double-stranded cdna Search Results


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  • 99
    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 7122 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa primescript first strand 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.
    Primescript First Strand Cdna Synthesis Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 2799 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Illumina Inc double stranded cdna
    Methods for total RNA-Seq Shown are the salient details for five protocols for total RNA-Seq. DSN-lite (Duplex-specific nuclease, low C 0 t normalization), RNase H, and <t>Ribo-Zero</t> were tested for low quality samples; SMART was tested for low quantity samples. NuGEN, which generates double-stranded <t>cDNA</t> amplified using Ribo-SPIA (Single Primer Isothermal Amplification), was tested for both types of samples: (NuGEN 100f for low quality; NuGEN 1i for low quantity, and NuGEN 1f for low quantity and low quality). In each case, RNA and matching cDNA are in black, adaptors and primers in color, and rRNA is in grey.
    Double Stranded Cdna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 92/100, based on 2694 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher maxima h minus double stranded 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 Double Stranded Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 8962 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher double stranded cdna
    Semi-quantitative RT-PCR analysis of differentially expressed genes PCR-analysis was performed for six genes on <t>cDNA</t> generated from normal skin tissues or different AIDS-KS tissues. Expression level indicated the 10-fold dilution step starting with 500 ng total <t>RNA/DNase</t> treated. Error bars are shown. * P
    Double Stranded Cdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 13429 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Agilent technologies double stranded cdna
    Differential expression of genes by TWEAK in C2C12 myotubes. A). Distribution curve of differentially expressed genes in response to TWEAK treatment detected by <t>cDNA</t> microarray analysis. The normalized fold changes were plotted on y-axis on logarithmic scale. B C). C2C12 myotubes were treated with 10 ng/ml of TWEAK for 18h followed by isolation of total <t>RNA</t> and QRT-PCR. Untreated cells under similar conditions were taken as control. The relative expression values from the QRT-PCR analysis were plotted for each gene are mean ± SD (n = 3). The numbers above the bar represents the fold changes with TWEAK treatment against control, and ‘*’ represents the statistical significance (p-value ≤0.01). Data presented here show that mRNA levels of Nfkbia, Nfkb2, cyclinD1, Map3k14, and Mmp9 was significantly increased whereas the levels of Notch1, Pgam2, Ankrd2, TCap, Mhc4, Mmp2, and Timp2 are reduced in TWEAK-treated C2C12 cells. The relative expression values from the QRT-PCR analysis were plotted for each gene are mean ± SD (n = 3). The numbers above the bar represents the fold changes with TWEAK treatment against control, and ‘*’ represents the statistical significance (p-value ≤0.01). D). Differential expression of NF-κB2, MMP-9, Notch1, and MMP-2. C2C12 myotubes were treated with 10 ng/ml of TWEAK for 18h following isolation of total protein for Western blotting. All the samples were quantified and equal amounts of proteins were loaded on 10% SDS-PAGE gel. Representative immunoblots from three independent experiments (n = 3) presented here showed that TWEAK treatment increases the protein levels of NF-κB2 and MMP-9 and reduces the levels of Notch1 and MMP-2.
    Double Stranded Cdna, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 648 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Illumina Inc double stranded cdnas
    Differential expression of genes by TWEAK in C2C12 myotubes. A). Distribution curve of differentially expressed genes in response to TWEAK treatment detected by <t>cDNA</t> microarray analysis. The normalized fold changes were plotted on y-axis on logarithmic scale. B C). C2C12 myotubes were treated with 10 ng/ml of TWEAK for 18h followed by isolation of total <t>RNA</t> and QRT-PCR. Untreated cells under similar conditions were taken as control. The relative expression values from the QRT-PCR analysis were plotted for each gene are mean ± SD (n = 3). The numbers above the bar represents the fold changes with TWEAK treatment against control, and ‘*’ represents the statistical significance (p-value ≤0.01). Data presented here show that mRNA levels of Nfkbia, Nfkb2, cyclinD1, Map3k14, and Mmp9 was significantly increased whereas the levels of Notch1, Pgam2, Ankrd2, TCap, Mhc4, Mmp2, and Timp2 are reduced in TWEAK-treated C2C12 cells. The relative expression values from the QRT-PCR analysis were plotted for each gene are mean ± SD (n = 3). The numbers above the bar represents the fold changes with TWEAK treatment against control, and ‘*’ represents the statistical significance (p-value ≤0.01). D). Differential expression of NF-κB2, MMP-9, Notch1, and MMP-2. C2C12 myotubes were treated with 10 ng/ml of TWEAK for 18h following isolation of total protein for Western blotting. All the samples were quantified and equal amounts of proteins were loaded on 10% SDS-PAGE gel. Representative immunoblots from three independent experiments (n = 3) presented here showed that TWEAK treatment increases the protein levels of NF-κB2 and MMP-9 and reduces the levels of Notch1 and MMP-2.
