superscript system  (Thermo Fisher)


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  • 93
    Name:
    SuperScript Plasmid System
    Description:
    The SuperScript Plasmid System is designed for synthesis of double stranded cDNA from a purified mRNA population The cDNA is suitable for directional cloning into the pSPORT1 vector or the pCMV• SPORT6 plasmid vector for subsequent generation of cDNA libraries and use of Gateway Technology The SuperScript Plasmid System • Results in greater cDNA yields and increased full length cDNAs with SuperScript II RT• Offers a simplified primer adapter strategy for directional cloning• Offers one tube format for first and second strand reactions improving cDNA yields• Fractionates cDNA using efficient and convenient pre packed columns• Ligates inserts into a pre cut Not I Sal I vector prepared to minimize background
    Catalog Number:
    19625011
    Price:
    None
    Applications:
    Cloning|cDNA Libraries & Library Construction
    Category:
    Kits and Assays
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    Structured Review

    Thermo Fisher superscript system
    The SuperScript Plasmid System is designed for synthesis of double stranded cDNA from a purified mRNA population The cDNA is suitable for directional cloning into the pSPORT1 vector or the pCMV• SPORT6 plasmid vector for subsequent generation of cDNA libraries and use of Gateway Technology The SuperScript Plasmid System • Results in greater cDNA yields and increased full length cDNAs with SuperScript II RT• Offers a simplified primer adapter strategy for directional cloning• Offers one tube format for first and second strand reactions improving cDNA yields• Fractionates cDNA using efficient and convenient pre packed columns• Ligates inserts into a pre cut Not I Sal I vector prepared to minimize background
    https://www.bioz.com/result/superscript system/product/Thermo Fisher
    Average 93 stars, based on 34 article reviews
    Price from $9.99 to $1999.99
    superscript system - by Bioz Stars, 2020-08
    93/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Overexpression of C16orf74 is involved in aggressive pancreatic cancers
    Article Snippet: .. cDNA library screening We constructed a cDNA library using a SuperscriptTM plasmid system with GatewayTM technology for cDNA synthesis, a cloning kit (Invitrogen) and poly(A)+ RNA obtained from pancreatic cancer cell line Capan-1. .. We screened 3×106 independent clones of this library using the same cDNA probes used for Northern blot analysis.

    Article Title: CD8+ T Cells from SIV Elite Controller Macaques Recognize Mamu-B*08-Bound Epitopes and Select for Widespread Viral Variation
    Article Snippet: .. One microgram of mRNA from each animal served as the template for first strand cDNA synthesis, using the SuperScript plasmid system for cDNA synthesis and cloning (Invitrogen, Carlsbad, CA) by following the manufacturer's instructions. .. Size-fractionated cDNA containing SalI and NotI restriction endonuclease cohesive ends was ligated into the multiple cloning site of pCMV.SPORT6 and used to transform DH5α chemically competent E. coli (Invitrogen).

    Article Title: Venom gland transcriptomes of two elapid snakes (Bungarus multicinctus and Naja atra) and evolution of toxin genes
    Article Snippet: .. The purified mRNA was used to make cDNA library, following the instructions in the SuperScript Plasmid System for cDNA Synthesis and Cloning Kit (plasmid used: pCMV-SPORT6 for B. multicinctus , pSPORT1 for N. atra ) (Invitrogen, USA). .. Plasmids were purified using the QIAprep spin miniprep kit (Qiagen).

    Article Title: Selection of novel mediators of E2F1-induced apoptosis through retroviral expression of an antisense cDNA library
    Article Snippet: .. The cDNA library was constructed mainly based on SUPERSCRIPT™ Plasmid System with GATEWAY™ Technology for cDNA Synthesis and Cloning (Invitrogen). .. Instead of using pSPORT 1 plasmid provided with the kit, a self-inactivating oncoretroviral plasmid, pCAMS/U3EPac , was utilized to express the antisense mRNA transcripts.

