow with a +1, and the gfp coding sequence is in green. (C) Two pUC18 plasmids carrying the minor 5′ RACE product (RACE4 and -5) were analyzed as described for the major product.
    Mircute Mirna First Strand Cdna Synthesis Kit, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 92/100, based on 1063 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    qPCR analyses of candidate circRNA expression. Cells were treated with DMSO (control) and cisplatin as explained in Materials and Methods. RNAse R-treated RNA samples were used to prepare cDNAs using ProtoScript ® first strand cDNA synthesis kit (NEB, United States) with random primers. GoTaq q-PCR Master Mix (Promega, United States) was used for qPCR analyses. Data collected in three biological replicates were normalized against DMSO control and GAPDH. ∗∗ and ∗∗∗ denote p ≤ 0.01 and p ≤ 0.001, respectively. (A) HeLa cells; (B) MCF and Jurkat cells.

    Journal: Frontiers in Genetics

    Article Title: Transcriptomics Analysis of Circular RNAs Differentially Expressed in Apoptotic HeLa Cells

    doi: 10.3389/fgene.2019.00176

    Figure Lengend Snippet: qPCR analyses of candidate circRNA expression. Cells were treated with DMSO (control) and cisplatin as explained in Materials and Methods. RNAse R-treated RNA samples were used to prepare cDNAs using ProtoScript ® first strand cDNA synthesis kit (NEB, United States) with random primers. GoTaq q-PCR Master Mix (Promega, United States) was used for qPCR analyses. Data collected in three biological replicates were normalized against DMSO control and GAPDH. ∗∗ and ∗∗∗ denote p ≤ 0.01 and p ≤ 0.001, respectively. (A) HeLa cells; (B) MCF and Jurkat cells.

    Article Snippet: Subsequently, RNA clean-up was performed with Nucleospin RNA kit (Macherey Nagel, Germany) according to the manufacturer’s instructions. cDNAs were prepared from RNAse R+ and RNAseR- RNA samples with ProtoScript® first strand cDNA synthesis kit (NEB, United States) with random primers in a total volume of 50 ul according to the manufacturer’s instructions.

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Polymerase Chain Reaction

    IL-37 + T-cells play a protective role in DSS-induced chronic colitis in mice. ( A ) Relative weight curves of WT mice, DSS-induced WT mice, DSS-induced WT mice treated with PBS, and DSS-induced WT mice injected with IL-37 + T-cells in chronic colitis. The statistical significance between the relative weights of DSS-induced mice injected with IL-37 + T-cells versus DSS-induced mice treated with PBS at the end of the experiment is shown. ( B ) The disease activity index (DAI) was determined at day 63 of the study period, based on body weight loss (0, none; 1, 1–5%; 2, 5–10%; 3, 10–20%; 4, > 20%), stool consistency (0, normal; 2, loose stool; 4, diarrhea), and stool blood (0, negative; 2, fecal occult blood test positive; 4, gross bleeding). ( C ) Representative pictures of the colon from indicated treatment cohorts. ( D ) Macroscopic damage score of the colon in the four groups. ( E ) Representative haematoxylin and eosin (H E) staining of colon sections of mice. ( F ) Histological inflammation score. This score comprises the sum of architecture of the bowel, infiltration of neutrophils and mononuclear cells, gobleT-cell depletion, and epithelial cell erosion. Three sections per animal were evaluated. ( G ) Expressions of IFN-γ, IL-1β, TNF-α, and IL-10 mRNAs within colonic tissues in the four groups. Total RNA with the RNAiso Plusi Kit. cDNAs were synthesized by using the RevertAid First Strand cDNA Synthesis Kit according to the manufacturer’s instructions. qPCR amplification reactions were prepared with the SYBR Green PCR Kit and performed using the CFX96 Real-Time System. Data are means ± SEM ( n = 6). * p

    Journal: International Journal of Molecular Sciences

    Article Title: Anti-Inflammatory Effect of IL-37-Producing T-Cell Population in DSS-Induced Chronic Inflammatory Bowel Disease in Mice

    doi: 10.3390/ijms19123884

    Figure Lengend Snippet: IL-37 + T-cells play a protective role in DSS-induced chronic colitis in mice. ( A ) Relative weight curves of WT mice, DSS-induced WT mice, DSS-induced WT mice treated with PBS, and DSS-induced WT mice injected with IL-37 + T-cells in chronic colitis. The statistical significance between the relative weights of DSS-induced mice injected with IL-37 + T-cells versus DSS-induced mice treated with PBS at the end of the experiment is shown. ( B ) The disease activity index (DAI) was determined at day 63 of the study period, based on body weight loss (0, none; 1, 1–5%; 2, 5–10%; 3, 10–20%; 4, > 20%), stool consistency (0, normal; 2, loose stool; 4, diarrhea), and stool blood (0, negative; 2, fecal occult blood test positive; 4, gross bleeding). ( C ) Representative pictures of the colon from indicated treatment cohorts. ( D ) Macroscopic damage score of the colon in the four groups. ( E ) Representative haematoxylin and eosin (H E) staining of colon sections of mice. ( F ) Histological inflammation score. This score comprises the sum of architecture of the bowel, infiltration of neutrophils and mononuclear cells, gobleT-cell depletion, and epithelial cell erosion. Three sections per animal were evaluated. ( G ) Expressions of IFN-γ, IL-1β, TNF-α, and IL-10 mRNAs within colonic tissues in the four groups. Total RNA with the RNAiso Plusi Kit. cDNAs were synthesized by using the RevertAid First Strand cDNA Synthesis Kit according to the manufacturer’s instructions. qPCR amplification reactions were prepared with the SYBR Green PCR Kit and performed using the CFX96 Real-Time System. Data are means ± SEM ( n = 6). * p

    Article Snippet: The Nanodrop spectrophotometer (Thermo Scientific, Waltham, MA, USA) was used to measure absorbance. cDNAs were synthesized by using the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher, Waltham, MA, USA) according to manufacturer’s instructions.