    Double Stranded Cdnas, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 93/100, based on 556 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Stratagene double stranded cdna
    ( A ) Quantification of RCA–RCA amplification-fold in <t>cDNA.</t> QRT-PCR was performed either directly from unamplified BT474 cDNA or from the RCA–RCA-amplified product for three single copy genes. Curves 1–3 CYC, GAPDH, HER2 respectively, using RCA–RCA-amplified DNA as starting material. Curves 4–6 CYC, GAPDH, and HER2 respectively, using unamplified DNA as starting material. ( B ) Detection of relative gene expression of BT-474 versus reference human mammary epithelial cells before and after RCA–RCA-amplification of cDNA, using Taqman realtime PCR. cDNA used for RCA–RCA amplification was obtained from the reverse transcription of ∼25 ng total <t>RNA</t> starting material.
    Double Stranded Cdna, supplied by Stratagene, used in various techniques. Bioz Stars score: 93/100, based on 321 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Eppendorf AG double stranded cdna
    ( A ) Quantification of RCA–RCA amplification-fold in <t>cDNA.</t> QRT-PCR was performed either directly from unamplified BT474 cDNA or from the RCA–RCA-amplified product for three single copy genes. Curves 1–3 CYC, GAPDH, HER2 respectively, using RCA–RCA-amplified DNA as starting material. Curves 4–6 CYC, GAPDH, and HER2 respectively, using unamplified DNA as starting material. ( B ) Detection of relative gene expression of BT-474 versus reference human mammary epithelial cells before and after RCA–RCA-amplification of cDNA, using Taqman realtime PCR. cDNA used for RCA–RCA amplification was obtained from the reverse transcription of ∼25 ng total <t>RNA</t> starting material.
    Double Stranded Cdna, supplied by Eppendorf AG, used in various techniques. Bioz Stars score: 92/100, based on 244 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Enzo Biochem double stranded cdna
    ( A ) Quantification of RCA–RCA amplification-fold in <t>cDNA.</t> QRT-PCR was performed either directly from unamplified BT474 cDNA or from the RCA–RCA-amplified product for three single copy genes. Curves 1–3 CYC, GAPDH, HER2 respectively, using RCA–RCA-amplified DNA as starting material. Curves 4–6 CYC, GAPDH, and HER2 respectively, using unamplified DNA as starting material. ( B ) Detection of relative gene expression of BT-474 versus reference human mammary epithelial cells before and after RCA–RCA-amplification of cDNA, using Taqman realtime PCR. cDNA used for RCA–RCA amplification was obtained from the reverse transcription of ∼25 ng total <t>RNA</t> starting material.
    Double Stranded Cdna, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 92/100, based on 291 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Illumina Inc double stranded cdna synthesis
    Fig fruit developmental stages and tissues used to extract <t>RNA</t> and construct <t>cDNA</t> libraries for the Illumina high-throughput sequencing. (A) Developmental stages of parthenocarpic (top rows of each set) and pollinated (lower rows of each set) fruit displaying pulp and inflorescence; 8WAP – 8 weeks after pollination; 9WAP – 9 weeks after pollination; 10% – 10% fruit ripening; 60% – 60% fruit ripening; 100% – 100% fruit ripening. (B) Parthenocarpic sampled tissues that were used in this study. ( C ) Pollinated sampled tissues that were used in this study.
    Double Stranded Cdna Synthesis, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 91/100, based on 122 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Axygen double stranded cdna
    Fig fruit developmental stages and tissues used to extract <t>RNA</t> and construct <t>cDNA</t> libraries for the Illumina high-throughput sequencing. (A) Developmental stages of parthenocarpic (top rows of each set) and pollinated (lower rows of each set) fruit displaying pulp and inflorescence; 8WAP – 8 weeks after pollination; 9WAP – 9 weeks after pollination; 10% – 10% fruit ripening; 60% – 60% fruit ripening; 100% – 100% fruit ripening. (B) Parthenocarpic sampled tissues that were used in this study. ( C ) Pollinated sampled tissues that were used in this study.
    Double Stranded Cdna, supplied by Axygen, used in various techniques. Bioz Stars score: 90/100, based on 213 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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