    In Vitro:

    Article Title: Directional cDNA library construction assisted by the in vitro recombination reaction
    Article Snippet: .. cDNA synthesis from in vitro synthesized β-spectrin III (βSp3) mRNA was carried out according to the supplier’s instructions in the SuperScriptTM Plasmid System for cDNA synthesis (Life Technologies). ..

    Synthesized:

    Article Title: Directional cDNA library construction assisted by the in vitro recombination reaction
    Article Snippet: .. cDNA synthesis from in vitro synthesized β-spectrin III (βSp3) mRNA was carried out according to the supplier’s instructions in the SuperScriptTM Plasmid System for cDNA synthesis (Life Technologies). ..

    Isolation:

    Article Title: Na+- and Cl–-coupled active transport of nitric oxide synthase inhibitors via amino acid transport system B0,+
    Article Snippet: .. The SuperScript plasmid system (Life Technologies Inc., Rockville, Maryland, USA) was used to establish a unidirectional cDNA library with poly(A)+ RNA isolated from mouse colon as described previously ( – ). .. The probe for library screening was prepared by RT-PCR using primers specific for mouse ATB0,+ cDNA reported in the GenBank (accession no. ).

    Construct:

    Article Title: Overexpression of C16orf74 is involved in aggressive pancreatic cancers
    Article Snippet: .. cDNA library screening We constructed a cDNA library using a SuperscriptTM plasmid system with GatewayTM technology for cDNA synthesis, a cloning kit (Invitrogen) and poly(A)+ RNA obtained from pancreatic cancer cell line Capan-1. .. We screened 3×106 independent clones of this library using the same cDNA probes used for Northern blot analysis.

    Article Title: Selection of novel mediators of E2F1-induced apoptosis through retroviral expression of an antisense cDNA library
    Article Snippet: .. The cDNA library was constructed mainly based on SUPERSCRIPT™ Plasmid System with GATEWAY™ Technology for cDNA Synthesis and Cloning (Invitrogen). .. Instead of using pSPORT 1 plasmid provided with the kit, a self-inactivating oncoretroviral plasmid, pCAMS/U3EPac , was utilized to express the antisense mRNA transcripts.

    Purification:

    Article Title: Venom gland transcriptomes of two elapid snakes (Bungarus multicinctus and Naja atra) and evolution of toxin genes
    Article Snippet: .. The purified mRNA was used to make cDNA library, following the instructions in the SuperScript Plasmid System for cDNA Synthesis and Cloning Kit (plasmid used: pCMV-SPORT6 for B. multicinctus , pSPORT1 for N. atra ) (Invitrogen, USA). .. Plasmids were purified using the QIAprep spin miniprep kit (Qiagen).

    Produced:

    Article Title: A novel high-affinity inhibitor against the human ATP-sensitive Kir6.2 channel
    Article Snippet: .. The total mRNAs were extracted from the venom glands by using TRIzol (Ambion); their cDNAs were produced by using the SuperScript plasmid system (Invitrogen). .. The cDNA corresponding to the purified inhibitor protein was amplified by PCR, where the 5′ end is primed with degenerate oligonucleotides corresponding to the N-terminal amino acid sequence of the inhibitor protein, whereas the 3′ end is primed with a poly-A oligonucleotide.

    cDNA Library Assay:

    Article Title: Overexpression of C16orf74 is involved in aggressive pancreatic cancers
    Article Snippet: .. cDNA library screening We constructed a cDNA library using a SuperscriptTM plasmid system with GatewayTM technology for cDNA synthesis, a cloning kit (Invitrogen) and poly(A)+ RNA obtained from pancreatic cancer cell line Capan-1. .. We screened 3×106 independent clones of this library using the same cDNA probes used for Northern blot analysis.

    Article Title: Na+- and Cl–-coupled active transport of nitric oxide synthase inhibitors via amino acid transport system B0,+
    Article Snippet: .. The SuperScript plasmid system (Life Technologies Inc., Rockville, Maryland, USA) was used to establish a unidirectional cDNA library with poly(A)+ RNA isolated from mouse colon as described previously ( – ). .. The probe for library screening was prepared by RT-PCR using primers specific for mouse ATB0,+ cDNA reported in the GenBank (accession no. ).