    Techniques: Mouse Assay, Injection, Activity Assay, Staining, Synthesized, Real-time Polymerase Chain Reaction, Amplification, SYBR Green Assay, Polymerase Chain Reaction

    Long non-coding RNA LINC01021 was downregulated by Da0324 treatment in gastric cancer cells. A. Volcano plots of lncRNAs differentially expressed between the control and Da0324 treatment groups. SGC7901 cells were treated with DMSO (control) or 4 μM Da0324 for 48 h and high-throughput sequencing assay was performed. The X-axis represents log 2 of fold changes. The Y-axis represents -log 10 of P values. Red spots denote upregulated lncRNAs, blue spots denote downregulated lncRNAs. B. Heatmap of the top 40 lncRNAs most significantly differentially expressed in SGC7901 cells with Da0324 treatment. C. Ten differentially expressed lncRNAs regulated by Da0324 were validated by qRT-PCR. SGC7901 cells were treated with DMSO (control) or 4 μM Da0324 for 48 h. RNAs were isolated and converted to cDNA. Quantitative real-time PCR was performed to determine the expression level of lncRNAs. GAPDH was used as a housekeeping gene. All bars represent relative expression levels and data represent mean ± SD. ***, P

    Journal: American Journal of Translational Research

    Article Title: Downregulation of LINC01021 by curcumin analog Da0324 inhibits gastric cancer progression through activation of P53

    doi:

    Figure Lengend Snippet: Long non-coding RNA LINC01021 was downregulated by Da0324 treatment in gastric cancer cells. A. Volcano plots of lncRNAs differentially expressed between the control and Da0324 treatment groups. SGC7901 cells were treated with DMSO (control) or 4 μM Da0324 for 48 h and high-throughput sequencing assay was performed. The X-axis represents log 2 of fold changes. The Y-axis represents -log 10 of P values. Red spots denote upregulated lncRNAs, blue spots denote downregulated lncRNAs. B. Heatmap of the top 40 lncRNAs most significantly differentially expressed in SGC7901 cells with Da0324 treatment. C. Ten differentially expressed lncRNAs regulated by Da0324 were validated by qRT-PCR. SGC7901 cells were treated with DMSO (control) or 4 μM Da0324 for 48 h. RNAs were isolated and converted to cDNA. Quantitative real-time PCR was performed to determine the expression level of lncRNAs. GAPDH was used as a housekeeping gene. All bars represent relative expression levels and data represent mean ± SD. ***, P

    Article Snippet: Total RNA was reverse transcribed to cDNA using the Revert Aid First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific).

    Techniques: Next-Generation Sequencing, Quantitative RT-PCR, Isolation, Real-time Polymerase Chain Reaction, Expressing

    Elevated gene 50 transcription is accompanied by hypomethylation of the gene 50 promoter at day 18. (A) Quantitative RT-PCR for total gene 50 (G50) (exon 2) transcripts on cDNA generated from total splenocyte RNA. Transcript levels are elevated in Cre-positive

    Journal: Journal of Virology

    Article Title: The De Novo Methyltransferases DNMT3a and DNMT3b Target the Murine Gammaherpesvirus Immediate-Early Gene 50 Promoter during Establishment of Latency ▿

    doi: 10.1128/JVI.00060-10

    Figure Lengend Snippet: Elevated gene 50 transcription is accompanied by hypomethylation of the gene 50 promoter at day 18. (A) Quantitative RT-PCR for total gene 50 (G50) (exon 2) transcripts on cDNA generated from total splenocyte RNA. Transcript levels are elevated in Cre-positive

    Article Snippet: Twenty microliters of DNase-treated RNA was subsequently used for first-strand cDNA synthesis by use of SuperScript II reverse transcriptase (Invitrogen).

    Techniques: Quantitative RT-PCR, Generated

    Calponin-3-GFP is expressed from the endogenous Cnn3 locus. A. RT-PCR analysis of the Cnn3 mRNA splicing in targeted cells. Total RNA from the pre-B cell line Oct as well as from pre-B cells derived from a Cnn3 ki f/f mouse was transcribed into cDNA and analyzed by PCR. Primer pairs for PCRs 1 and 2 are depicted in the schematic illustration of the targeted allele. A sample lacking cDNA served as a control. B. Western blot analysis for expression of the calponin-3-GFP fusion protein from the targeted Cnn3 locus. Pre-B cells derived from a Cnn3 ki f/f mouse or a non-targeted littermate were lysed and subjected to SDS-PAGE and blotting. Oct pre-B cells expressing GFP and calponin-3-GFP were used as a control. Equal loading was demonstrated by anti-eIF4α. C. Flow cytometric analysis of cells from a Cnn3 ki f/f mouse or a non-targeted +/+ littermate. Cells were isolated from the bone marrow of the respective mice, stained with an anti-IgM antibody and analyzed for expression of IgM and GFP by flow cytometry. Numbers indicate the percentage of cells in the respective gate.

