first strand complementary dna  (Thermo Fisher)


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    Thermo Fisher first strand complementary dna
    Validation of AS events in different tissues by RT-PCR. Twenty-two multi-exon genes that underwent AS were validated by RT-PCR. RT-PCR analysis was performed on multiple tissues including those used for <t>RNA-seq.</t> eIF4α6 was used as control. Sl, S.lycopersicum cv Heinz1706; Sp, S.pimpinellifolium LA1589. noRT, negative control using reaction mixture without reverse transcriptase added as templates. Genomic, genomic <t>DNA</t> of LA1589 as PCR templates. M, DNA marker 2 K Plus II (Transgen, Beijing). IR, intron retention; AA, alternative acceptor; AD, alternative donor; ES, exon skipping; Others, AS events with more than one of the four basic types. Function description and primer information for the 22 genes can be found in Additional file 8 : Table S5
    First Strand Complementary Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 217 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/first strand complementary dna/product/Thermo Fisher
    Average 94 stars, based on 217 article reviews
    Price from $9.99 to $1999.99
    first strand complementary dna - by Bioz Stars, 2020-08
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    Images

    1) Product Images from "Identification of alternative splicing events by RNA sequencing in early growth tomato fruits"

    Article Title: Identification of alternative splicing events by RNA sequencing in early growth tomato fruits

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-2128-6

    Validation of AS events in different tissues by RT-PCR. Twenty-two multi-exon genes that underwent AS were validated by RT-PCR. RT-PCR analysis was performed on multiple tissues including those used for RNA-seq. eIF4α6 was used as control. Sl, S.lycopersicum cv Heinz1706; Sp, S.pimpinellifolium LA1589. noRT, negative control using reaction mixture without reverse transcriptase added as templates. Genomic, genomic DNA of LA1589 as PCR templates. M, DNA marker 2 K Plus II (Transgen, Beijing). IR, intron retention; AA, alternative acceptor; AD, alternative donor; ES, exon skipping; Others, AS events with more than one of the four basic types. Function description and primer information for the 22 genes can be found in Additional file 8 : Table S5
    Figure Legend Snippet: Validation of AS events in different tissues by RT-PCR. Twenty-two multi-exon genes that underwent AS were validated by RT-PCR. RT-PCR analysis was performed on multiple tissues including those used for RNA-seq. eIF4α6 was used as control. Sl, S.lycopersicum cv Heinz1706; Sp, S.pimpinellifolium LA1589. noRT, negative control using reaction mixture without reverse transcriptase added as templates. Genomic, genomic DNA of LA1589 as PCR templates. M, DNA marker 2 K Plus II (Transgen, Beijing). IR, intron retention; AA, alternative acceptor; AD, alternative donor; ES, exon skipping; Others, AS events with more than one of the four basic types. Function description and primer information for the 22 genes can be found in Additional file 8 : Table S5

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, RNA Sequencing Assay, Negative Control, Polymerase Chain Reaction, Marker

    2) Product Images from "Microbiota maintain colonic homeostasis by activating TLR2/MyD88/PI3K signaling in IL-10–producing regulatory B cells"

    Article Title: Microbiota maintain colonic homeostasis by activating TLR2/MyD88/PI3K signaling in IL-10–producing regulatory B cells

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI93820

    Bacterial products predominantly expand GFP + B cells and innate immune cells ex vivo. Unfractionated colonic LP cells from GF Il10 +/EGFP reporter mice were cultured without (–, media only) or with 200 ng/mL LPS, 50 ng/mL Pam3csk (Pam), 1 nM CpG-DNA (CpG), 10 μg/mL lysates of E . coli LF82 (Ec), E . faecalis (Ef), or R . gnavus (Rg) or a mixture of 17 strains of Clostridia species (Clo). ( A ) Frequencies of GFP-expressing cell types were determined by flow cytometry using antibodies to cell surface markers as described in Methods. Data are presented as median of 4 separate cell cultures, with cells in each culture pooled from 2–4 mice; * P
    Figure Legend Snippet: Bacterial products predominantly expand GFP + B cells and innate immune cells ex vivo. Unfractionated colonic LP cells from GF Il10 +/EGFP reporter mice were cultured without (–, media only) or with 200 ng/mL LPS, 50 ng/mL Pam3csk (Pam), 1 nM CpG-DNA (CpG), 10 μg/mL lysates of E . coli LF82 (Ec), E . faecalis (Ef), or R . gnavus (Rg) or a mixture of 17 strains of Clostridia species (Clo). ( A ) Frequencies of GFP-expressing cell types were determined by flow cytometry using antibodies to cell surface markers as described in Methods. Data are presented as median of 4 separate cell cultures, with cells in each culture pooled from 2–4 mice; * P

    Techniques Used: Ex Vivo, Mouse Assay, Cell Culture, Expressing, Flow Cytometry, Cytometry

    3) Product Images from "An important role for peroxiredoxin II in survival of A549 lung cancer cells resistant to gefitinib"

    Article Title: An important role for peroxiredoxin II in survival of A549 lung cancer cells resistant to gefitinib