    Article Title: Venom gland transcriptomes of two elapid snakes (Bungarus multicinctus and Naja atra) and evolution of toxin genes
    Article Snippet: .. The purified mRNA was used to make cDNA library, following the instructions in the SuperScript Plasmid System for cDNA Synthesis and Cloning Kit (plasmid used: pCMV-SPORT6 for B. multicinctus , pSPORT1 for N. atra ) (Invitrogen, USA). .. Plasmids were purified using the QIAprep spin miniprep kit (Qiagen).

    Article Title: Selection of novel mediators of E2F1-induced apoptosis through retroviral expression of an antisense cDNA library
    Article Snippet: .. The cDNA library was constructed mainly based on SUPERSCRIPT™ Plasmid System with GATEWAY™ Technology for cDNA Synthesis and Cloning (Invitrogen). .. Instead of using pSPORT 1 plasmid provided with the kit, a self-inactivating oncoretroviral plasmid, pCAMS/U3EPac , was utilized to express the antisense mRNA transcripts.

    Plasmid Preparation:

    Article Title: Antimelanoma CTL recognizes peptides derived from an ORF transcribed from the antisense strand of the 3′ untranslated region of TRIT1
    Article Snippet: .. In brief, polyA+ RNA (5 µg) from WM793 cells were reverse transcribed using the cDNA synthesis superscript plasmid system (Life Technologies). ..

    Article Title: Overexpression of C16orf74 is involved in aggressive pancreatic cancers
    Article Snippet: .. cDNA library screening We constructed a cDNA library using a SuperscriptTM plasmid system with GatewayTM technology for cDNA synthesis, a cloning kit (Invitrogen) and poly(A)+ RNA obtained from pancreatic cancer cell line Capan-1. .. We screened 3×106 independent clones of this library using the same cDNA probes used for Northern blot analysis.

    Article Title: Na+- and Cl–-coupled active transport of nitric oxide synthase inhibitors via amino acid transport system B0,+
    Article Snippet: .. The SuperScript plasmid system (Life Technologies Inc., Rockville, Maryland, USA) was used to establish a unidirectional cDNA library with poly(A)+ RNA isolated from mouse colon as described previously ( – ). .. The probe for library screening was prepared by RT-PCR using primers specific for mouse ATB0,+ cDNA reported in the GenBank (accession no. ).

    Article Title: CD8+ T Cells from SIV Elite Controller Macaques Recognize Mamu-B*08-Bound Epitopes and Select for Widespread Viral Variation
    Article Snippet: .. One microgram of mRNA from each animal served as the template for first strand cDNA synthesis, using the SuperScript plasmid system for cDNA synthesis and cloning (Invitrogen, Carlsbad, CA) by following the manufacturer's instructions. .. Size-fractionated cDNA containing SalI and NotI restriction endonuclease cohesive ends was ligated into the multiple cloning site of pCMV.SPORT6 and used to transform DH5α chemically competent E. coli (Invitrogen).

    Article Title: Venom gland transcriptomes of two elapid snakes (Bungarus multicinctus and Naja atra) and evolution of toxin genes
    Article Snippet: .. The purified mRNA was used to make cDNA library, following the instructions in the SuperScript Plasmid System for cDNA Synthesis and Cloning Kit (plasmid used: pCMV-SPORT6 for B. multicinctus , pSPORT1 for N. atra ) (Invitrogen, USA). .. Plasmids were purified using the QIAprep spin miniprep kit (Qiagen).

    Article Title: Selection of novel mediators of E2F1-induced apoptosis through retroviral expression of an antisense cDNA library
    Article Snippet: .. The cDNA library was constructed mainly based on SUPERSCRIPT™ Plasmid System with GATEWAY™ Technology for cDNA Synthesis and Cloning (Invitrogen). .. Instead of using pSPORT 1 plasmid provided with the kit, a self-inactivating oncoretroviral plasmid, pCAMS/U3EPac , was utilized to express the antisense mRNA transcripts.