    Journal: PLoS ONE

    Article Title: A Conditional Knockout Mouse Model Reveals That Calponin-3 Is Dispensable for Early B Cell Development

    doi: 10.1371/journal.pone.0128385

    Figure Lengend Snippet: Calponin-3-GFP is expressed from the endogenous Cnn3 locus. A. RT-PCR analysis of the Cnn3 mRNA splicing in targeted cells. Total RNA from the pre-B cell line Oct as well as from pre-B cells derived from a Cnn3 ki f/f mouse was transcribed into cDNA and analyzed by PCR. Primer pairs for PCRs 1 and 2 are depicted in the schematic illustration of the targeted allele. A sample lacking cDNA served as a control. B. Western blot analysis for expression of the calponin-3-GFP fusion protein from the targeted Cnn3 locus. Pre-B cells derived from a Cnn3 ki f/f mouse or a non-targeted littermate were lysed and subjected to SDS-PAGE and blotting. Oct pre-B cells expressing GFP and calponin-3-GFP were used as a control. Equal loading was demonstrated by anti-eIF4α. C. Flow cytometric analysis of cells from a Cnn3 ki f/f mouse or a non-targeted +/+ littermate. Cells were isolated from the bone marrow of the respective mice, stained with an anti-IgM antibody and analyzed for expression of IgM and GFP by flow cytometry. Numbers indicate the percentage of cells in the respective gate.

    Article Snippet: 1 μg of RNA was reverse-transcribed into cDNA using the First Strand cDNA Synthesis Kit (Fermentas) according to the manufacturers’ instructions.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Polymerase Chain Reaction, Western Blot, Expressing, SDS Page, Flow Cytometry, Isolation, Mouse Assay, Staining, Cytometry

    Genetic organization of PeWBVYV. (A) PeWBVYV ORFs and validation strategy using RT-PCR amplification. UTR, untranslated region. (B) Strategy for assembly of cDNA corresponding to the PeWBVYV RNA genome from overlapping fragments by overlap extension PCR. (C) PCR amplification of cDNA corresponding to the PeWBVYV RNA genome by overlap PCR.

    Journal: Journal of Virology

    Article Title: Transmission of a New Polerovirus Infecting Pepper by the Whitefly Bemisia tabaci

    doi: 10.1128/JVI.00488-19

    Figure Lengend Snippet: Genetic organization of PeWBVYV. (A) PeWBVYV ORFs and validation strategy using RT-PCR amplification. UTR, untranslated region. (B) Strategy for assembly of cDNA corresponding to the PeWBVYV RNA genome from overlapping fragments by overlap extension PCR. (C) PCR amplification of cDNA corresponding to the PeWBVYV RNA genome by overlap PCR.

    Article Snippet: Treatment with DNase I and synthesis of first-strand cDNA with 2 μg RNA and random hexamers were done using a Maxima first-strand cDNA synthesis kit (Thermo Fisher Scientific).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction

    Effect of TLR9 deletion on cytokine mRNA expression pattern in leukocytes isolated from C. neoformans infected lungs. Lung leukocytes were isolated from uninfected (week 0) and infected TLR9+/+ and TLR9−/−mice at 3 wpi. Total leukocyte RNA was extracted, converted to cDNA, and analyzed by real-time RT-PCR for the expression of selected “polarizing” cytokines. The values were normalized to GAPDH mRNA levels and were expressed as relative gene expression ± SEM. Data were pooled from two parallel experiments. N = 3 for uninfected controls and at least seven for infected mice at each of the analyzed time points. For the comparisons between TLR9+/+ versus TLR9−/− mice, * P

    Journal: The American Journal of Pathology

    Article Title: TLR9 Signaling Is Required for Generation of the Adaptive Immune Protection in Cryptococcus neoformans-Infected Lungs

    doi: 10.2353/ajpath.2010.091104

    Figure Lengend Snippet: Effect of TLR9 deletion on cytokine mRNA expression pattern in leukocytes isolated from C. neoformans infected lungs. Lung leukocytes were isolated from uninfected (week 0) and infected TLR9+/+ and TLR9−/−mice at 3 wpi. Total leukocyte RNA was extracted, converted to cDNA, and analyzed by real-time RT-PCR for the expression of selected “polarizing” cytokines. The values were normalized to GAPDH mRNA levels and were expressed as relative gene expression ± SEM. Data were pooled from two parallel experiments. N = 3 for uninfected controls and at least seven for infected mice at each of the analyzed time points. For the comparisons between TLR9+/+ versus TLR9−/− mice, * P

    Article Snippet: Total RNA was prepared using RNeasy Plus Mini Kit (Qiagen, Valencia, CA) and first-strand cDNA was synthesized using SuperScriptIII (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

    Techniques: Expressing, Isolation, Infection, Mouse Assay, Quantitative RT-PCR

    Effect of TLR9 deletion on pulmonary lymph node and spleen polarization. A: Pulmonary lymph nodes were collected from uninfected (week 0) and infected TLR9+/+ and TLR9−/−mice at 3 wpi. Total RNA was extracted, converted to cDNA, and analyzed by real-time RT-PCR for the expression of selected “polarizing” cytokines. The values were normalized to GAPDH mRNA levels and were expressed as relative mRNA expression ± SEM. Data were pooled from two parallel experiments. N = 2 for uninfected controls and 7 for infected mice; * P

    Journal: The American Journal of Pathology

    Article Title: TLR9 Signaling Is Required for Generation of the Adaptive Immune Protection in Cryptococcus neoformans-Infected Lungs

    doi: 10.2353/ajpath.2010.091104

    Figure Lengend Snippet: Effect of TLR9 deletion on pulmonary lymph node and spleen polarization. A: Pulmonary lymph nodes were collected from uninfected (week 0) and infected TLR9+/+ and TLR9−/−mice at 3 wpi. Total RNA was extracted, converted to cDNA, and analyzed by real-time RT-PCR for the expression of selected “polarizing” cytokines. The values were normalized to GAPDH mRNA levels and were expressed as relative mRNA expression ± SEM. Data were pooled from two parallel experiments. N = 2 for uninfected controls and 7 for infected mice; * P