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/emm.2015.24

    Expression of antioxidant enzymes and DNA methylation state at peroxiredoxin II (Prx II) promoter in NSCLC cells. ( A ) Western blot analysis of Prxs in A549 gefitinib- and EGFR-TKI-resistant cells. ( B ) Quantitative reverse transcriptase-PCR analysis of Prxs and other antioxidant enzymes in A549/GR cells. ( C ) Sequence information of the Prx II proximal promoter region used for DNA methylation analysis. The HpaII/MspI recognition sequence (5′-CCGG-3′) is italicized and boxed (a, b-1, b-2, c and d). The forward primers (F1–F6) are underlined, and the reverse primers (R1–R6) are italicized. Transcription start site, +1. ( D ) DNA methylation analysis using restriction enzyme digestion. Genomic DNA was extracted from the indicated cell lines and digested with either HpaII (HII) or MspI (MI). Both enzymes recognize 5′-CCGG-3′, but HpaII is unable to cut DNA when the internal cytosine is methylated. The combinations of primers used in amplifying each of the HpaII/MspI sites (a–d) are indicated. The data are the mean±s.e.m. ( n =16) * P
    Figure Legend Snippet: Expression of antioxidant enzymes and DNA methylation state at peroxiredoxin II (Prx II) promoter in NSCLC cells. ( A ) Western blot analysis of Prxs in A549 gefitinib- and EGFR-TKI-resistant cells. ( B ) Quantitative reverse transcriptase-PCR analysis of Prxs and other antioxidant enzymes in A549/GR cells. ( C ) Sequence information of the Prx II proximal promoter region used for DNA methylation analysis. The HpaII/MspI recognition sequence (5′-CCGG-3′) is italicized and boxed (a, b-1, b-2, c and d). The forward primers (F1–F6) are underlined, and the reverse primers (R1–R6) are italicized. Transcription start site, +1. ( D ) DNA methylation analysis using restriction enzyme digestion. Genomic DNA was extracted from the indicated cell lines and digested with either HpaII (HII) or MspI (MI). Both enzymes recognize 5′-CCGG-3′, but HpaII is unable to cut DNA when the internal cytosine is methylated. The combinations of primers used in amplifying each of the HpaII/MspI sites (a–d) are indicated. The data are the mean±s.e.m. ( n =16) * P

    Techniques Used: Expressing, DNA Methylation Assay, Western Blot, Polymerase Chain Reaction, Sequencing, Methylation

    4) Product Images from "Targeting RNA-Mediated Toxicity in C9orf72 ALS and/or FTD by RNAi-Based Gene Therapy"

    Article Title: Targeting RNA-Mediated Toxicity in C9orf72 ALS and/or FTD by RNAi-Based Gene Therapy

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2019.02.001

    Reduction of C9orf72 in C9BAC Mice (A) Vector copy distribution of AAV5 upon intrastriatal injection. The 3-month-old Tg( C9orf72 _3) line 112 mice were injected with AAV5-GFP (5e10 GC), AAV5-miC32 (5e10 GC), and AAV5-miC46 (1e10 GC) bilaterally in the striatum. All mice were sacrificed 6 weeks after surgeries, and frontal cortex, striatum, midbrain, cerebellum, and spinal cord were collected. DNA was isolated from the tissues, and qPCR was performed with primers amplifying a 95-bp fragment from the CAG promoter region. The genome copies per tissue were calculated using a standard curve. (B) Expressions of mature miC32 and miC46 guide strands in the cortex and striatum of Tg( C9orf72 _3) line 112 mice. Performed as described in (A), total RNA was isolated from the cortex and striatum for small RNA TaqMan. MicroRNA input levels were normalized to U6 small nuclear RNA and set relative to AAV-GFP-treated mice. (C and D) Lowering of total (C) and intronic C9orf72 (D) by miC in Tg( C9orf72 _3) line 112 mice. Performed as described in (A), total RNA was isolated from the cortex and striatum, and qRT-PCR was performed using primers for total C9ORF72 mRNA and sense intronic transcripts. RNA input levels were normalized to GAPDH and set relative to AAV-GFP mice. (E) Processing of miC32 and miC46 in mice. Small RNA NGS was performed on RNA isolated from the striatum to determine the length and ratio of guide and passenger strands. (F) detection of RNA foci in the cortex of Tg( C9orf72 _3) line 112 mice. Mouse brain was frozen and sectioned in OCT. RNA FISH was performed using a TYE563-(CCCCGG) 3 LNA probe to detect the sense foci. Sense foci (shown as white spots) were detected in Tg( C9orf72 _3) line 112 mice (C9+), but not in control littermates (C9−). (G and H) Reduction of RNA foci in frontal cortex. Cells with 0, 1–5, or > 5 foci were counted in control (AAV5-GFP) and treated groups (AAV5-miC32 and AAV5-miC46). Red arrows show the cells that contain RNA foci (G). The percentages of cells containing 0, 1–5, or > 5 foci were calculated from 6 different images per treatment group (H) (n = 3). Data were evaluated using a one-way ANOVA with Dunnett’s multiple comparison test (*p
    Figure Legend Snippet: Reduction of C9orf72 in C9BAC Mice (A) Vector copy distribution of AAV5 upon intrastriatal injection. The 3-month-old Tg( C9orf72 _3) line 112 mice were injected with AAV5-GFP (5e10 GC), AAV5-miC32 (5e10 GC), and AAV5-miC46 (1e10 GC) bilaterally in the striatum. All mice were sacrificed 6 weeks after surgeries, and frontal cortex, striatum, midbrain, cerebellum, and spinal cord were collected. DNA was isolated from the tissues, and qPCR was performed with primers amplifying a 95-bp fragment from the CAG promoter region. The genome copies per tissue were calculated using a standard curve. (B) Expressions of mature miC32 and miC46 guide strands in the cortex and striatum of Tg( C9orf72 _3) line 112 mice. Performed as described in (A), total RNA was isolated from the cortex and striatum for small RNA TaqMan. MicroRNA input levels were normalized to U6 small nuclear RNA and set relative to AAV-GFP-treated mice. (C and D) Lowering of total (C) and intronic C9orf72 (D) by miC in Tg( C9orf72 _3) line 112 mice. Performed as described in (A), total RNA was isolated from the cortex and striatum, and qRT-PCR was performed using primers for total C9ORF72 mRNA and sense intronic transcripts. RNA input levels were normalized to GAPDH and set relative to AAV-GFP mice. (E) Processing of miC32 and miC46 in mice. Small RNA NGS was performed on RNA isolated from the striatum to determine the length and ratio of guide and passenger strands. (F) detection of RNA foci in the cortex of Tg( C9orf72 _3) line 112 mice. Mouse brain was frozen and sectioned in OCT. RNA FISH was performed using a TYE563-(CCCCGG) 3 LNA probe to detect the sense foci. Sense foci (shown as white spots) were detected in Tg( C9orf72 _3) line 112 mice (C9+), but not in control littermates (C9−). (G and H) Reduction of RNA foci in frontal cortex. Cells with 0, 1–5, or > 5 foci were counted in control (AAV5-GFP) and treated groups (AAV5-miC32 and AAV5-miC46). Red arrows show the cells that contain RNA foci (G). The percentages of cells containing 0, 1–5, or > 5 foci were calculated from 6 different images per treatment group (H) (n = 3). Data were evaluated using a one-way ANOVA with Dunnett’s multiple comparison test (*p