    Article Title: Directional cDNA library construction assisted by the in vitro recombination reaction
    Article Snippet: .. cDNA synthesis from in vitro synthesized β-spectrin III (βSp3) mRNA was carried out according to the supplier’s instructions in the SuperScriptTM Plasmid System for cDNA synthesis (Life Technologies). ..

    Article Title: A novel high-affinity inhibitor against the human ATP-sensitive Kir6.2 channel
    Article Snippet: .. The total mRNAs were extracted from the venom glands by using TRIzol (Ambion); their cDNAs were produced by using the SuperScript plasmid system (Invitrogen). .. The cDNA corresponding to the purified inhibitor protein was amplified by PCR, where the 5′ end is primed with degenerate oligonucleotides corresponding to the N-terminal amino acid sequence of the inhibitor protein, whereas the 3′ end is primed with a poly-A oligonucleotide.

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  • 94
    Thermo Fisher superscript iii
    Validation of antisense transcripts. a Nanostring nCounter assays: controls. <t>RNA</t> was isolated from the <t>three</t> indicated cell lines at different timepoints post-infection and used for Nanostring nCounter assays. Experiments were performed with one measurement each from two biological replicates and scaled to the 2 h post-infection (hpi) timepoint after normalization using the provided control spike-ins. Values for HSV-1 mRNAs are shown as log(10) transformed normalized Nanostring counts. Error bars represent standard deviations. b Nanostring nCounter assays: antisense transcripts and corresponding sense mRNAs. Assays were performed as in a . c Absolute counts of SLC27A4 antisense and sense. To compare the three cell lines, absolute counts after technical normalization using the provided control spike-ins are shown. Error bars represent standard deviations. d Expression of antisense transcripts in total NET-seq data. Shown is the distribution of antisense transcripts and protein coding genes as lines, with the values of the 12 selected antisense transcripts and housekeeping genes marked on top
    Superscript Iii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 254 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii/product/Thermo Fisher
    Average 94 stars, based on 254 article reviews
    Price from $9.99 to $1999.99
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    94
    Thermo Fisher superscript iii reverse transcriptase kit
    Downstream targets of wheat PBF . The downstream target genes of PBF were identified using a GENIE3 network constructed using 850 <t>RNA-seq</t> samples ( Ramírez-González et al., 2018 ). A. A comparison of the target genes of each of the wheat PBF homoeologs. The number of predicted target genes common to all <t>three</t> homoeologs is 226. B. The 226 common target genes were analysed for evidence of enrichment with respect to molecular function. The top-ranking functional categories (adjusted p value
    Superscript Iii Reverse Transcriptase Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 296 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii reverse transcriptase kit/product/Thermo Fisher
    Average 94 stars, based on 296 article reviews
    Price from $9.99 to $1999.99
    superscript iii reverse transcriptase kit - by Bioz Stars, 2020-08
    94/100 stars
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    94
    Thermo Fisher superscript iii platinum one step qrt pcr kit
    Virus profiling by tandem mass spectrometry. (A) Kinetic of viral production by LC-MS/MS. Relative abundance at each time points represents the mean ± standard deviation of the technical replicates at the two MOI tested. Dashed curves indicate results obtained at MOI 0.01 while solid curves refer to MOI 0.001. (B) Comparison between viral peptides identified at the different time points. Sets of intersections are visualized using the UpSet matrix layout and plotted horizontally. Each column corresponds to an exclusive intersection that contains the elements of the sets represented by the dark circles. Sets are represented by the different peptides assigned to viral proteins at each time point. J2, J3, J4 and J7 refer to the different time points analysed: Day 2, 3, 4 and 7, respectively. (C) Correlation between viral counts (copies/mL) obtained by <t>qRT-PCR</t> and the abundance of viral proteins measured by LC-MS/MS across time points. Dashed curves indicate results obtained at MOI 0.01 while solid curves refer to MOI 0.001.
    Superscript Iii Platinum One Step Qrt Pcr Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 318 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii platinum one step qrt pcr kit/product/Thermo Fisher
    Average 94 stars, based on 318 article reviews
    Price from $9.99 to $1999.99
    superscript iii platinum one step qrt pcr kit - by Bioz Stars, 2020-08
    94/100 stars
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    93
    Thermo Fisher superscript iii first strand synthesis supermix for qrt pcr
    Moesin knockdown impacts Aβ phagocytosis and pro-inflammatory cytokine production by microglia. A Overview of in-vitro knockdown studies in primary mouse microglia treated with Msn siRNA or sham siRNA under resting and LPS-stimulated conditions. B Results from <t>qRT‐PCR</t> experiments demonstrating efficiency of Msn knockdown by siRNA as compared to sham siRNA. Y‐axis represents relative mRNA expression (2‐ΔΔCt method) normalized to Gapdh as housekeeping gene <t>(three</t> independent biological replicate experiments were performed per condition). C In‐vitro phagocytic capacity of primary microglia for fAβ42 HiLyte488 following exposure to sham siRNA or Msn siRNA under resting and LPS-stimulated conditions. Phagocytic uptake of fAβ42 was measured as proportion of CD45 + microglia using untreated microglia as negative controls. For each sample, at least 2,000 live CD45 + microglial events were captured. N =□3 independently performed biological replicate experiments. D-I Bar graphs displaying cytokine/chemokine data, shown as fluorescence intensity in arbitrary units (A.U.), obtained from microglial culture supernatants after siRNA sham or siRNA Msn treatment under resting and LPS-stimulated conditions: D IL10, E IL6, F TNF, G G-CSF, H CXCL10, I CCL3. Error bars represent ± SEM. Unpaired t-test: * p
    Superscript Iii First Strand Synthesis Supermix For Qrt Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii first strand synthesis supermix for qrt pcr/product/Thermo Fisher
    Average 93 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    superscript iii first strand synthesis supermix for qrt pcr - by Bioz Stars, 2020-08
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    Image Search Results


