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    Thermo Fisher superscript double stranded complementary dna
    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 Complementary Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 11290 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher superscript iii complimentary dna 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 Iii Complimentary Dna Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    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

    Deletion of pilA does not affect masB transcription. RNA was harvested from 1×10 8 cells and mRNA was used to generate cDNA using a random hexanucleotide primer. Gene specific primers for masB and the 16S gene (endogenous control) were used in the amplification reaction with cDNA. masB expression was amplified from cDNA using 50, 5 and 0.5 ng template, while 16 S expression was amplified using 200, 20 and 2 pg cDNA. Both the WT and pilA strain express masB (black band in lanes 1 and 4 respectively). Hence, deletion of pilA does not adversely affect the expression of masB in M. xanthus . Deletion of masABK abolished masB expression (lanes 7–9).

    Journal: PLoS ONE

    Article Title: MasABK Proteins Interact with Proteins of the Type IV Pilin System to Affect Social Motility of Myxococcus xanthus

    doi: 10.1371/journal.pone.0054557

    Figure Lengend Snippet: Deletion of pilA does not affect masB transcription. RNA was harvested from 1×10 8 cells and mRNA was used to generate cDNA using a random hexanucleotide primer. Gene specific primers for masB and the 16S gene (endogenous control) were used in the amplification reaction with cDNA. masB expression was amplified from cDNA using 50, 5 and 0.5 ng template, while 16 S expression was amplified using 200, 20 and 2 pg cDNA. Both the WT and pilA strain express masB (black band in lanes 1 and 4 respectively). Hence, deletion of pilA does not adversely affect the expression of masB in M. xanthus . Deletion of masABK abolished masB expression (lanes 7–9).

    Article Snippet: Double-stranded cDNAs were synthesized using Invitrogen's SuperScript cDNA Synthesis Kit with the standard protocol using random hexanucleotide primers (Invitrogen, Carlsbad, CA).

    Techniques: Amplification, Expressing

    mRNA expression analysis in Nf1 mutant mouse Schwann cells. (A) Microarray analysis was used to compare genome-wide expression levels between normal mouse Schwann cells and Nf1 mutant Schwann cells. The control for each comparison was Cy3-labeled cDNA generated from normal mouse Schwann cell mRNA. For each of four Nf1 mutant Schwann cell samples ( Nf1 +/− , Nf1 −/− , Nf1 −/− TXF, and Nf1 −/− TXF treated with FTI), mRNA was used as a template to synthesize Cy5-labeled cDNA. Cy3- and Cy5-labeled cDNA probes were hybridized simultaneously to the Incyte Genomics MouseGEM 1.0 cDNA microarray. Relative intensities of Cy3 versus Cy5 fluorescent signals for each cDNA target sequence were analyzed with GeneSpring software. The most changes were observed in the Nf1 −/− TXF cells (genes upregulated in Nf1 −/− TXF are red; genes downregulated in Nf1 −/− TXF are green). Expression of one target cDNA, BLBP (black line), was 26-fold above normal in the Nf1 −/− TXF cells and not normalized by FTI treatment. (B) RT-PCR analysis confirmedthe microarray result of elevated BLBP expression in Nf1 −/− TXF cells. Reverse transcriptase (RT) was omitted from duplicate samples to control for DNA contamination. Primers for BLBP (∼200-bp amplicon) and actin control primers (∼500-bp amplicon) were included in the mixture for each 40-cycle reaction. The plasmid positive control for BLBP amplification is the UniGEM clone (Incyte Genomics) containing the BLBP cDNA insert spotted on the microarray. (C) Quantitative real-time PCR of BLBP normalized to GAPDH resulted in a 145-fold change over expression in Nf1 −/− TXF cells compared to wild-type mouse Schwann cells. Rn, fluorescent signal intensity; horizontal starred line, chosen threshold at geometric phase of amplification.

    Journal: Molecular and Cellular Biology

    Article Title: Brain Lipid Binding Protein in Axon-Schwann Cell Interactions and Peripheral Nerve Tumorigenesis

    doi: 10.1128/MCB.23.6.2213-2224.2003

    Figure Lengend Snippet: mRNA expression analysis in Nf1 mutant mouse Schwann cells. (A) Microarray analysis was used to compare genome-wide expression levels between normal mouse Schwann cells and Nf1 mutant Schwann cells. The control for each comparison was Cy3-labeled cDNA generated from normal mouse Schwann cell mRNA. For each of four Nf1 mutant Schwann cell samples ( Nf1 +/− , Nf1 −/− , Nf1 −/− TXF, and Nf1 −/− TXF treated with FTI), mRNA was used as a template to synthesize Cy5-labeled cDNA. Cy3- and Cy5-labeled cDNA probes were hybridized simultaneously to the Incyte Genomics MouseGEM 1.0 cDNA microarray. Relative intensities of Cy3 versus Cy5 fluorescent signals for each cDNA target sequence were analyzed with GeneSpring software. The most changes were observed in the Nf1 −/− TXF cells (genes upregulated in Nf1 −/− TXF are red; genes downregulated in Nf1 −/− TXF are green). Expression of one target cDNA, BLBP (black line), was 26-fold above normal in the Nf1 −/− TXF cells and not normalized by FTI treatment. (B) RT-PCR analysis confirmedthe microarray result of elevated BLBP expression in Nf1 −/− TXF cells. Reverse transcriptase (RT) was omitted from duplicate samples to control for DNA contamination. Primers for BLBP (∼200-bp amplicon) and actin control primers (∼500-bp amplicon) were included in the mixture for each 40-cycle reaction. The plasmid positive control for BLBP amplification is the UniGEM clone (Incyte Genomics) containing the BLBP cDNA insert spotted on the microarray. (C) Quantitative real-time PCR of BLBP normalized to GAPDH resulted in a 145-fold change over expression in Nf1 −/− TXF cells compared to wild-type mouse Schwann cells. Rn, fluorescent signal intensity; horizontal starred line, chosen threshold at geometric phase of amplification.