    Techniques Used: Mouse Assay, Plasmid Preparation, Injection, Isolation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Next-Generation Sequencing, Fluorescence In Situ Hybridization

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Release and extracellular metabolism of ATP by ecto-nucleotidase eNTPDase 1-2 in hypothalamic and pituitary cells
    Article Snippet: .. The amplification was conducted in a Robocycler Thermal Cycler (Stratagene, La Jolla, CA) in a 50-ml reaction volume containing 1 µl of the first-strand cDNA as template, 1 unit of Taq DNA polymerase (Invitrogen), 0.5 µM concentration of each primer, 0.2 mM dNTP, and 1 × PCR buffer (2 mM MgCl2 , 50 mM KCl, 20 mM TrisYHCl, pH 8.4). .. Amplification of DNA templates was initiated by a denaturation step at 94 °C for 180 s, followed by 35 cycles of denaturing at 94 °C for 45 s, annealing at 52–60 °C for 30 s, and extension at 72 °C for 60 s. The reaction was then terminated by a final extension step at 72 °C for 10 min. After PCR, a 10-ml aliquot of PCR products was size-fractionated by electrophoresis in a 1.2% agarose gel and visualized by ethidium bromide staining.

    Article Title: CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport: relevance to pathophysiology of Dent's disease
    Article Snippet: .. PCR was performed on first-strand cDNA generated from total RNA of HEK293 cells, OK cells, human kidney (Ambion Europe), mouse kidney and mouse proximal renal tubular cells (mPTCs) using avian myeloblastosis virus RT and primers specific for cubilin, megalin, KIF3B, calmodulin, caveolin-1, caveolin-2, and GAPDH (primer sequences available on request), using methods previously described ( ). ..

    Article Title: Three novel ABCC5 splice variants in human retina and their role as regulators of ABCC5 gene expression
    Article Snippet: .. First-strand cDNA was generated from RNA samples by reverse transcription using Superscript II (Invitrogen) and served as a template for subsequent PCR assays. .. Real-time quantitative RT-PCR (qRT-PCR) was performed as described previously (Krämer et al., 2004).

    Concentration Assay:

    Article Title: Release and extracellular metabolism of ATP by ecto-nucleotidase eNTPDase 1-2 in hypothalamic and pituitary cells
    Article Snippet: .. The amplification was conducted in a Robocycler Thermal Cycler (Stratagene, La Jolla, CA) in a 50-ml reaction volume containing 1 µl of the first-strand cDNA as template, 1 unit of Taq DNA polymerase (Invitrogen), 0.5 µM concentration of each primer, 0.2 mM dNTP, and 1 × PCR buffer (2 mM MgCl2 , 50 mM KCl, 20 mM TrisYHCl, pH 8.4). .. Amplification of DNA templates was initiated by a denaturation step at 94 °C for 180 s, followed by 35 cycles of denaturing at 94 °C for 45 s, annealing at 52–60 °C for 30 s, and extension at 72 °C for 60 s. The reaction was then terminated by a final extension step at 72 °C for 10 min. After PCR, a 10-ml aliquot of PCR products was size-fractionated by electrophoresis in a 1.2% agarose gel and visualized by ethidium bromide staining.

    Generated:

    Article Title: CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport: relevance to pathophysiology of Dent's disease
    Article Snippet: .. PCR was performed on first-strand cDNA generated from total RNA of HEK293 cells, OK cells, human kidney (Ambion Europe), mouse kidney and mouse proximal renal tubular cells (mPTCs) using avian myeloblastosis virus RT and primers specific for cubilin, megalin, KIF3B, calmodulin, caveolin-1, caveolin-2, and GAPDH (primer sequences available on request), using methods previously described ( ). ..

    Article Title: Three novel ABCC5 splice variants in human retina and their role as regulators of ABCC5 gene expression
    Article Snippet: .. First-strand cDNA was generated from RNA samples by reverse transcription using Superscript II (Invitrogen) and served as a template for subsequent PCR assays. .. Real-time quantitative RT-PCR (qRT-PCR) was performed as described previously (Krämer et al., 2004).

    Amplification:

    Article Title: Release and extracellular metabolism of ATP by ecto-nucleotidase eNTPDase 1-2 in hypothalamic and pituitary cells
    Article Snippet: .. The amplification was conducted in a Robocycler Thermal Cycler (Stratagene, La Jolla, CA) in a 50-ml reaction volume containing 1 µl of the first-strand cDNA as template, 1 unit of Taq DNA polymerase (Invitrogen), 0.5 µM concentration of each primer, 0.2 mM dNTP, and 1 × PCR buffer (2 mM MgCl2 , 50 mM KCl, 20 mM TrisYHCl, pH 8.4). .. Amplification of DNA templates was initiated by a denaturation step at 94 °C for 180 s, followed by 35 cycles of denaturing at 94 °C for 45 s, annealing at 52–60 °C for 30 s, and extension at 72 °C for 60 s. The reaction was then terminated by a final extension step at 72 °C for 10 min. After PCR, a 10-ml aliquot of PCR products was size-fractionated by electrophoresis in a 1.2% agarose gel and visualized by ethidium bromide staining.