    Upregulation of expression of the Grg2 (RDA1) and Grg6 (RDA3) genes induced by E2A-HLF. (A) Northern blot analysis of poly(A) + RNA (1 μg per lane) prepared from FL5.12 cells stably transfected with the pMT empty vector (lanes 1 to 4) or with metallothionein promoter-regulated constructs containing cDNAs for E2A-HLF (lanes 5 to 8), the ΔAD1 E2A-HLF mutant lacking the AD1 transactivation domain of E2A (lanes 9 and 10), the BX E2A-HLF mutant with a disabled HLF DNA-binding domain (lanes 11 and 12), E2A (E12 [lanes 13 and 14] and E47 [lanes 15 and 16]), HLF (lanes 17 and 18), or TEF (lanes 19 and 20). Cells were cultured in IL-3-containing medium with (+) or without (−) 100 μM ZnSO 4 for 18 h to induce expression of the transfected cDNA and then continued in the same concentration of zinc and either maintained in IL-3 (+) or deprived of the cytokine (−) for an additional 12 h before RNA extraction. The blot was hybridized with the Grg2 (RDA1), Grg6 (RDA3), or GAPDH cDNA probe as indicated. The mobilities of the 18S and 28S rRNAs are shown. (B) Immunoblot analysis of FL5.12 cells transfected with E2A-HLF, E12, E47, HLF, or TEF protein. To verify that expression of the indicated genes was upregulated by zinc addition, lysates were prepared from FL5.12 cells and analyzed by immunoblotting with E2A rabbit antisera (αE2A) (top) and HLF(C) rabbit antisera (αHLF) (bottom). Cells were stably transfected with the pMT empty vector (pMT-CB6+) or with metallothionein promoter-regulated constructs containing cDNAs for E2A-HLF, E2A (E12 and E47), HLF, or TEF, with or without the addition of zinc as described for panel A.

    Journal: Molecular and Cellular Biology

    Article Title: The E2A-HLF Oncoprotein Activates Groucho-Related Genes and Suppresses Runx1

    doi: 10.1128/MCB.21.17.5935-5945.2001

    Figure Lengend Snippet: Upregulation of expression of the Grg2 (RDA1) and Grg6 (RDA3) genes induced by E2A-HLF. (A) Northern blot analysis of poly(A) + RNA (1 μg per lane) prepared from FL5.12 cells stably transfected with the pMT empty vector (lanes 1 to 4) or with metallothionein promoter-regulated constructs containing cDNAs for E2A-HLF (lanes 5 to 8), the ΔAD1 E2A-HLF mutant lacking the AD1 transactivation domain of E2A (lanes 9 and 10), the BX E2A-HLF mutant with a disabled HLF DNA-binding domain (lanes 11 and 12), E2A (E12 [lanes 13 and 14] and E47 [lanes 15 and 16]), HLF (lanes 17 and 18), or TEF (lanes 19 and 20). Cells were cultured in IL-3-containing medium with (+) or without (−) 100 μM ZnSO 4 for 18 h to induce expression of the transfected cDNA and then continued in the same concentration of zinc and either maintained in IL-3 (+) or deprived of the cytokine (−) for an additional 12 h before RNA extraction. The blot was hybridized with the Grg2 (RDA1), Grg6 (RDA3), or GAPDH cDNA probe as indicated. The mobilities of the 18S and 28S rRNAs are shown. (B) Immunoblot analysis of FL5.12 cells transfected with E2A-HLF, E12, E47, HLF, or TEF protein. To verify that expression of the indicated genes was upregulated by zinc addition, lysates were prepared from FL5.12 cells and analyzed by immunoblotting with E2A rabbit antisera (αE2A) (top) and HLF(C) rabbit antisera (αHLF) (bottom). Cells were stably transfected with the pMT empty vector (pMT-CB6+) or with metallothionein promoter-regulated constructs containing cDNAs for E2A-HLF, E2A (E12 and E47), HLF, or TEF, with or without the addition of zinc as described for panel A.

    Article Snippet: Double-stranded cDNAs were synthesized with the Superscript cDNA synthesis kit (GIBCO-BRL) according to the manufacturer's protocol.