    Validation of antisense transcripts. a Nanostring nCounter assays: controls. RNA was isolated from the three indicated cell lines at different timepoints post-infection and used for Nanostring nCounter assays. Experiments were performed with one measurement each from two biological replicates and scaled to the 2 h post-infection (hpi) timepoint after normalization using the provided control spike-ins. Values for HSV-1 mRNAs are shown as log(10) transformed normalized Nanostring counts. Error bars represent standard deviations. b Nanostring nCounter assays: antisense transcripts and corresponding sense mRNAs. Assays were performed as in a . c Absolute counts of SLC27A4 antisense and sense. To compare the three cell lines, absolute counts after technical normalization using the provided control spike-ins are shown. Error bars represent standard deviations. d Expression of antisense transcripts in total NET-seq data. Shown is the distribution of antisense transcripts and protein coding genes as lines, with the values of the 12 selected antisense transcripts and housekeeping genes marked on top

    Journal: Genome Biology

    Article Title: Widespread activation of antisense transcription of the host genome during herpes simplex virus 1 infection

    doi: 10.1186/s13059-017-1329-5

    Figure Lengend Snippet: Validation of antisense transcripts. a Nanostring nCounter assays: controls. RNA was isolated from the three indicated cell lines at different timepoints post-infection and used for Nanostring nCounter assays. Experiments were performed with one measurement each from two biological replicates and scaled to the 2 h post-infection (hpi) timepoint after normalization using the provided control spike-ins. Values for HSV-1 mRNAs are shown as log(10) transformed normalized Nanostring counts. Error bars represent standard deviations. b Nanostring nCounter assays: antisense transcripts and corresponding sense mRNAs. Assays were performed as in a . c Absolute counts of SLC27A4 antisense and sense. To compare the three cell lines, absolute counts after technical normalization using the provided control spike-ins are shown. Error bars represent standard deviations. d Expression of antisense transcripts in total NET-seq data. Shown is the distribution of antisense transcripts and protein coding genes as lines, with the values of the 12 selected antisense transcripts and housekeeping genes marked on top

    Article Snippet: Per reaction, 1 μg total RNA was used and transcribed using SuperScript III with random hexamer primers (both Thermo Fisher Scientific) according to the manufacturer’s protocol.

