superscript first strand synthesis system  (Thermo Fisher)


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    Name:
    SuperScript First Strand Synthesis System for RT PCR
    Description:
    The SuperScript First Strand Synthesis System for RT PCR is optimized to synthesize first strand cDNA from purified poly A or total RNA The system can be used with as little as 1 ng or as much as 5 µg of total RNA After synthesis the cDNA can be amplified with specific primers by PCR without intermediate organic extractions or ethanol precipitations In conjunction with PCR the system can be used to detect the presence of rare messages to quantitate the amount of specific mRNA from small numbers of cells or to clone specific cDNAs without constructing an entire cDNA library The system is flexible allowing the use of any PCR enzyme Combine with AccuPrime Taq DNA Polymerase or Platinum Taq DNA Polymerase for higher specificity PCR or with AccuPrime Pfx DNA Polymerase for high fidelity cloning applications SuperScript II RTThe first strand cDNA synthesis reaction is catalyzed by SuperScript II Reverse Transcriptase RT This enzyme has been engineered to reduce the RNase H activity that degrades mRNA during the first strand reaction resulting in greater full length cDNA synthesis and higher yields of first strand cDNA than obtained with RNase H RTs Because SuperScript II RT is not inhibited significantly by ribosomal and transfer RNA it may be used effectively to synthesize first strand cDNA from a total RNA preparation The enzyme exhibits increased thermal stability and may be used at temperatures up to 50°C Using the SuperScript First Strand Synthesis System for RT PCRThis system has been optimized to synthesize first strand cDNA from varying amounts of starting material The SuperScript II RT concentration has been lowered and RNaseOUT Recombinant RNase Inhibitor has been added to the system as part of this optimization process Additionally reaction conditions have been modified to further increase the sensitivity of the system Using the kit you synthesize first strand cDNA using either total RNA or poly A selected RNA primed with oligo dT random primers or a gene specific primer Then you perform PCR in a separate tube using primers specific for the gene of interest
    Catalog Number:
    11904018
    Price:
    None
    Applications:
    PCR & Real-Time PCR|Reverse Transcription
    Category:
    Kits and Assays
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    Structured Review

    Thermo Fisher superscript first strand synthesis system
    Midkine expression was induced by FBS, growth factors and cytokines. A . LNCaP cells were cultured in serum-free DMEM and treated for 48 h with 10% FBS and the indicated agents: 10 ng/ml insulin, 10 ng/ml IGF-I, 10 ng/ml EGF, 10 ng/ml HGF, 10 ng/ml bFGF, 20 ng/ml T3, 10 nM R1881, 10 nM DHT, 10 ng/ml TNFα, 10 ng/ml IL-1β, 50 ng/ml IL-6, 50 ng/ml IL-17, and 33.3 μM RA. B . LNCaP cells were treated with different dosages (1 to 50 ng/ml) of TNFα for 48 h. C . LNCaP cells were also treated with 20 ng/ml TNFα for different time periods (8 to 48 h). 20 μl of each medium supernatant was subjected to Western blot analysis of midkine expression using rabbit anti-midkine antibodies, horseradish peroxidase-conjungated secondary antibodies and enhanced chemiluminescence reagents. D . Total RNA was extracted from LNCaP cells not treated or treated with 20 ng/ml TNFα, using RNeasy Mini Kit; cDNA was made from total RNA using <t>Superscript™</t> <t>First-Strand</t> <t>Synthesis</t> <t>System</t> with oligo dT primers; real-time quantitative PCR was done in triplicates with Sybr-Green reagents; results were normalized to GAPDH levels as described in Methods; the data (mean ± standard deviation of three independent experiments) were presented as fold change of midkine mRNA compared to the LNCaP cells without treatment for 8 h, where fold = 2 ΔΔCt ; solid bar, TNFα treated; open bar, control; * P
    The SuperScript First Strand Synthesis System for RT PCR is optimized to synthesize first strand cDNA from purified poly A or total RNA The system can be used with as little as 1 ng or as much as 5 µg of total RNA After synthesis the cDNA can be amplified with specific primers by PCR without intermediate organic extractions or ethanol precipitations In conjunction with PCR the system can be used to detect the presence of rare messages to quantitate the amount of specific mRNA from small numbers of cells or to clone specific cDNAs without constructing an entire cDNA library The system is flexible allowing the use of any PCR enzyme Combine with AccuPrime Taq DNA Polymerase or Platinum Taq DNA Polymerase for higher specificity PCR or with AccuPrime Pfx DNA Polymerase for high fidelity cloning applications SuperScript II RTThe first strand cDNA synthesis reaction is catalyzed by SuperScript II Reverse Transcriptase RT This enzyme has been engineered to reduce the RNase H activity that degrades mRNA during the first strand reaction resulting in greater full length cDNA synthesis and higher yields of first strand cDNA than obtained with RNase H RTs Because SuperScript II RT is not inhibited significantly by ribosomal and transfer RNA it may be used effectively to synthesize first strand cDNA from a total RNA preparation The enzyme exhibits increased thermal stability and may be used at temperatures up to 50°C Using the SuperScript First Strand Synthesis System for RT PCRThis system has been optimized to synthesize first strand cDNA from varying amounts of starting material The SuperScript II RT concentration has been lowered and RNaseOUT Recombinant RNase Inhibitor has been added to the system as part of this optimization process Additionally reaction conditions have been modified to further increase the sensitivity of the system Using the kit you synthesize first strand cDNA using either total RNA or poly A selected RNA primed with oligo dT random primers or a gene specific primer Then you perform PCR in a separate tube using primers specific for the gene of interest
    https://www.bioz.com/result/superscript first strand synthesis system/product/Thermo Fisher
    Average 99 stars, based on 2730 article reviews
    Price from $9.99 to $1999.99
    superscript first strand synthesis system - by Bioz Stars, 2021-01
    99/100 stars