    Techniques: Expressing, Northern Blot, Stable Transfection, Transfection, Plasmid Preparation, Construct, Mutagenesis, Binding Assay, Cell Culture, Concentration Assay, RNA Extraction

    Agarose gel electrophoresis of RDA products from RNA extracted from bovine parainfluenza virus 3-infected cells. Double-stranded cDNA was synthesized from RNA of bovine parainfluenza virus 3-infected MDBK cells and subjected to RDA. Mock-infected cells were used for the synthesis of driver amplicons for RDA. One-twentieth of the volume of the amplified products was separated on 3% agarose gel and stained with ethidium bromide. RDA product from the uninfected control cells was used as a negative control.

    Journal: Nucleic Acids Research

    Article Title: Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription

    doi: 10.1093/nar/gni064

    Figure Lengend Snippet: Agarose gel electrophoresis of RDA products from RNA extracted from bovine parainfluenza virus 3-infected cells. Double-stranded cDNA was synthesized from RNA of bovine parainfluenza virus 3-infected MDBK cells and subjected to RDA. Mock-infected cells were used for the synthesis of driver amplicons for RDA. One-twentieth of the volume of the amplified products was separated on 3% agarose gel and stained with ethidium bromide. RDA product from the uninfected control cells was used as a negative control.

    Article Snippet: cDNA RDA First-strand cDNA was synthesized from the mixed RNA with non-ribosomal hexanucleotides by using a double-stranded cDNA synthesis kit (Invitrogen) according to the manufacturer's protocol, i.e. the total RNA was diluted to 1 μg per μl and mixed with dNTPs, the non-ribosomal hexanucleotides, 5× reaction buffer, 0.1 M DTT and an RNase inhibitor.

    Techniques: Agarose Gel Electrophoresis, Infection, Synthesized, Amplification, Staining, Negative Control

    Autoradiogram of 32 P-labelled double-stranded cDNA synthesized from mixtures consisting of artificial RNA and total cellular RNA. In vitro transcribed RNA was synthesized from pCIneo plasmid and mixed with total cellular RNA extracted from rat2 cells in weight proportions 1:0 (lanes 1 and 8), 1:1 (lanes 2 and 9), 1:10 (lanes 3 and 10), 1:100 (lanes 4 and 11), 1:300 (lanes 5 and 12), 1:1000 (lanes 6 and 13) and 0:1 (lanes 7 and 14). One microgram of mixed RNA was reverse transcribed using random (lanes 1–7) or non-ribosomal (lanes 8–14) hexanucleotides and a second-strand cDNA was then synthesized with RNaseH, DNA polymerase and DNA ligase according to the method described in Materials and Methods. One-tenth of the volume of synthesized cDNAs was loaded on agarose gel ( A ). Loaded volumes were corrected to include the same amounts of 32 P in each sample ( B ). Positions and sizes (bp) of markers are present on the left.

    Journal: Nucleic Acids Research

    Article Title: Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription

    doi: 10.1093/nar/gni064

    Figure Lengend Snippet: Autoradiogram of 32 P-labelled double-stranded cDNA synthesized from mixtures consisting of artificial RNA and total cellular RNA. In vitro transcribed RNA was synthesized from pCIneo plasmid and mixed with total cellular RNA extracted from rat2 cells in weight proportions 1:0 (lanes 1 and 8), 1:1 (lanes 2 and 9), 1:10 (lanes 3 and 10), 1:100 (lanes 4 and 11), 1:300 (lanes 5 and 12), 1:1000 (lanes 6 and 13) and 0:1 (lanes 7 and 14). One microgram of mixed RNA was reverse transcribed using random (lanes 1–7) or non-ribosomal (lanes 8–14) hexanucleotides and a second-strand cDNA was then synthesized with RNaseH, DNA polymerase and DNA ligase according to the method described in Materials and Methods. One-tenth of the volume of synthesized cDNAs was loaded on agarose gel ( A ). Loaded volumes were corrected to include the same amounts of 32 P in each sample ( B ). Positions and sizes (bp) of markers are present on the left.

    Article Snippet: cDNA RDA First-strand cDNA was synthesized from the mixed RNA with non-ribosomal hexanucleotides by using a double-stranded cDNA synthesis kit (Invitrogen) according to the manufacturer's protocol, i.e. the total RNA was diluted to 1 μg per μl and mixed with dNTPs, the non-ribosomal hexanucleotides, 5× reaction buffer, 0.1 M DTT and an RNase inhibitor.