    Synthesized:

    Article Title: Calcium/calmodulin‐dependent kinase 2 mediates Epac‐induced spontaneous transient outward currents in rat vascular smooth muscle
    Article Snippet: .. Extracted RNA was treated with DNase I by incubating 8 μl of total RNA, 1 μl of 10 × DNase I buffer and 1 μl of DNase I (1 U μl−1 ; Invitrogen, Carlsbad, CA, USA) at room temperature for 15 min. Then, 1 μl of EDTA (25 mmol l−1 ) was added and the reaction heated at 65°C for 10 min. First‐strand cDNA was synthesized using SuperScript® III reverse transcriptase (Invitrogen) in accordance with the manufacturer's instructions. .. The purity and concentration of the resulting cDNA template was determined by measuring absorbance at 260 and 280 nm using a NanoDrop spectrophotometer (Thermo Fisher Scientific Inc.).

    Article Title: Generation of primitive neural stem cells from human fibroblasts using a defined set of factors
    Article Snippet: .. First-strand cDNA was synthesized from 2 μg RNA using SuperScript III reverse transcriptase and an oligo-dT primer (Life Technologies) according to the manufacturer protocols. .. PCR reactions were performed using TaKaRa Ex Taq Hot Start (Takara Bio Inc., Shiga, Japan).

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    Thermo Fisher first strand complementary dna
    Validation of AS events in different tissues by RT-PCR. Twenty-two multi-exon genes that underwent AS were validated by RT-PCR. RT-PCR analysis was performed on multiple tissues including those used for <t>RNA-seq.</t> eIF4α6 was used as control. Sl, S.lycopersicum cv Heinz1706; Sp, S.pimpinellifolium LA1589. noRT, negative control using reaction mixture without reverse transcriptase added as templates. Genomic, genomic <t>DNA</t> of LA1589 as PCR templates. M, DNA marker 2 K Plus II (Transgen, Beijing). IR, intron retention; AA, alternative acceptor; AD, alternative donor; ES, exon skipping; Others, AS events with more than one of the four basic types. Function description and primer information for the 22 genes can be found in Additional file 8 : Table S5
    First Strand Complementary Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 8840 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/first strand complementary dna/product/Thermo Fisher
    Average 94 stars, based on 8840 article reviews
    Price from $9.99 to $1999.99
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    94/100 stars
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    Thermo Fisher cdna synthesis the revertaid first strand cdna synthesis kit thermo fisher
    Effects of siRNAs specific to CXXC5 on cellular growth. <t>MCF7</t> cells grown in 10% CD-FBS containing medium for 48 h were transiently transfected without (UT) or with 10 nM CtS or siRNA#10 in the absence (0.01% ethanol) or the presence of 10 −8 M E2 up to 72 h. ( a ) Cells were subjected to cell counting using a hemocytometer. Results, as the mean ± S.E. of three independent determinations, depict fold change in cellular growth compared with those observed with cells at time 0 in the absence of E2, which is set to 1. In ( a–f ) the asterisk with a superscript “a” indicates significant difference from the corresponding ethanol-treated group (-E2); whereas superscript “b” depicts a significant difference from CtS transfected cells in the presence of E2. ( b ) The <t>cDNA</t> library generated from total RNA (50 ng) isolated from transfected cells for 48 h was subjected to qPCR using a primer set specific to CXXC5. ( c ) Nuclear extracts from untransfected (UT) or transfected cells were subjected to WB using an antibody for CXXC5, ERα or HDAC1. NS denotes non-specific protein; Molecular mass in KDa is indicated. Transfected cells were subjected to cell counting ( d ) and MTT assay ( e ). In ( b , d , e) , results depicting the mean ± S.E. of three independent determinations indicate fold change in mRNA levels ( b ) or cell numbers ( d , e ) compared with CtS in the absence of E2, which is set to 1. Transfected MCF7 cells were subjected to flow cytometry ( f ) with results depicting the percent of cells in the G1, G2 and S phases, or to Annexin V assay followed by flow cytometry ( g ). In ( f , g ), results are the mean ± SEM of three independent experiments. ( h i ) Untransfected (UT) or transfected cells were also subjected to the cell death inducer camptothecin (2 nM) for 24 h without (−E2) or with E2 (E2). Cells were then subjected to Annexin V assay ( h ) with results as the mean ± S.E. of three independent experiments depicting percent change in apoptotic (upper right quadrant) and death (upper left quadrant) cells, or to WB ( i ) using a PARP1-specific antibody. P and CP denote the uncut and cut PARP1, respectively.
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    Thermo Fisher maxima first strand cdna synthesis kit
    A Zinc-dependent metalloproteinase of B. abortus is an operon that forms a putative type II Toxin-Antitoxin. Identification of the transcriptional unit (operon) constituted by the ORF BAB1_0270 and a transcriptional regulator in B. abortus 2308 expressed in A) the genomic <t>DNA</t> and B) the <t>cDNA</t> from total RNA. MW: Molecular weight; lane 1: Transcriptional regulator (357 bp); lane 2: BAB1_0270 (549 bp); and lane 3: operon constituted by ORF BAB1_0270-transcriptional regulator (906 bp). C) Hypothetic Type II Toxin-Antitoxin (TA) model for operon constitute by Zn-dependent metalloproteinase and transcriptional regulator. Toxin (Zinc-dependent metalloproteinase) and anti-toxin (transcriptional regulator) are transcribed to mRNA together. Proteases are activated under stress condition, cleaving anti-toxin, which increases the levels of Toxin free, inducing diverse biological functions in bacteria. Predicted promoter at site −35 and −10 binding by RNA polymerase sigma factor rpoD. ATG A: nucleotides share between final part of transcriptional factor and metalloproteinase codified by the BAB1_0270 ORF.
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    Validation of AS events in different tissues by RT-PCR. Twenty-two multi-exon genes that underwent AS were validated by RT-PCR. RT-PCR analysis was performed on multiple tissues including those used for RNA-seq. eIF4α6 was used as control. Sl, S.lycopersicum cv Heinz1706; Sp, S.pimpinellifolium LA1589. noRT, negative control using reaction mixture without reverse transcriptase added as templates. Genomic, genomic DNA of LA1589 as PCR templates. M, DNA marker 2 K Plus II (Transgen, Beijing). IR, intron retention; AA, alternative acceptor; AD, alternative donor; ES, exon skipping; Others, AS events with more than one of the four basic types. Function description and primer information for the 22 genes can be found in Additional file 8 : Table S5