    Article Snippet: Total RNA was prepared using RNeasy Plus Mini Kit (Qiagen, Valencia, CA) and first-strand cDNA was synthesized using SuperScriptIII (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

    Techniques: Infection, Mouse Assay, Quantitative RT-PCR, Expressing

    Overexpression of GMF in mouse neuroblastoma N18 cells after transfection with GMF/adenovirus construct (GMF-V). Mouse N18 cells were plated into 24-well plates (1×10 5 cells per well) and grown in DMEM/F12 containing 5% fetal bovine serum (complete medium). Replication-defective human adenovirus vector containing a full length GMF cDNA (GMF-V) or cytoplasmic lacZ cDNA (LacZ) at 20 MOI (multiplicity of infectivity) were added to cells in serum-free and antibiotic free DMEM/F12 medium for 4 h. Cells were rinsed and allowed to grow in fresh complete medium for another 24 h. Mock-transfected controls were processed identically in the absence of virus. Cell lysates (20 μg protein per lane) were subjected to SDS-polyacrylamide gel electrophoresis followed by electroblotting. The blots were probed with anti-GMF (G2-09) antibody and anti-β-actin antibody. Actin served as internal marker showing equal sample loading. For siRNA mediated down-regulation of GMF expression in N18, cells were transfected with duplex siRNA targeted against GMF (GsiRNA 10 nM), or with a control scrambled sequence (CsiRNA 10 nM). Cells were transfected with siRNA 4 h prior to adenovirus transfections. Lane 1, mock infection; lane 2, lacZ; lane 3, GMF-V; lane 4, CsiRNA; and lane 5, GsiRNA. (A) Western blot. (B) RT-PCR. The data shown are representative of at least three experiments.

    Journal: Brain research

    Article Title: Glia maturation factor overexpression in neuroblastoma cells activates glycogen synthase kinase-3? and caspase-3

    doi: 10.1016/j.brainres.2007.11.011

    Figure Lengend Snippet: Overexpression of GMF in mouse neuroblastoma N18 cells after transfection with GMF/adenovirus construct (GMF-V). Mouse N18 cells were plated into 24-well plates (1×10 5 cells per well) and grown in DMEM/F12 containing 5% fetal bovine serum (complete medium). Replication-defective human adenovirus vector containing a full length GMF cDNA (GMF-V) or cytoplasmic lacZ cDNA (LacZ) at 20 MOI (multiplicity of infectivity) were added to cells in serum-free and antibiotic free DMEM/F12 medium for 4 h. Cells were rinsed and allowed to grow in fresh complete medium for another 24 h. Mock-transfected controls were processed identically in the absence of virus. Cell lysates (20 μg protein per lane) were subjected to SDS-polyacrylamide gel electrophoresis followed by electroblotting. The blots were probed with anti-GMF (G2-09) antibody and anti-β-actin antibody. Actin served as internal marker showing equal sample loading. For siRNA mediated down-regulation of GMF expression in N18, cells were transfected with duplex siRNA targeted against GMF (GsiRNA 10 nM), or with a control scrambled sequence (CsiRNA 10 nM). Cells were transfected with siRNA 4 h prior to adenovirus transfections. Lane 1, mock infection; lane 2, lacZ; lane 3, GMF-V; lane 4, CsiRNA; and lane 5, GsiRNA. (A) Western blot. (B) RT-PCR. The data shown are representative of at least three experiments.

    Article Snippet: The first strand cDNA synthesis was carried out, using a ThermoScript RT-PCR system kit (GibcoBRL, Life Technologies).

    Techniques: Over Expression, Transfection, Construct, Plasmid Preparation, Infection, Polyacrylamide Gel Electrophoresis, Marker, Expressing, Sequencing, Western Blot, Reverse Transcription Polymerase Chain Reaction

    Topoisomerase-I-specific CD4+ T cells show a Th17 polarized functional phenotype. a CD4+ T cell subsets were identified by flow cytometry using the combined surface expression of chemokine receptors CCR6, CXCR3, and CCR4. b After sorting, the different T helper subsets were stimulated with anti-CD3/CD28 beads for 48 h and secretion of IFNγ, IL-4, and IL-17A was measured in their supernatants by enzyme-linked immunosorbent assay ( bars represent mean ± SE, n = 3). c The distribution of polarized T helper subsets within topo-I-specific CD4+ T cells ( closed circles ) is shown in comparison to the general CD4+ population ( open circles ). T test with Bonferroni correction was used for multiple comparisons. Lines indicate mean ± SD. d After sorting topo-I-specific CD4+ T cells according to their polarized T helper phenotype (range 16–2064 cells), mRNA expression levels of IFNγ, IL-4, and IL-17A were measured by qPCR using the Arcturus® PicoPure® RNA Isolation system, which is designed to recover high-quality RNA from a very low number of cells (10–500 cells) and to accomplish cDNA synthesis from minimal amounts of cDNA. IFNγ and IL-17A expression was calculated as fold change in reference to a mix of topo-I-reactive CD4+ T cells from three patients; IL-4 expression is reported as delta Ct from actin expression since it was not detectable in the reference population. Data are representative of four separate patients (mean ± SE)