    Techniques: Isolation, Infection, Transformation Assay, Expressing

    Downstream targets of wheat PBF . The downstream target genes of PBF were identified using a GENIE3 network constructed using 850 RNA-seq samples ( Ramírez-González et al., 2018 ). A. A comparison of the target genes of each of the wheat PBF homoeologs. The number of predicted target genes common to all three homoeologs is 226. B. The 226 common target genes were analysed for evidence of enrichment with respect to molecular function. The top-ranking functional categories (adjusted p value

    Journal: Journal of Cereal Science

    Article Title: LYS3 encodes a prolamin-box-binding transcription factor that controls embryo growth in barley and wheat

    doi: 10.1016/j.jcs.2020.102965

    Figure Lengend Snippet: Downstream targets of wheat PBF . The downstream target genes of PBF were identified using a GENIE3 network constructed using 850 RNA-seq samples ( Ramírez-González et al., 2018 ). A. A comparison of the target genes of each of the wheat PBF homoeologs. The number of predicted target genes common to all three homoeologs is 226. B. The 226 common target genes were analysed for evidence of enrichment with respect to molecular function. The top-ranking functional categories (adjusted p value

    Article Snippet: An aliquot containing 4 μg RNA was treated for 45 min with 2 μl of DNase RQ1 (1 μg/μl) at 37 °C (Promega, UK), and purified using an RNAeasy spin column (Qiagen, UK). cDNA was prepared from 0.5 μg RNA using a SuperScript III reverse transcriptase kit and oligo (dT)18 primers (Thermo Fisher Scientific, UK) according to the manufacturer's instructions and stored at −20 °C prior to PCR.

    Techniques: Construct, RNA Sequencing Assay, Functional Assay

    Patterns of expression of PBF/LYS3 in barley . A. Tissue-specific expression according to the Barley eFP Browser 2.0 ( www.bar.utoronto ). Samples are from barley cv. Morex. Caryopsis (no Emb) means caryopsis without embryo. B. Temporal and tissue-specific patterns of LYS3 / PBF expression in developing grains of wild type (Bomi) assessed by RT–PCR. Each cDNA sample was from a pool of 10–75 tissue samples each from an individual grain. Samples were collected from at least five spikes, each from a different plant. Amplicons were visualized on 1% agarose gels stained with SYBR Safe (Invitrogen, UK). Numbers above panels are DAF. PBF = Prolamin Binding Factor (HORVU5Hr1G048700). ACT = ACTIN .(HORVU1Hr1G047440), a constitutively expressed gene. Control reactions varied from the test reactions as follows. C1: contained cDNA from the barley PBF deletion mutant Risø18 at 26 DAF. No product was observed showing that the primers used were specific for PBF. C2: no DNase, no reverse transcriptase (RT). C3: no DNase. C4: no RT. C5: contained water used for PCR instead of cDNA. C. In situ localization of PBF in developing wild type (Bomi) grains at 8 and 12 DAF. ( i ) Longitudinal section of 8 DAF grain stained with antisense probe. PBF is expressed mostly in the starchy endosperm (En) cells. ( ii ) Longitudinal section of 12 DAF grain (including the embryo) stained with an antisense probe. PBF is expressed in the scutellum (Sc), coleoptile (Co) and coleorhiza tip (Cr). PBF is not expressed in the radicle (Ra). ( iii ) Longitudinal section of 12 DAF as in (ii) but stained with a sense probe (negative control). Scale bars are 0.5 mm (i) and 20 mm (ii and iii). D. Embryo fresh weight in developing grains of wild type (Bomi) and lys3 mutant Risø19. Embryos were extracted from developing grains and immediately weighed. Values are means ± SE (n > 4) for 10 embryos extracted from the middle of at least three spikes. Values are significantly different (p