    Journal: BMC Genomics

    Article Title: Identification of alternative splicing events by RNA sequencing in early growth tomato fruits

    doi: 10.1186/s12864-015-2128-6

    Figure Lengend Snippet: Validation of AS events in different tissues by RT-PCR. Twenty-two multi-exon genes that underwent AS were validated by RT-PCR. RT-PCR analysis was performed on multiple tissues including those used for RNA-seq. eIF4α6 was used as control. Sl, S.lycopersicum cv Heinz1706; Sp, S.pimpinellifolium LA1589. noRT, negative control using reaction mixture without reverse transcriptase added as templates. Genomic, genomic DNA of LA1589 as PCR templates. M, DNA marker 2 K Plus II (Transgen, Beijing). IR, intron retention; AA, alternative acceptor; AD, alternative donor; ES, exon skipping; Others, AS events with more than one of the four basic types. Function description and primer information for the 22 genes can be found in Additional file 8 : Table S5

    Article Snippet: After genomic DNA in these RNA samples was removed by RNase-free DNase according to the manufacturer’s protocol (New England BioLabs, USA), the total RNA (1 μg per sample) was used to synthesize first strand complementary DNA using the First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA).

    Techniques: Reverse Transcription Polymerase Chain Reaction, RNA Sequencing Assay, Negative Control, Polymerase Chain Reaction, Marker

    CHME-3 cells were infected with S- PA and MDR- PA , with an MOI of 10:1. At indicated time points, the cells were collected and processed for RNA isolation and cDNA synthesis, followed by RT-qPCR. Human microglia were infected with clinical MDR- PA and S- PA strains. The MDR- PA strain exhibited persistently higher inflammatory mediators compared to S- PA . Student’s t -test was used for statistical analysis. The data was shown as the mean ± SE from three sets of independent experiments. *** p

    Journal: Microorganisms

    Article Title: Multidrug-Resistant Pseudomonas aeruginosa Evokes Differential Inflammatory Responses in Human Microglial and Retinal Pigment Epithelial Cells

    doi: 10.3390/microorganisms8050735

    Figure Lengend Snippet: CHME-3 cells were infected with S- PA and MDR- PA , with an MOI of 10:1. At indicated time points, the cells were collected and processed for RNA isolation and cDNA synthesis, followed by RT-qPCR. Human microglia were infected with clinical MDR- PA and S- PA strains. The MDR- PA strain exhibited persistently higher inflammatory mediators compared to S- PA . Student’s t -test was used for statistical analysis. The data was shown as the mean ± SE from three sets of independent experiments. *** p

    Article Snippet: First-strand cDNA was synthesized from equal RNA amounts (2 μg) using the Verso cDNA Synthesis Kit (Thermo scientific). cDNA was further amplified using primers for human Toll-like receptors 1–7 (TLR1–7; TLR8 and TLR10), interleukin (IL)-1α, IL-1β, tumor necrosis factor (TNF)-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, IL-8, IL-10, matrix metalloproteinase (MMP)-2 and -9, tissue inhibitor of metalloproteinases (TIMP-1), and β-actin (internal control), as described previously [ , ], using SYBR Green Master Mix (Thermo scientific) in a Step-One Plus real-time PCR system (Applied Biosystems, Foster City, CA, United States), with the following amplification conditions: initial denaturation of 10 min at 95 °C, followed by 40 cycles of 50° for 2 min, 95 °C for 10 min, 95 °C for 15 s, and 60 °C for 1 min. To confirm the specificity of the reaction, melting curve analysis was carried out.

    Techniques: Infection, Isolation, Quantitative RT-PCR

    CHME-3 cells were infected with S- PA and MDR- PA , with an MOI of 10:1. At the indicated time points, the cells were collected and processed for RNA isolation and cDNA synthesis, followed by RT-qPCR. RT-qPCR results shows the differential expression of Toll-like receptor (TLR)4 ( A ), TLR5 ( B ), and TLR9 ( C ) by human microglia infected with clinical strains of MDR- PA and S- PA at different time points. The data was shown as the mean ± SE from three sets of independent experiments. ** p

    Journal: Microorganisms

    Article Title: Multidrug-Resistant Pseudomonas aeruginosa Evokes Differential Inflammatory Responses in Human Microglial and Retinal Pigment Epithelial Cells

    doi: 10.3390/microorganisms8050735

    Figure Lengend Snippet: CHME-3 cells were infected with S- PA and MDR- PA , with an MOI of 10:1. At the indicated time points, the cells were collected and processed for RNA isolation and cDNA synthesis, followed by RT-qPCR. RT-qPCR results shows the differential expression of Toll-like receptor (TLR)4 ( A ), TLR5 ( B ), and TLR9 ( C ) by human microglia infected with clinical strains of MDR- PA and S- PA at different time points. The data was shown as the mean ± SE from three sets of independent experiments. ** p