    Journal: Arthritis Research & Therapy

    Article Title: Frequency of circulating topoisomerase-I-specific CD4 T cells predicts presence and progression of interstitial lung disease in scleroderma

    doi: 10.1186/s13075-016-0993-2

    Figure Lengend Snippet: Topoisomerase-I-specific CD4+ T cells show a Th17 polarized functional phenotype. a CD4+ T cell subsets were identified by flow cytometry using the combined surface expression of chemokine receptors CCR6, CXCR3, and CCR4. b After sorting, the different T helper subsets were stimulated with anti-CD3/CD28 beads for 48 h and secretion of IFNγ, IL-4, and IL-17A was measured in their supernatants by enzyme-linked immunosorbent assay ( bars represent mean ± SE, n = 3). c The distribution of polarized T helper subsets within topo-I-specific CD4+ T cells ( closed circles ) is shown in comparison to the general CD4+ population ( open circles ). T test with Bonferroni correction was used for multiple comparisons. Lines indicate mean ± SD. d After sorting topo-I-specific CD4+ T cells according to their polarized T helper phenotype (range 16–2064 cells), mRNA expression levels of IFNγ, IL-4, and IL-17A were measured by qPCR using the Arcturus® PicoPure® RNA Isolation system, which is designed to recover high-quality RNA from a very low number of cells (10–500 cells) and to accomplish cDNA synthesis from minimal amounts of cDNA. IFNγ and IL-17A expression was calculated as fold change in reference to a mix of topo-I-reactive CD4+ T cells from three patients; IL-4 expression is reported as delta Ct from actin expression since it was not detectable in the reference population. Data are representative of four separate patients (mean ± SE)

    Article Snippet: Single-strand cDNAs were synthesized with the Superscript III first-strand cDNA synthesis kit following the manufacturer’s protocol (Invitrogen, Life Technologies, Waltham, MA, USA).

    Techniques: Functional Assay, Flow Cytometry, Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Isolation

    Effects of Arf6 mutants on Tac recycling in MDA-MB-231 cells. MDA-MB-231 cells were transfected with Tac cDNA, together with Arf6(Q67L) or Arf6(T27N) cDNAs as described, and subjected to internalization ( A ) and recycling-back ( B ) assays of Tac antigen. The control included cotransfection of Tac with EGFP cDNA (mock). Results are means ± SEM from three independent experiments.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Requirement for Arf6 in breast cancer invasive activities

    doi: 10.1073/pnas.0401753101

    Figure Lengend Snippet: Effects of Arf6 mutants on Tac recycling in MDA-MB-231 cells. MDA-MB-231 cells were transfected with Tac cDNA, together with Arf6(Q67L) or Arf6(T27N) cDNAs as described, and subjected to internalization ( A ) and recycling-back ( B ) assays of Tac antigen. The control included cotransfection of Tac with EGFP cDNA (mock). Results are means ± SEM from three independent experiments.

    Article Snippet: Random-primed cDNAs, prepared from 2 μg of cellular RNAs by using the SuperScript first-strand cDNA synthesis kit (Invitrogen), were subjected to real-time PCR amplification analysis using the LightCycler FastStart DNA Master SYBR Green I (Roche) and the LightCycler (Roche), according to the manufacturer's instructions.

    Techniques: Multiple Displacement Amplification, Transfection, Cotransfection

    Northern blot analysis of rasGEFM expression in parental and mutant strains . ( A ) Total RNA was extracted from AX2 cells developed in suspension for 0 to 9 hours or on filters. In the latter case the cells were harvested at mound (12 hours), first finger (16 hours) and preculminant (20 hours) stages. The membrane was hybridized to a radiolabelled rasGEFM specific probe (probe a ) corresponding to bp 760–1518 of the cDNA clone and to the actin gene used as a loading control. ( B ) Total RNA was extracted from different developmental Dictyostelium mutants starved in shaking suspension up to 6 hours. LW6 (G protein β subunit minus), HSB1 (PIA ts -mutant, defective in the G-protein adenylyl cyclase activation) and HSB50 (mutant blocked at mound stage).

    Journal: BMC Cell Biology

    Article Title: A novel Dictyostelium RasGEF required for chemotaxis and development

    doi: 10.1186/1471-2121-6-43

    Figure Lengend Snippet: Northern blot analysis of rasGEFM expression in parental and mutant strains . ( A ) Total RNA was extracted from AX2 cells developed in suspension for 0 to 9 hours or on filters. In the latter case the cells were harvested at mound (12 hours), first finger (16 hours) and preculminant (20 hours) stages. The membrane was hybridized to a radiolabelled rasGEFM specific probe (probe a ) corresponding to bp 760–1518 of the cDNA clone and to the actin gene used as a loading control. ( B ) Total RNA was extracted from different developmental Dictyostelium mutants starved in shaking suspension up to 6 hours. LW6 (G protein β subunit minus), HSB1 (PIA ts -mutant, defective in the G-protein adenylyl cyclase activation) and HSB50 (mutant blocked at mound stage).

    Article Snippet: As template, cDNA isolated with "First strand cDNA synthesis kit" (Amersham Pharmacia Biotech), from RNA of cells developed for 5 hours on solid substrata, was used.

    Techniques: Northern Blot, Expressing, Mutagenesis, Activation Assay

    Disruption of the rasGEFM gene . ( A ) Southern blot of genomic DNA from parental strain (AX2) and rasGEFM null mutant (HSB61). Genomic DNA was digested with Eco RI, separated in 0.8% agarose gel, blotted onto nylon membrane and probed with probe a ( rasGEFM N-terminal fragment corresponding to bp 0–617 of the cDNA clone), probe b (corresponding to bp 760–1518 of the rasGEFM cDNA clone) and probe c ( bsr cassette). Three different probes were used to characterize the genomic locus of the mutant and the parental strain. In the rasGEFM null strain the central part of the gene (recognized by probe b ), was replaced by the blasticidin cassette (recognized by probe c ), which has the same size of the replaced fragment. Because of that, the size of the locus remains unchanged, but the central part is recognized specifically by bsr (probe c ) or probe b in HSB61 or AX2, respectively. Both AX2 and HSB61 genomic loci are recognized by the probe a . ( B ) Northern blot of total RNA extracted from HSB61 cells at the indicated time points of growth and development. The membrane was hybridized to the rasGEFM and to the actin gene.