    Techniques: Synthesized, In Vitro, Plasmid Preparation, Agarose Gel Electrophoresis

    Analysis of MV up-regulated genes by Northern blot hybridization. Twenty micrograms of total RNA from control and MV-treated seedlings harvested at the indicated times after the onset of MV treatment were fractionated on formaldehyde–agarose gels and transferred to nylon membranes. Membranes were hybridized with 32 P-labelled cDNA fragments of the indicated genes and their MIPS identifiers are indicated

    Journal: Plant Molecular Biology

    Article Title: Generation of superoxide anion in chloroplasts of Arabidopsis thaliana during active photosynthesis: a focus on rapidly induced genes

    doi: 10.1007/s11103-007-9274-4

    Figure Lengend Snippet: Analysis of MV up-regulated genes by Northern blot hybridization. Twenty micrograms of total RNA from control and MV-treated seedlings harvested at the indicated times after the onset of MV treatment were fractionated on formaldehyde–agarose gels and transferred to nylon membranes. Membranes were hybridized with 32 P-labelled cDNA fragments of the indicated genes and their MIPS identifiers are indicated

    Article Snippet: Twenty μg of total RNA were used as template for double-stranded cDNA synthesis (SuperScript Choice system, Gibco/BRL, Carlsbad, California).

    Techniques: Northern Blot, Hybridization

    Bioenergetics analysis of macrophages challenged with S. aureus Mouse BMDM (5 × 10 4 ) were plated in XF 96 V3-PS cell culture plates and treated with AICAR (1mM) one hour before challenge with S. aureus (MOI 10:1) (A) and HKSA (10 8 cfu) (B) for 8h. The extracellular acidification rate (ECAR) was measured by the sequential addition of glucose, oligomycin and 2DG. In a separate experiment, macrophages were plated in 6 well plate (5× 10 6 cells/well) and treated with AICAR (1mM) one hour before S. aureus infection for 8h. Cells were collected, RNA was extracted, cDNA was synthesized, and qRT PCR was performed for the glycolytic pathway genes HK2 and Glut1 and fold change was calculated using GAPDH ( C ). Each data point represents mean ± SD (n = 6/condition/experiment), and results are representative of at least 3 independent experiments. Statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple-comparison test. * P

    Journal: Cellular microbiology

    Article Title: AICAR-mediated AMPK activation induces protective innate responses in bacterial endophthalmitis

    doi: 10.1111/cmi.12625

    Figure Lengend Snippet: Bioenergetics analysis of macrophages challenged with S. aureus Mouse BMDM (5 × 10 4 ) were plated in XF 96 V3-PS cell culture plates and treated with AICAR (1mM) one hour before challenge with S. aureus (MOI 10:1) (A) and HKSA (10 8 cfu) (B) for 8h. The extracellular acidification rate (ECAR) was measured by the sequential addition of glucose, oligomycin and 2DG. In a separate experiment, macrophages were plated in 6 well plate (5× 10 6 cells/well) and treated with AICAR (1mM) one hour before S. aureus infection for 8h. Cells were collected, RNA was extracted, cDNA was synthesized, and qRT PCR was performed for the glycolytic pathway genes HK2 and Glut1 and fold change was calculated using GAPDH ( C ). Each data point represents mean ± SD (n = 6/condition/experiment), and results are representative of at least 3 independent experiments. Statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple-comparison test. * P

    Article Snippet: RNA was reversed transcribed to cDNA using a cDNA synthesis kit (Superscript, Invitrogen Carlsbad, CA, USA).