    Article Snippet: First-strand cDNA was synthesized from equal RNA amounts (2 μg) using the Verso cDNA Synthesis Kit (Thermo scientific). cDNA was further amplified using primers for human Toll-like receptors 1–7 (TLR1–7; TLR8 and TLR10), interleukin (IL)-1α, IL-1β, tumor necrosis factor (TNF)-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, IL-8, IL-10, matrix metalloproteinase (MMP)-2 and -9, tissue inhibitor of metalloproteinases (TIMP-1), and β-actin (internal control), as described previously [ , ], using SYBR Green Master Mix (Thermo scientific) in a Step-One Plus real-time PCR system (Applied Biosystems, Foster City, CA, United States), with the following amplification conditions: initial denaturation of 10 min at 95 °C, followed by 40 cycles of 50° for 2 min, 95 °C for 10 min, 95 °C for 15 s, and 60 °C for 1 min. To confirm the specificity of the reaction, melting curve analysis was carried out.

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

    CHME-3 and ARPE-19 cells were challenged with S- PA and MDR- PA , with a MOI of 10:1. At indicated time points, the cells were collected and processed for RNA isolation; cDNA synthesis followed by RT-qPCR. The bar graphs show that there was no significant expression of MMP-2 ( A , D ) or tissue inhibitor of metalloproteinases (TIMP)-1 ( B , E ) in either CHME-3 or ARPE-19 cells. ( C , F ) Significantly elevated expression of MMP-9 was observed after infection of CHME-3 and ARPE-19 cells with MDR- PA and S- PA strains. The data are shown as the mean ± SE from three sets of independent experiments; ** p

    Journal: Microorganisms

    Article Title: Multidrug-Resistant Pseudomonas aeruginosa Evokes Differential Inflammatory Responses in Human Microglial and Retinal Pigment Epithelial Cells

    doi: 10.3390/microorganisms8050735

    Figure Lengend Snippet: CHME-3 and ARPE-19 cells were challenged with S- PA and MDR- PA , with a MOI of 10:1. At indicated time points, the cells were collected and processed for RNA isolation; cDNA synthesis followed by RT-qPCR. The bar graphs show that there was no significant expression of MMP-2 ( A , D ) or tissue inhibitor of metalloproteinases (TIMP)-1 ( B , E ) in either CHME-3 or ARPE-19 cells. ( C , F ) Significantly elevated expression of MMP-9 was observed after infection of CHME-3 and ARPE-19 cells with MDR- PA and S- PA strains. The data are shown as the mean ± SE from three sets of independent experiments; ** p

    Article Snippet: First-strand cDNA was synthesized from equal RNA amounts (2 μg) using the Verso cDNA Synthesis Kit (Thermo scientific). cDNA was further amplified using primers for human Toll-like receptors 1–7 (TLR1–7; TLR8 and TLR10), interleukin (IL)-1α, IL-1β, tumor necrosis factor (TNF)-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, IL-8, IL-10, matrix metalloproteinase (MMP)-2 and -9, tissue inhibitor of metalloproteinases (TIMP-1), and β-actin (internal control), as described previously [ , ], using SYBR Green Master Mix (Thermo scientific) in a Step-One Plus real-time PCR system (Applied Biosystems, Foster City, CA, United States), with the following amplification conditions: initial denaturation of 10 min at 95 °C, followed by 40 cycles of 50° for 2 min, 95 °C for 10 min, 95 °C for 15 s, and 60 °C for 1 min. To confirm the specificity of the reaction, melting curve analysis was carried out.

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

    Effects of siRNAs specific to CXXC5 on cellular growth. MCF7 cells grown in 10% CD-FBS containing medium for 48 h were transiently transfected without (UT) or with 10 nM CtS or siRNA#10 in the absence (0.01% ethanol) or the presence of 10 −8 M E2 up to 72 h. ( a ) Cells were subjected to cell counting using a hemocytometer. Results, as the mean ± S.E. of three independent determinations, depict fold change in cellular growth compared with those observed with cells at time 0 in the absence of E2, which is set to 1. In ( a–f ) the asterisk with a superscript “a” indicates significant difference from the corresponding ethanol-treated group (-E2); whereas superscript “b” depicts a significant difference from CtS transfected cells in the presence of E2. ( b ) The cDNA library generated from total RNA (50 ng) isolated from transfected cells for 48 h was subjected to qPCR using a primer set specific to CXXC5. ( c ) Nuclear extracts from untransfected (UT) or transfected cells were subjected to WB using an antibody for CXXC5, ERα or HDAC1. NS denotes non-specific protein; Molecular mass in KDa is indicated. Transfected cells were subjected to cell counting ( d ) and MTT assay ( e ). In ( b , d , e) , results depicting the mean ± S.E. of three independent determinations indicate fold change in mRNA levels ( b ) or cell numbers ( d , e ) compared with CtS in the absence of E2, which is set to 1. Transfected MCF7 cells were subjected to flow cytometry ( f ) with results depicting the percent of cells in the G1, G2 and S phases, or to Annexin V assay followed by flow cytometry ( g ). In ( f , g ), results are the mean ± SEM of three independent experiments. ( h i ) Untransfected (UT) or transfected cells were also subjected to the cell death inducer camptothecin (2 nM) for 24 h without (−E2) or with E2 (E2). Cells were then subjected to Annexin V assay ( h ) with results as the mean ± S.E. of three independent experiments depicting percent change in apoptotic (upper right quadrant) and death (upper left quadrant) cells, or to WB ( i ) using a PARP1-specific antibody. P and CP denote the uncut and cut PARP1, respectively.