    Journal: BMC Cell Biology

    Article Title: A novel Dictyostelium RasGEF required for chemotaxis and development

    doi: 10.1186/1471-2121-6-43

    Figure Lengend Snippet: Disruption of the rasGEFM gene . ( A ) Southern blot of genomic DNA from parental strain (AX2) and rasGEFM null mutant (HSB61). Genomic DNA was digested with Eco RI, separated in 0.8% agarose gel, blotted onto nylon membrane and probed with probe a ( rasGEFM N-terminal fragment corresponding to bp 0–617 of the cDNA clone), probe b (corresponding to bp 760–1518 of the rasGEFM cDNA clone) and probe c ( bsr cassette). Three different probes were used to characterize the genomic locus of the mutant and the parental strain. In the rasGEFM null strain the central part of the gene (recognized by probe b ), was replaced by the blasticidin cassette (recognized by probe c ), which has the same size of the replaced fragment. Because of that, the size of the locus remains unchanged, but the central part is recognized specifically by bsr (probe c ) or probe b in HSB61 or AX2, respectively. Both AX2 and HSB61 genomic loci are recognized by the probe a . ( B ) Northern blot of total RNA extracted from HSB61 cells at the indicated time points of growth and development. The membrane was hybridized to the rasGEFM and to the actin gene.

    Article Snippet: As template, cDNA isolated with "First strand cDNA synthesis kit" (Amersham Pharmacia Biotech), from RNA of cells developed for 5 hours on solid substrata, was used.

    Techniques: Southern Blot, Mutagenesis, Agarose Gel Electrophoresis, Northern Blot

    Overview of Cufflinks. The algorithm takes as input cDNA fragment sequences that have been ( a ) aligned to the genome by software capable of producing spliced alignments, such as TopHat. With paired-end RNA-Seq, Cufflinks treats each pair of fragment reads as a single alignment. The algorithm assembles overlapping ‘bundles’ of fragment alignments ( b-c ) separately, which reduces running time and memory use because each bundle typically contains the fragments from no more than a few genes. Cufflinks then estimates the abundances of the assembled transcripts ( d-e ). ( b ) The first step in fragment assembly is to identify pairs of ‘incompatible’ fragments that must have originated from distinct spliced mRNA isoforms. Fragments are connected in an ‘overlap graph’ when they are compatible and their alignments overlap in the genome. Each fragment has one node in the graph, and an edge, directed from left to right along the genome, is placed between each pair of compatible fragments. In this example, the yellow, blue, and red fragments must have originated from separate isoforms, but any other fragment could have come from the same transcript as one of these three. ( c ) Assembling isoforms from the overlap graph. Paths through the graph correspond to sets of mutually compatible fragments that could be merged into complete isoforms. The overlap graph here can be minimally ‘covered’ by three paths, each representing a different isoform. Dilworth's Theorem states that the number of mutually incompatible reads is the same as the minimum number of transcripts needed to “explain” all the fragments. Cufflinks implements a proof of Dilworth's Theorem that produces a minimal set of paths that cover all the fragments in the overlap graph by finding the largest set of reads with the property that no two could have originated from the same isoform. ( d ) Estimating transcript abundance. Fragments are matched (denoted here using color) to the transcripts from which they could have originated. The violet fragment could have originated from the blue or red isoform. Gray fragments could have come from any of the three shown. Cufflinks estimates transcript abundances using a statistical model in which the probability of observing each fragment is a linear function of the abundances of the transcripts from which it could have originated. Because only the ends of each fragment are sequenced, the length of each may be unknown. Assigning a fragment to different isoforms often implies a different length for it. Cufflinks can incorporate the distribution of fragment lengths to help assign fragments to isoforms. For example, the violet fragment would be much longer, and very improbable according to Cufflinks' model, if it were to come from the red isoform instead of the blue isoform. ( e ) The program then numerically maximizes a function that assigns a likelihood to all possible sets of relative abundances of the yellow, red and blue isoforms (γ 1 ,γ 2 ,γ 3 ), producing the abundances that best explain the observed fragments, shown as a pie chart.

    Journal: Nature biotechnology

    Article Title: Transcript assembly and abundance estimation from RNA-Seq reveals thousands of new transcripts and switching among isoforms