    Techniques: Cell Culture, Infection, Synthesized, Quantitative RT-PCR

    S. aureus -infected microglia and retinal tissue showed increased glycolysis and AICAR treatment inhibited this response BV2 cells (3 × 10 4 cells/well) were plated in XF 96 V3-PS 96 cell culture plates (Seahorse Bioscience) and treated with AICAR (1mM) one hour before challenged with live S. aureus (MOI 10:1) (A) or HKSA (10 8 cfu) for 8h (B). The extracellular acidification rate (ECAR) was measured by the sequential addition of glucose, oligomycin and 2 deoxyglucose (2-DG). To examine the contribution of bacteria alone in the total response of the cells, live S. aureus was added in media only. In the another experiment, BV2 mouse microglia cells were plated in 6 well plate (1× 10 6 cells/well) and treated with AICAR (1mM) one hour before S. aureus infection for 8h. Cells were collected, RNA was extracted, cDNA was synthesized, and qRT-PCR was performed for glycolytic pathway genes HK2 and Glut1 and fold changes were calculated using housekeeping gene GAPDH ( C ). Eyes of WT (C57BL/6) (n = 5) were infected with S. aureus (5000 cfu/eye) followed by AICAR treatment (30 μg/eye) for 24h. Retinas were removed and pooled for RNA extraction. cDNA was prepared, and qPCR was performed for the glycolytic pathway genes HK2 and Glut1 and fold change was calculated using GAPDH (D) . Each data point represents mean ± SD (n = 6/condition/experiment), and results are representative of at least 3 independent experiments. The results represent the mean ± SD of triplicates from three independent experiments. Statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple-comparison test. * P

    Journal: Cellular microbiology

    Article Title: AICAR-mediated AMPK activation induces protective innate responses in bacterial endophthalmitis

    doi: 10.1111/cmi.12625

    Figure Lengend Snippet: S. aureus -infected microglia and retinal tissue showed increased glycolysis and AICAR treatment inhibited this response BV2 cells (3 × 10 4 cells/well) were plated in XF 96 V3-PS 96 cell culture plates (Seahorse Bioscience) and treated with AICAR (1mM) one hour before challenged with live S. aureus (MOI 10:1) (A) or HKSA (10 8 cfu) for 8h (B). The extracellular acidification rate (ECAR) was measured by the sequential addition of glucose, oligomycin and 2 deoxyglucose (2-DG). To examine the contribution of bacteria alone in the total response of the cells, live S. aureus was added in media only. In the another experiment, BV2 mouse microglia cells were plated in 6 well plate (1× 10 6 cells/well) and treated with AICAR (1mM) one hour before S. aureus infection for 8h. Cells were collected, RNA was extracted, cDNA was synthesized, and qRT-PCR was performed for glycolytic pathway genes HK2 and Glut1 and fold changes were calculated using housekeeping gene GAPDH ( C ). Eyes of WT (C57BL/6) (n = 5) were infected with S. aureus (5000 cfu/eye) followed by AICAR treatment (30 μg/eye) for 24h. Retinas were removed and pooled for RNA extraction. cDNA was prepared, and qPCR was performed for the glycolytic pathway genes HK2 and Glut1 and fold change was calculated using GAPDH (D) . Each data point represents mean ± SD (n = 6/condition/experiment), and results are representative of at least 3 independent experiments. The results represent the mean ± SD of triplicates from three independent experiments. Statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple-comparison test. * P

    Article Snippet: RNA was reversed transcribed to cDNA using a cDNA synthesis kit (Superscript, Invitrogen Carlsbad, CA, USA).

    Techniques: Infection, Cell Culture, Synthesized, Quantitative RT-PCR, RNA Extraction, Real-time Polymerase Chain Reaction

    Methods for total RNA-Seq Shown are the salient details for five protocols for total RNA-Seq. DSN-lite (Duplex-specific nuclease, low C 0 t normalization), RNase H, and Ribo-Zero were tested for low quality samples; SMART was tested for low quantity samples. NuGEN, which generates double-stranded cDNA amplified using Ribo-SPIA (Single Primer Isothermal Amplification), was tested for both types of samples: (NuGEN 100f for low quality; NuGEN 1i for low quantity, and NuGEN 1f for low quantity and low quality). In each case, RNA and matching cDNA are in black, adaptors and primers in color, and rRNA is in grey.

    Journal: Nature methods

    Article Title: Comprehensive comparative analysis of RNA sequencing methods for degraded or low input samples

    doi: 10.1038/nmeth.2483

    Figure Lengend Snippet: Methods for total RNA-Seq Shown are the salient details for five protocols for total RNA-Seq. DSN-lite (Duplex-specific nuclease, low C 0 t normalization), RNase H, and Ribo-Zero were tested for low quality samples; SMART was tested for low quantity samples. NuGEN, which generates double-stranded cDNA amplified using Ribo-SPIA (Single Primer Isothermal Amplification), was tested for both types of samples: (NuGEN 100f for low quality; NuGEN 1i for low quantity, and NuGEN 1f for low quantity and low quality). In each case, RNA and matching cDNA are in black, adaptors and primers in color, and rRNA is in grey.