    Journal: Scientific Reports

    Article Title: CXXC5 as an unmethylated CpG dinucleotide binding protein contributes to estrogen-mediated cellular proliferation

    doi: 10.1038/s41598-020-62912-0

    Figure Lengend Snippet: Effects of siRNAs specific to CXXC5 on cellular growth. MCF7 cells grown in 10% CD-FBS containing medium for 48 h were transiently transfected without (UT) or with 10 nM CtS or siRNA#10 in the absence (0.01% ethanol) or the presence of 10 −8 M E2 up to 72 h. ( a ) Cells were subjected to cell counting using a hemocytometer. Results, as the mean ± S.E. of three independent determinations, depict fold change in cellular growth compared with those observed with cells at time 0 in the absence of E2, which is set to 1. In ( a–f ) the asterisk with a superscript “a” indicates significant difference from the corresponding ethanol-treated group (-E2); whereas superscript “b” depicts a significant difference from CtS transfected cells in the presence of E2. ( b ) The cDNA library generated from total RNA (50 ng) isolated from transfected cells for 48 h was subjected to qPCR using a primer set specific to CXXC5. ( c ) Nuclear extracts from untransfected (UT) or transfected cells were subjected to WB using an antibody for CXXC5, ERα or HDAC1. NS denotes non-specific protein; Molecular mass in KDa is indicated. Transfected cells were subjected to cell counting ( d ) and MTT assay ( e ). In ( b , d , e) , results depicting the mean ± S.E. of three independent determinations indicate fold change in mRNA levels ( b ) or cell numbers ( d , e ) compared with CtS in the absence of E2, which is set to 1. Transfected MCF7 cells were subjected to flow cytometry ( f ) with results depicting the percent of cells in the G1, G2 and S phases, or to Annexin V assay followed by flow cytometry ( g ). In ( f , g ), results are the mean ± SEM of three independent experiments. ( h i ) Untransfected (UT) or transfected cells were also subjected to the cell death inducer camptothecin (2 nM) for 24 h without (−E2) or with E2 (E2). Cells were then subjected to Annexin V assay ( h ) with results as the mean ± S.E. of three independent experiments depicting percent change in apoptotic (upper right quadrant) and death (upper left quadrant) cells, or to WB ( i ) using a PARP1-specific antibody. P and CP denote the uncut and cut PARP1, respectively.

    Article Snippet: For RT-qPCR, total RNA isolated from MCF7 cells, treated as described for PanCancer Pathway Panel, was used for cDNA synthesis (The RevertAid First Strand cDNA Synthesis Kit, Thermo-Fisher).

    Techniques: Transfection, Cell Counting, cDNA Library Assay, Generated, Isolation, Real-time Polymerase Chain Reaction, Western Blot, MTT Assay, Flow Cytometry, Annexin V Assay

    Electrophoretic mobility shift (EMSA) and reporter assay. ( a ) To assess the ability of the recombinant FL-CXXC5 or its CXXC domain (CXXC-D), 50 μM DNA with the central unmethylated ( CG ) or methylated ( mCG ) CpG dinucleotides was mixed with FL-CXXC5 at 1:0.5, 1:1, 1:2, 1:4 and 1:8 molar ratios or with CCCX-D at 1:4 molar ratios. Samples were run onto 5% native TBE gel, stained and visualized with UV spectrometry. “M” represents the molecular marker. “Free” denotes unbound DNA; “Shifted” indicates DNA bound protein. A representative experiment performed with two independent times is shown. (b) To assess the intrinsic transcription regulatory function of FL-CXXC5, pGal4-RE Luciferase Reporter vector (pGal4RE-Luc, 125 ng) containing tandem Gal4 response elements (Gal4-RE) juxtaposed to a simple TATA box promoter that drives the expression of the firefly Luciferase enzyme cDNA together with an expression vector (75 ng) bearing Gal4 DBD , VP16, FL-CXXC5, WT-MeCP2 or ERα-EF domain cDNA or Gal4 DBD -VP16, Gal4 DBD -CXXC5, Gal4 DBD -MeCP2 or Gal4 DBD -ERαEF cDNA transfected into MCF7 cells. MCF7 cells grown in 10% CD-FBS for 48 h were transfected with expression vector bearing ERαEF cDNA or Gal4 DBD -ERαEF and were treated without (%0.01 ethanol) or with 10 −8 M E2 for 24 h. Transfection efficiency was monitored with the co-expression of a reporter plasmid bearing the Renilla Luciferase enzyme cDNA (0.5 ng). Results indicating relative firefly/ Renilla luciferase activity determined using a dual luciferase assay kit are the mean ± S.E. of three independent experiments performed in duplicate. The asterisk denotes significant differences compared to responses observed with Gal4-RE, which is set to 1. (c) Cells were also transfected with pGal4RE-Luc together with the expression vector bearing none (empty vector, EV), Gal4 DBD -VP16 and/or Gal4 DBD -CXXC5 cDNA. In transfections, we used a total of 300 ng expression vector (1 denotes 75 ng), for which appropriate amounts of the parent expression vector (EV) were supplemented to equalize the total plasmid DNA amount. Results presented as relative firefly/ Renilla luciferase levels indicate percent change and are the mean ± S.E. of three independent experiments performed in duplicate. The asterisk denotes significant differences from responses observed with Gal4 DBD -VP16, which was set to 100%.