    doi: 10.1038/nbt.1621

    Figure Lengend Snippet: Overview of Cufflinks. The algorithm takes as input cDNA fragment sequences that have been ( a ) aligned to the genome by software capable of producing spliced alignments, such as TopHat. With paired-end RNA-Seq, Cufflinks treats each pair of fragment reads as a single alignment. The algorithm assembles overlapping ‘bundles’ of fragment alignments ( b-c ) separately, which reduces running time and memory use because each bundle typically contains the fragments from no more than a few genes. Cufflinks then estimates the abundances of the assembled transcripts ( d-e ). ( b ) The first step in fragment assembly is to identify pairs of ‘incompatible’ fragments that must have originated from distinct spliced mRNA isoforms. Fragments are connected in an ‘overlap graph’ when they are compatible and their alignments overlap in the genome. Each fragment has one node in the graph, and an edge, directed from left to right along the genome, is placed between each pair of compatible fragments. In this example, the yellow, blue, and red fragments must have originated from separate isoforms, but any other fragment could have come from the same transcript as one of these three. ( c ) Assembling isoforms from the overlap graph. Paths through the graph correspond to sets of mutually compatible fragments that could be merged into complete isoforms. The overlap graph here can be minimally ‘covered’ by three paths, each representing a different isoform. Dilworth's Theorem states that the number of mutually incompatible reads is the same as the minimum number of transcripts needed to “explain” all the fragments. Cufflinks implements a proof of Dilworth's Theorem that produces a minimal set of paths that cover all the fragments in the overlap graph by finding the largest set of reads with the property that no two could have originated from the same isoform. ( d ) Estimating transcript abundance. Fragments are matched (denoted here using color) to the transcripts from which they could have originated. The violet fragment could have originated from the blue or red isoform. Gray fragments could have come from any of the three shown. Cufflinks estimates transcript abundances using a statistical model in which the probability of observing each fragment is a linear function of the abundances of the transcripts from which it could have originated. Because only the ends of each fragment are sequenced, the length of each may be unknown. Assigning a fragment to different isoforms often implies a different length for it. Cufflinks can incorporate the distribution of fragment lengths to help assign fragments to isoforms. For example, the violet fragment would be much longer, and very improbable according to Cufflinks' model, if it were to come from the red isoform instead of the blue isoform. ( e ) The program then numerically maximizes a function that assigns a likelihood to all possible sets of relative abundances of the yellow, red and blue isoforms (γ 1 ,γ 2 ,γ 3 ), producing the abundances that best explain the observed fragments, shown as a pie chart.

    Article Snippet: After removal of hydrolysis ions by G50 Sephadex filtration (USA Scientific catalog # 1415-1602), the fragmented mRNA was random primed with hexamers and reverse-transcribed using the Super Script II cDNA synthesis kit (Invitrogen catalog # 11917010).

    Techniques: Software, RNA Sequencing Assay

    Basic characteristics of pJAB1. ( a ) The PCR result of the pJAB1 gene. M, 5000 bp DNA marker; 1, the pJAB1 gene; 2, blank control. ( b ) The exon–intron structure. Eight exons were found in the cDNA sequence. ( c ) The secondary structure showing chromo, MPN and DEAD domains in the pJAB1 protein. ( d ) Comparison of the nucleotide sequences of pJAB1 and hJAB1. Short bars represent the identical nucleotides among the two sequences. Different regions were indicated by lowercase letters

    Journal: Cell Death & Disease

    Article Title: Porcine JAB1 significantly enhances apoptosis induced by staurosporine

    doi: 10.1038/cddis.2013.357

    Figure Lengend Snippet: Basic characteristics of pJAB1. ( a ) The PCR result of the pJAB1 gene. M, 5000 bp DNA marker; 1, the pJAB1 gene; 2, blank control. ( b ) The exon–intron structure. Eight exons were found in the cDNA sequence. ( c ) The secondary structure showing chromo, MPN and DEAD domains in the pJAB1 protein. ( d ) Comparison of the nucleotide sequences of pJAB1 and hJAB1. Short bars represent the identical nucleotides among the two sequences. Different regions were indicated by lowercase letters

    Article Snippet: Extracted RNAs were subjected to RT-PCR using first-strand cDNA synthesis kit (Takara Bio., Dalian, China).

    Techniques: Polymerase Chain Reaction, Marker, Sequencing

    Footprint of a putative regulatory protein bound to the 5’ UTR of psbA : A , Predicted mRNA secondary structure of the psbA translation initiation region in low light (LL) using normalized DMS reactivities ( Figure 1A ) as constrains. The white box marks the position of the primer used to amplify the cDNA. For this region no DMS reactivities could be obtained. The green box marks the footprint of a RNA binding protein ( McDermott et al., 2019 ) (Supplemental Figure S11A-D), the grey boxes indicate the Shine-Dalgarno sequence (AGGA) and the start codon (AUG). For each nucleotide, the normalized DMS reactivity is shown in a color code. The kcal mol −1 value for the strength of the RNA structure is given. B , Predicted mRNA secondary structure in high light (HL) using normalized DMS reactivities ( Figure 1A ) and the protein binding site (forced to be single-stranded) as constrains. For the structure predictions for in vitro -folded RNA see Supplemental Figure S11G. C , Normalized DMS reactivities of the nucleotides predicted to form base pairs in low light ( A ) between the region of the footprint (between nucleotides 35-48) and the region including the start codon and the Shine-Dalgarno sequence (SD) (between nucleotides 69-86). The average normalized DMS reactivities are shown separately for both regions. Nucleotides in these regions predicted not to be paired are excluded. DMS reactivities at the SD side significantly increase in high light indicating a shift to single-stranded RNA. This suggests that in low light a stem loop structure is formed ( A ), whereas in high light a protein is bound to the psbA translation initiation region making the SD and the start codon accessible ( B ). Asterisks indicate statistically significant changes compared to LL ( P -values calculated with the Wilcoxon rank sum test; *** = P