    Article Snippet: For the Ribo-Zero libraries, we synthesized double-stranded cDNA and prepared an indexed Illumina library as described for the RNase H libraries.

    Techniques: RNA Sequencing Assay, Amplification

    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

    Semi-quantitative RT-PCR analysis of differentially expressed genes PCR-analysis was performed for six genes on cDNA generated from normal skin tissues or different AIDS-KS tissues. Expression level indicated the 10-fold dilution step starting with 500 ng total RNA/DNase treated. Error bars are shown. * P

    Journal: BMC Cancer

    Article Title: Gene expression profile of AIDS-related Kaposi's sarcoma

    doi: 10.1186/1471-2407-3-7

    Figure Lengend Snippet: Semi-quantitative RT-PCR analysis of differentially expressed genes PCR-analysis was performed for six genes on cDNA generated from normal skin tissues or different AIDS-KS tissues. Expression level indicated the 10-fold dilution step starting with 500 ng total RNA/DNase treated. Error bars are shown. * P

    Article Snippet: In brief, poly A+ RNA was converted to double-stranded cDNA with Superscript II RNAse H- Reverse Transcriptase (Life Technologies, San Diego CA, USA) and a primer biotin-5'-T18-3'.

    Techniques: Quantitative RT-PCR, Polymerase Chain Reaction, Generated, Expressing

    Differential expression of genes by TWEAK in C2C12 myotubes. A). Distribution curve of differentially expressed genes in response to TWEAK treatment detected by cDNA microarray analysis. The normalized fold changes were plotted on y-axis on logarithmic scale. B C). C2C12 myotubes were treated with 10 ng/ml of TWEAK for 18h followed by isolation of total RNA and QRT-PCR. Untreated cells under similar conditions were taken as control. The relative expression values from the QRT-PCR analysis were plotted for each gene are mean ± SD (n = 3). The numbers above the bar represents the fold changes with TWEAK treatment against control, and ‘*’ represents the statistical significance (p-value ≤0.01). Data presented here show that mRNA levels of Nfkbia, Nfkb2, cyclinD1, Map3k14, and Mmp9 was significantly increased whereas the levels of Notch1, Pgam2, Ankrd2, TCap, Mhc4, Mmp2, and Timp2 are reduced in TWEAK-treated C2C12 cells. The relative expression values from the QRT-PCR analysis were plotted for each gene are mean ± SD (n = 3). The numbers above the bar represents the fold changes with TWEAK treatment against control, and ‘*’ represents the statistical significance (p-value ≤0.01). D). Differential expression of NF-κB2, MMP-9, Notch1, and MMP-2. C2C12 myotubes were treated with 10 ng/ml of TWEAK for 18h following isolation of total protein for Western blotting. All the samples were quantified and equal amounts of proteins were loaded on 10% SDS-PAGE gel. Representative immunoblots from three independent experiments (n = 3) presented here showed that TWEAK treatment increases the protein levels of NF-κB2 and MMP-9 and reduces the levels of Notch1 and MMP-2.

    Journal: PLoS ONE

    Article Title: Genomic Profiling of Messenger RNAs and MicroRNAs Reveals Potential Mechanisms of TWEAK-Induced Skeletal Muscle Wasting in Mice

    doi: 10.1371/journal.pone.0008760

    Figure Lengend Snippet: Differential expression of genes by TWEAK in C2C12 myotubes. A). Distribution curve of differentially expressed genes in response to TWEAK treatment detected by cDNA microarray analysis. The normalized fold changes were plotted on y-axis on logarithmic scale. B C). C2C12 myotubes were treated with 10 ng/ml of TWEAK for 18h followed by isolation of total RNA and QRT-PCR. Untreated cells under similar conditions were taken as control. The relative expression values from the QRT-PCR analysis were plotted for each gene are mean ± SD (n = 3). The numbers above the bar represents the fold changes with TWEAK treatment against control, and ‘*’ represents the statistical significance (p-value ≤0.01). Data presented here show that mRNA levels of Nfkbia, Nfkb2, cyclinD1, Map3k14, and Mmp9 was significantly increased whereas the levels of Notch1, Pgam2, Ankrd2, TCap, Mhc4, Mmp2, and Timp2 are reduced in TWEAK-treated C2C12 cells. The relative expression values from the QRT-PCR analysis were plotted for each gene are mean ± SD (n = 3). The numbers above the bar represents the fold changes with TWEAK treatment against control, and ‘*’ represents the statistical significance (p-value ≤0.01). D). Differential expression of NF-κB2, MMP-9, Notch1, and MMP-2. C2C12 myotubes were treated with 10 ng/ml of TWEAK for 18h following isolation of total protein for Western blotting. All the samples were quantified and equal amounts of proteins were loaded on 10% SDS-PAGE gel. Representative immunoblots from three independent experiments (n = 3) presented here showed that TWEAK treatment increases the protein levels of NF-κB2 and MMP-9 and reduces the levels of Notch1 and MMP-2.