    Journal: Journal of Cereal Science

    Article Title: LYS3 encodes a prolamin-box-binding transcription factor that controls embryo growth in barley and wheat

    doi: 10.1016/j.jcs.2020.102965

    Figure Lengend Snippet: Patterns of expression of PBF/LYS3 in barley . A. Tissue-specific expression according to the Barley eFP Browser 2.0 ( www.bar.utoronto ). Samples are from barley cv. Morex. Caryopsis (no Emb) means caryopsis without embryo. B. Temporal and tissue-specific patterns of LYS3 / PBF expression in developing grains of wild type (Bomi) assessed by RT–PCR. Each cDNA sample was from a pool of 10–75 tissue samples each from an individual grain. Samples were collected from at least five spikes, each from a different plant. Amplicons were visualized on 1% agarose gels stained with SYBR Safe (Invitrogen, UK). Numbers above panels are DAF. PBF = Prolamin Binding Factor (HORVU5Hr1G048700). ACT = ACTIN .(HORVU1Hr1G047440), a constitutively expressed gene. Control reactions varied from the test reactions as follows. C1: contained cDNA from the barley PBF deletion mutant Risø18 at 26 DAF. No product was observed showing that the primers used were specific for PBF. C2: no DNase, no reverse transcriptase (RT). C3: no DNase. C4: no RT. C5: contained water used for PCR instead of cDNA. C. In situ localization of PBF in developing wild type (Bomi) grains at 8 and 12 DAF. ( i ) Longitudinal section of 8 DAF grain stained with antisense probe. PBF is expressed mostly in the starchy endosperm (En) cells. ( ii ) Longitudinal section of 12 DAF grain (including the embryo) stained with an antisense probe. PBF is expressed in the scutellum (Sc), coleoptile (Co) and coleorhiza tip (Cr). PBF is not expressed in the radicle (Ra). ( iii ) Longitudinal section of 12 DAF as in (ii) but stained with a sense probe (negative control). Scale bars are 0.5 mm (i) and 20 mm (ii and iii). D. Embryo fresh weight in developing grains of wild type (Bomi) and lys3 mutant Risø19. Embryos were extracted from developing grains and immediately weighed. Values are means ± SE (n > 4) for 10 embryos extracted from the middle of at least three spikes. Values are significantly different (p

    Article Snippet: An aliquot containing 4 μg RNA was treated for 45 min with 2 μl of DNase RQ1 (1 μg/μl) at 37 °C (Promega, UK), and purified using an RNAeasy spin column (Qiagen, UK). cDNA was prepared from 0.5 μg RNA using a SuperScript III reverse transcriptase kit and oligo (dT)18 primers (Thermo Fisher Scientific, UK) according to the manufacturer's instructions and stored at −20 °C prior to PCR.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Binding Assay, Mutagenesis, Polymerase Chain Reaction, In Situ, Negative Control

    Virus profiling by tandem mass spectrometry. (A) Kinetic of viral production by LC-MS/MS. Relative abundance at each time points represents the mean ± standard deviation of the technical replicates at the two MOI tested. Dashed curves indicate results obtained at MOI 0.01 while solid curves refer to MOI 0.001. (B) Comparison between viral peptides identified at the different time points. Sets of intersections are visualized using the UpSet matrix layout and plotted horizontally. Each column corresponds to an exclusive intersection that contains the elements of the sets represented by the dark circles. Sets are represented by the different peptides assigned to viral proteins at each time point. J2, J3, J4 and J7 refer to the different time points analysed: Day 2, 3, 4 and 7, respectively. (C) Correlation between viral counts (copies/mL) obtained by qRT-PCR and the abundance of viral proteins measured by LC-MS/MS across time points. Dashed curves indicate results obtained at MOI 0.01 while solid curves refer to MOI 0.001.