    Article Snippet: mRNA isolated from mouse Schwann cells (MicroFastTrack 2.0 kit) was used as a template to create double-stranded cDNA (Superscript preamplification system; Gibco-BRL).

    Techniques: Expressing, Mutagenesis, Microarray, Genome Wide, Labeling, Generated, Sequencing, Software, Reverse Transcription Polymerase Chain Reaction, Amplification, Plasmid Preparation, Positive Control, Real-time Polymerase Chain Reaction, Over Expression

    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

    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

    Schematic representation of the IVV selection procedure. (1) A cDNA library is transcribed and ligated with a Fluoro-PEG Puro spacer. (2) The IVV template RNA library is translated in a wheat germ cell-free translation system. (3) The constructed IVV library is purified with anti-FLAG M2 antibody-immobilized beads to remove untranslated mRNA and impurities contained in the wheat germ mixture. (4) The purified IVV are (5) subjected to affinity selection with mutated bait DNA-immobilized beads to eliminate non-specific binders to DNA. (6) Unbound IVV are then subjected to affinity selection with bait DNA-immobilized beads. (7) After washing, DNA-binding IVV are eluted by DNase I digestion. (8) The mRNA portions of the selected IVV are reverse-transcribed, PCR-amplified and (9) subjected to the next round of selection or (10) identified by cloning and sequencing.

    Journal: Nucleic Acids Research

    Article Title: Affinity selection of DNA-binding protein complexes using mRNA display

    doi: 10.1093/nar/gnj025

    Figure Lengend Snippet: Schematic representation of the IVV selection procedure. (1) A cDNA library is transcribed and ligated with a Fluoro-PEG Puro spacer. (2) The IVV template RNA library is translated in a wheat germ cell-free translation system. (3) The constructed IVV library is purified with anti-FLAG M2 antibody-immobilized beads to remove untranslated mRNA and impurities contained in the wheat germ mixture. (4) The purified IVV are (5) subjected to affinity selection with mutated bait DNA-immobilized beads to eliminate non-specific binders to DNA. (6) Unbound IVV are then subjected to affinity selection with bait DNA-immobilized beads. (7) After washing, DNA-binding IVV are eluted by DNase I digestion. (8) The mRNA portions of the selected IVV are reverse-transcribed, PCR-amplified and (9) subjected to the next round of selection or (10) identified by cloning and sequencing.

    Article Snippet: Briefly, 1 µg of mouse brain polyA+ mRNA (BD Biosciences Clontech) was reverse-transcribed using a SuperScript double-strand cDNA synthesis kit (Invitrogen) and 2 pmol of a 3′ random primer (Supplementary Table 1) according to the manufacturer's instructions.

    Techniques: Selection, cDNA Library Assay, Construct, Purification, Binding Assay, Polymerase Chain Reaction, Amplification, Clone Assay, Sequencing

    RNA isolation, cDNA synthesis, Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Real-Time PCR

    Journal: PLoS ONE

    Article Title: In silico analyses and global transcriptional profiling reveal novel putative targets for Pea3 transcription factor related to its function in neurons

    doi: 10.1371/journal.pone.0170585

    Figure Lengend Snippet: RNA isolation, cDNA synthesis, Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Real-Time PCR

    Article Snippet: Thereafter, RNA was converted to cDNA using the Superscript Double-stranded cDNA Synthesis (Invitrogen) Kit and labeled with NimbleGen One Color DNA Labeling (NimbleGen, Roche).

    Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    E. coli gene expression in voided urine from cystitis patients and culture in urine ex vivo . (A) Correlation of gene expression levels obtained by microarray and qPCR. Ct values determined by qPCR are plotted for 13 genes (see text) versus normalized microarray signal intensity for in vivo (left two panels) and in vitro (right two panels) cDNA samples from patient isolates 371 (first and third panel) and 151 (second and last panel). Correlation coefficient (r) values are shown and P

    Journal: PLoS Pathogens

    Article Title: Escherichia coli Global Gene Expression in Urine from Women with Urinary Tract Infection

    doi: 10.1371/journal.ppat.1001187

    Figure Lengend Snippet: E. coli gene expression in voided urine from cystitis patients and culture in urine ex vivo . (A) Correlation of gene expression levels obtained by microarray and qPCR. Ct values determined by qPCR are plotted for 13 genes (see text) versus normalized microarray signal intensity for in vivo (left two panels) and in vitro (right two panels) cDNA samples from patient isolates 371 (first and third panel) and 151 (second and last panel). Correlation coefficient (r) values are shown and P

    Article Snippet: cDNA synthesis, labeling and microarray hybridization cDNA was synthesized from total RNA isolated using the Superscript Double-Stranded cDNA Synthesis system (Invitrogen) according to the manufacturer's instructions.

    Techniques: Expressing, Ex Vivo, Microarray, Real-time Polymerase Chain Reaction, In Vivo, In Vitro