    Journal: Scientific Reports

    Article Title: CXXC5 as an unmethylated CpG dinucleotide binding protein contributes to estrogen-mediated cellular proliferation

    doi: 10.1038/s41598-020-62912-0

    Figure Lengend Snippet: Electrophoretic mobility shift (EMSA) and reporter assay. ( a ) To assess the ability of the recombinant FL-CXXC5 or its CXXC domain (CXXC-D), 50 μM DNA with the central unmethylated ( CG ) or methylated ( mCG ) CpG dinucleotides was mixed with FL-CXXC5 at 1:0.5, 1:1, 1:2, 1:4 and 1:8 molar ratios or with CCCX-D at 1:4 molar ratios. Samples were run onto 5% native TBE gel, stained and visualized with UV spectrometry. “M” represents the molecular marker. “Free” denotes unbound DNA; “Shifted” indicates DNA bound protein. A representative experiment performed with two independent times is shown. (b) To assess the intrinsic transcription regulatory function of FL-CXXC5, pGal4-RE Luciferase Reporter vector (pGal4RE-Luc, 125 ng) containing tandem Gal4 response elements (Gal4-RE) juxtaposed to a simple TATA box promoter that drives the expression of the firefly Luciferase enzyme cDNA together with an expression vector (75 ng) bearing Gal4 DBD , VP16, FL-CXXC5, WT-MeCP2 or ERα-EF domain cDNA or Gal4 DBD -VP16, Gal4 DBD -CXXC5, Gal4 DBD -MeCP2 or Gal4 DBD -ERαEF cDNA transfected into MCF7 cells. MCF7 cells grown in 10% CD-FBS for 48 h were transfected with expression vector bearing ERαEF cDNA or Gal4 DBD -ERαEF and were treated without (%0.01 ethanol) or with 10 −8 M E2 for 24 h. Transfection efficiency was monitored with the co-expression of a reporter plasmid bearing the Renilla Luciferase enzyme cDNA (0.5 ng). Results indicating relative firefly/ Renilla luciferase activity determined using a dual luciferase assay kit are the mean ± S.E. of three independent experiments performed in duplicate. The asterisk denotes significant differences compared to responses observed with Gal4-RE, which is set to 1. (c) Cells were also transfected with pGal4RE-Luc together with the expression vector bearing none (empty vector, EV), Gal4 DBD -VP16 and/or Gal4 DBD -CXXC5 cDNA. In transfections, we used a total of 300 ng expression vector (1 denotes 75 ng), for which appropriate amounts of the parent expression vector (EV) were supplemented to equalize the total plasmid DNA amount. Results presented as relative firefly/ Renilla luciferase levels indicate percent change and are the mean ± S.E. of three independent experiments performed in duplicate. The asterisk denotes significant differences from responses observed with Gal4 DBD -VP16, which was set to 100%.

    Article Snippet: For RT-qPCR, total RNA isolated from MCF7 cells, treated as described for PanCancer Pathway Panel, was used for cDNA synthesis (The RevertAid First Strand cDNA Synthesis Kit, Thermo-Fisher).

    Techniques: Electrophoretic Mobility Shift Assay, Reporter Assay, Recombinant, Methylation, Staining, Marker, Luciferase, Plasmid Preparation, Expressing, Transfection, Activity Assay

    A Zinc-dependent metalloproteinase of B. abortus is an operon that forms a putative type II Toxin-Antitoxin. Identification of the transcriptional unit (operon) constituted by the ORF BAB1_0270 and a transcriptional regulator in B. abortus 2308 expressed in A) the genomic DNA and B) the cDNA from total RNA. MW: Molecular weight; lane 1: Transcriptional regulator (357 bp); lane 2: BAB1_0270 (549 bp); and lane 3: operon constituted by ORF BAB1_0270-transcriptional regulator (906 bp). C) Hypothetic Type II Toxin-Antitoxin (TA) model for operon constitute by Zn-dependent metalloproteinase and transcriptional regulator. Toxin (Zinc-dependent metalloproteinase) and anti-toxin (transcriptional regulator) are transcribed to mRNA together. Proteases are activated under stress condition, cleaving anti-toxin, which increases the levels of Toxin free, inducing diverse biological functions in bacteria. Predicted promoter at site −35 and −10 binding by RNA polymerase sigma factor rpoD. ATG A: nucleotides share between final part of transcriptional factor and metalloproteinase codified by the BAB1_0270 ORF.

    Journal: bioRxiv

    Article Title: A Zinc-dependent metalloproteinase in the intracellular adaptation of Brucella abortus in macrophages

    doi: 10.1101/2020.04.17.046490

    Figure Lengend Snippet: A Zinc-dependent metalloproteinase of B. abortus is an operon that forms a putative type II Toxin-Antitoxin. Identification of the transcriptional unit (operon) constituted by the ORF BAB1_0270 and a transcriptional regulator in B. abortus 2308 expressed in A) the genomic DNA and B) the cDNA from total RNA. MW: Molecular weight; lane 1: Transcriptional regulator (357 bp); lane 2: BAB1_0270 (549 bp); and lane 3: operon constituted by ORF BAB1_0270-transcriptional regulator (906 bp). C) Hypothetic Type II Toxin-Antitoxin (TA) model for operon constitute by Zn-dependent metalloproteinase and transcriptional regulator. Toxin (Zinc-dependent metalloproteinase) and anti-toxin (transcriptional regulator) are transcribed to mRNA together. Proteases are activated under stress condition, cleaving anti-toxin, which increases the levels of Toxin free, inducing diverse biological functions in bacteria. Predicted promoter at site −35 and −10 binding by RNA polymerase sigma factor rpoD. ATG A: nucleotides share between final part of transcriptional factor and metalloproteinase codified by the BAB1_0270 ORF.

    Article Snippet: Complementary DNA (cDNA) was obtained from RNA by reverse transcription using the Maxima First Strand cDNA Synthesis kit for RT-PCR (ThermoFisher Scientific Inc, MA, USA) and the relative expression of the genes of interest ( ) was quantified using the Takyon q-PCR kit for SYBR assays by means of the AriaMx Real Time PCR system (Agilent Technologies, CA, USA). gyrA and 16s housekeeping genes were used as reference genes for all assays.

    Techniques: Molecular Weight, Binding Assay