    Journal: bioRxiv

    Article Title: Shotgun proteomics of SARS-CoV-2 infected cells and its application to the optimisation of whole viral particle antigen production for vaccines

    doi: 10.1101/2020.04.17.046193

    Figure Lengend Snippet: Virus profiling by tandem mass spectrometry. (A) Kinetic of viral production by LC-MS/MS. Relative abundance at each time points represents the mean ± standard deviation of the technical replicates at the two MOI tested. Dashed curves indicate results obtained at MOI 0.01 while solid curves refer to MOI 0.001. (B) Comparison between viral peptides identified at the different time points. Sets of intersections are visualized using the UpSet matrix layout and plotted horizontally. Each column corresponds to an exclusive intersection that contains the elements of the sets represented by the dark circles. Sets are represented by the different peptides assigned to viral proteins at each time point. J2, J3, J4 and J7 refer to the different time points analysed: Day 2, 3, 4 and 7, respectively. (C) Correlation between viral counts (copies/mL) obtained by qRT-PCR and the abundance of viral proteins measured by LC-MS/MS across time points. Dashed curves indicate results obtained at MOI 0.01 while solid curves refer to MOI 0.001.

    Article Snippet: RNA was subjected to qRT-PCR analysis using the SuperScript III Platinum One-Step qRT-PCR Kit (ThermoFisher) and a CFX96 Touch Real-Time PCR Detection System Thermal Cycler (BioRad).

    Techniques: Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Standard Deviation, Quantitative RT-PCR

    Moesin knockdown impacts Aβ phagocytosis and pro-inflammatory cytokine production by microglia. A Overview of in-vitro knockdown studies in primary mouse microglia treated with Msn siRNA or sham siRNA under resting and LPS-stimulated conditions. B Results from qRT‐PCR experiments demonstrating efficiency of Msn knockdown by siRNA as compared to sham siRNA. Y‐axis represents relative mRNA expression (2‐ΔΔCt method) normalized to Gapdh as housekeeping gene (three independent biological replicate experiments were performed per condition). C In‐vitro phagocytic capacity of primary microglia for fAβ42 HiLyte488 following exposure to sham siRNA or Msn siRNA under resting and LPS-stimulated conditions. Phagocytic uptake of fAβ42 was measured as proportion of CD45 + microglia using untreated microglia as negative controls. For each sample, at least 2,000 live CD45 + microglial events were captured. N =□3 independently performed biological replicate experiments. D-I Bar graphs displaying cytokine/chemokine data, shown as fluorescence intensity in arbitrary units (A.U.), obtained from microglial culture supernatants after siRNA sham or siRNA Msn treatment under resting and LPS-stimulated conditions: D IL10, E IL6, F TNF, G G-CSF, H CXCL10, I CCL3. Error bars represent ± SEM. Unpaired t-test: * p

    Journal: bioRxiv

    Article Title: Flow-cytometric microglial sorting coupled with quantitative proteomics identifies moesin as a highly-abundant microglial protein with relevance to Alzheimer’s disease

    doi: 10.1101/802694

    Figure Lengend Snippet: Moesin knockdown impacts Aβ phagocytosis and pro-inflammatory cytokine production by microglia. A Overview of in-vitro knockdown studies in primary mouse microglia treated with Msn siRNA or sham siRNA under resting and LPS-stimulated conditions. B Results from qRT‐PCR experiments demonstrating efficiency of Msn knockdown by siRNA as compared to sham siRNA. Y‐axis represents relative mRNA expression (2‐ΔΔCt method) normalized to Gapdh as housekeeping gene (three independent biological replicate experiments were performed per condition). C In‐vitro phagocytic capacity of primary microglia for fAβ42 HiLyte488 following exposure to sham siRNA or Msn siRNA under resting and LPS-stimulated conditions. Phagocytic uptake of fAβ42 was measured as proportion of CD45 + microglia using untreated microglia as negative controls. For each sample, at least 2,000 live CD45 + microglial events were captured. N =□3 independently performed biological replicate experiments. D-I Bar graphs displaying cytokine/chemokine data, shown as fluorescence intensity in arbitrary units (A.U.), obtained from microglial culture supernatants after siRNA sham or siRNA Msn treatment under resting and LPS-stimulated conditions: D IL10, E IL6, F TNF, G G-CSF, H CXCL10, I CCL3. Error bars represent ± SEM. Unpaired t-test: * p

    Article Snippet: After 48 hours, the efficiency of siRNA-mediated gene silencing was confirmed by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) ( ).

    Techniques: In Vitro, Quantitative RT-PCR, Expressing, Fluorescence