    Article Snippet: A total of 250 ng RNA was used to synthesize double stranded cDNA using the Low RNA Input Fluorescent Linear Application Kit (Agilent).

    Techniques: Expressing, Microarray, Isolation, Quantitative RT-PCR, Significance Assay, Western Blot, SDS Page

    ( A ) Quantification of RCA–RCA amplification-fold in cDNA. QRT-PCR was performed either directly from unamplified BT474 cDNA or from the RCA–RCA-amplified product for three single copy genes. Curves 1–3 CYC, GAPDH, HER2 respectively, using RCA–RCA-amplified DNA as starting material. Curves 4–6 CYC, GAPDH, and HER2 respectively, using unamplified DNA as starting material. ( B ) Detection of relative gene expression of BT-474 versus reference human mammary epithelial cells before and after RCA–RCA-amplification of cDNA, using Taqman realtime PCR. cDNA used for RCA–RCA amplification was obtained from the reverse transcription of ∼25 ng total RNA starting material.

    Journal: Genome Research

    Article Title: DNA amplification method tolerant to sample degradation

    doi: 10.1101/gr.2813404

    Figure Lengend Snippet: ( A ) Quantification of RCA–RCA amplification-fold in cDNA. QRT-PCR was performed either directly from unamplified BT474 cDNA or from the RCA–RCA-amplified product for three single copy genes. Curves 1–3 CYC, GAPDH, HER2 respectively, using RCA–RCA-amplified DNA as starting material. Curves 4–6 CYC, GAPDH, and HER2 respectively, using unamplified DNA as starting material. ( B ) Detection of relative gene expression of BT-474 versus reference human mammary epithelial cells before and after RCA–RCA-amplification of cDNA, using Taqman realtime PCR. cDNA used for RCA–RCA amplification was obtained from the reverse transcription of ∼25 ng total RNA starting material.

    Article Snippet: Double stranded cDNA was obtained from RNA extracted from reference breast epithelial cells (Stratagene) or from BT-474 cells using the Strategene kit.

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

    Fig fruit developmental stages and tissues used to extract RNA and construct cDNA libraries for the Illumina high-throughput sequencing. (A) Developmental stages of parthenocarpic (top rows of each set) and pollinated (lower rows of each set) fruit displaying pulp and inflorescence; 8WAP – 8 weeks after pollination; 9WAP – 9 weeks after pollination; 10% – 10% fruit ripening; 60% – 60% fruit ripening; 100% – 100% fruit ripening. (B) Parthenocarpic sampled tissues that were used in this study. ( C ) Pollinated sampled tissues that were used in this study.

    Journal: Frontiers in Plant Science

    Article Title: Tissue-Specific Transcriptome and Hormonal Regulation of Pollinated and Parthenocarpic Fig (Ficus carica L.) Fruit Suggest that Fruit Ripening Is Coordinated by the Reproductive Part of the Syconium

    doi: 10.3389/fpls.2016.01696

    Figure Lengend Snippet: Fig fruit developmental stages and tissues used to extract RNA and construct cDNA libraries for the Illumina high-throughput sequencing. (A) Developmental stages of parthenocarpic (top rows of each set) and pollinated (lower rows of each set) fruit displaying pulp and inflorescence; 8WAP – 8 weeks after pollination; 9WAP – 9 weeks after pollination; 10% – 10% fruit ripening; 60% – 60% fruit ripening; 100% – 100% fruit ripening. (B) Parthenocarpic sampled tissues that were used in this study. ( C ) Pollinated sampled tissues that were used in this study.

    Article Snippet: RNA fragmentation, double-stranded cDNA synthesis and adaptor ligation were performed using the TruseqTM RNA Sample Prep Kit-v2 (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions.

    Techniques: Construct, Next-Generation Sequencing