    Journal: bioRxiv

    Article Title: Light-dependent translation change of Arabidopsis psbA correlates with RNA structure alterations at the translation initiation region

    doi: 10.1101/2020.06.11.145870

    Figure Lengend Snippet: Footprint of a putative regulatory protein bound to the 5’ UTR of psbA : A , Predicted mRNA secondary structure of the psbA translation initiation region in low light (LL) using normalized DMS reactivities ( Figure 1A ) as constrains. The white box marks the position of the primer used to amplify the cDNA. For this region no DMS reactivities could be obtained. The green box marks the footprint of a RNA binding protein ( McDermott et al., 2019 ) (Supplemental Figure S11A-D), the grey boxes indicate the Shine-Dalgarno sequence (AGGA) and the start codon (AUG). For each nucleotide, the normalized DMS reactivity is shown in a color code. The kcal mol −1 value for the strength of the RNA structure is given. B , Predicted mRNA secondary structure in high light (HL) using normalized DMS reactivities ( Figure 1A ) and the protein binding site (forced to be single-stranded) as constrains. For the structure predictions for in vitro -folded RNA see Supplemental Figure S11G. C , Normalized DMS reactivities of the nucleotides predicted to form base pairs in low light ( A ) between the region of the footprint (between nucleotides 35-48) and the region including the start codon and the Shine-Dalgarno sequence (SD) (between nucleotides 69-86). The average normalized DMS reactivities are shown separately for both regions. Nucleotides in these regions predicted not to be paired are excluded. DMS reactivities at the SD side significantly increase in high light indicating a shift to single-stranded RNA. This suggests that in low light a stem loop structure is formed ( A ), whereas in high light a protein is bound to the psbA translation initiation region making the SD and the start codon accessible ( B ). Asterisks indicate statistically significant changes compared to LL ( P -values calculated with the Wilcoxon rank sum test; *** = P

    Article Snippet: RNA was recovered by ethanol precipitation. cDNA synthesis, library preparation and sequencing was done as described above.

    Techniques: RNA Binding Assay, Sequencing, Protein Binding, In Vitro

    Reverse transcriptase-PCR analysis demonstrates a polycistronic transcript of mtsABC mRNA . Total RNA from S. iniae HD-1 was reverse transcribed into cDNA, and PCR was performed with ORF-specific primers. Each box contains products with the same primer pairs. For PCR, S. iniae HD-1 genomic DNA was used as the template (on the left), and for reverse transcriptase-PCR, S. iniae HD-1 RNA was used as the template (on the right).

    Journal: BMC Microbiology

    Article Title: A solute-binding protein for iron transport in Streptococcus iniae

    doi: 10.1186/1471-2180-10-309

    Figure Lengend Snippet: Reverse transcriptase-PCR analysis demonstrates a polycistronic transcript of mtsABC mRNA . Total RNA from S. iniae HD-1 was reverse transcribed into cDNA, and PCR was performed with ORF-specific primers. Each box contains products with the same primer pairs. For PCR, S. iniae HD-1 genomic DNA was used as the template (on the left), and for reverse transcriptase-PCR, S. iniae HD-1 RNA was used as the template (on the right).

    Article Snippet: First-strand cDNA was synthesized from 1 μg total RNA using the first-strand cDNA synthesis kit with ReverTra Ace-α-reverse transcriptase (Toyobo Co., Ltd.).

    Techniques: Polymerase Chain Reaction

    Identification of the transcriptional start of ndvB by 5′ RACE. (A) RNA was extracted from PA14 or PA14/pSC01, and first-strand cDNA synthesis was performed with a gfp -specific primer. First-strand cDNA was incubated with dATP without or with TdT (−TdT and +TdT, respectively). The dA-tailed cDNA was subsequently amplified by two rounds of PCR as described in Materials and Methods. The 5′ RACE products were analyzed by electrophoresis on a 1.2% agarose gel. Two 5′ RACE products (major and minor) were obtained. Note that 5′ RACE products were observed only for the reaction that used RNA from PA14/pSC01 and that had been incubated with TdT, demonstrating the specificity of the gfp ). Sequence identity is shown by asterisks. The GTCGACTAGTAC(T) 17 sequence is derived from the 3′ RACE adapter primer. The −10 RpoS element is shown in blue, the TSS is shown in yellow with a +1, and the gfp coding sequence is in green. (C) Two pUC18 plasmids carrying the minor 5′ RACE product (RACE4 and -5) were analyzed as described for the major product.

    Journal: Applied and Environmental Microbiology

    Article Title: Pseudomonas aeruginosa Biofilm Antibiotic Resistance Gene ndvB Expression Requires the RpoS Stationary-Phase Sigma Factor

    doi: 10.1128/AEM.02762-17

    Figure Lengend Snippet: Identification of the transcriptional start of ndvB by 5′ RACE. (A) RNA was extracted from PA14 or PA14/pSC01, and first-strand cDNA synthesis was performed with a gfp -specific primer. First-strand cDNA was incubated with dATP without or with TdT (−TdT and +TdT, respectively). The dA-tailed cDNA was subsequently amplified by two rounds of PCR as described in Materials and Methods. The 5′ RACE products were analyzed by electrophoresis on a 1.2% agarose gel. Two 5′ RACE products (major and minor) were obtained. Note that 5′ RACE products were observed only for the reaction that used RNA from PA14/pSC01 and that had been incubated with TdT, demonstrating the specificity of the gfp ). Sequence identity is shown by asterisks. The GTCGACTAGTAC(T) 17 sequence is derived from the 3′ RACE adapter primer. The −10 RpoS element is shown in blue, the TSS is shown in yellow with a +1, and the gfp coding sequence is in green. (C) Two pUC18 plasmids carrying the minor 5′ RACE product (RACE4 and -5) were analyzed as described for the major product.

    Article Snippet: The iScript cDNA synthesis kit (Bio-Rad) was used for first-strand cDNA synthesis from 2 μg of RNA in a volume of 40 μl as recommended by the manufacturer. qPCR experiments were performed using a MyiQ single-color detection system (Bio-Rad) with 20-μl reaction mixtures containing SYBR green Supermix (Bio-Rad), 500 nM (each) forward and reverse gene-specific primers, and 2 μl of cDNA.

    Techniques: Incubation, Amplification, Polymerase Chain Reaction, Electrophoresis, Agarose Gel Electrophoresis, Sequencing, Derivative Assay