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    Images

    1) Product Images from "Midkine is a NF-?B-inducible gene that supports prostate cancer cell survival"

    Article Title: Midkine is a NF-?B-inducible gene that supports prostate cancer cell survival

    Journal: BMC Medical Genomics

    doi: 10.1186/1755-8794-1-6

    Midkine expression was induced by FBS, growth factors and cytokines. A . LNCaP cells were cultured in serum-free DMEM and treated for 48 h with 10% FBS and the indicated agents: 10 ng/ml insulin, 10 ng/ml IGF-I, 10 ng/ml EGF, 10 ng/ml HGF, 10 ng/ml bFGF, 20 ng/ml T3, 10 nM R1881, 10 nM DHT, 10 ng/ml TNFα, 10 ng/ml IL-1β, 50 ng/ml IL-6, 50 ng/ml IL-17, and 33.3 μM RA. B . LNCaP cells were treated with different dosages (1 to 50 ng/ml) of TNFα for 48 h. C . LNCaP cells were also treated with 20 ng/ml TNFα for different time periods (8 to 48 h). 20 μl of each medium supernatant was subjected to Western blot analysis of midkine expression using rabbit anti-midkine antibodies, horseradish peroxidase-conjungated secondary antibodies and enhanced chemiluminescence reagents. D . Total RNA was extracted from LNCaP cells not treated or treated with 20 ng/ml TNFα, using RNeasy Mini Kit; cDNA was made from total RNA using Superscript™ First-Strand Synthesis System with oligo dT primers; real-time quantitative PCR was done in triplicates with Sybr-Green reagents; results were normalized to GAPDH levels as described in Methods; the data (mean ± standard deviation of three independent experiments) were presented as fold change of midkine mRNA compared to the LNCaP cells without treatment for 8 h, where fold = 2 ΔΔCt ; solid bar, TNFα treated; open bar, control; * P
    Figure Legend Snippet: Midkine expression was induced by FBS, growth factors and cytokines. A . LNCaP cells were cultured in serum-free DMEM and treated for 48 h with 10% FBS and the indicated agents: 10 ng/ml insulin, 10 ng/ml IGF-I, 10 ng/ml EGF, 10 ng/ml HGF, 10 ng/ml bFGF, 20 ng/ml T3, 10 nM R1881, 10 nM DHT, 10 ng/ml TNFα, 10 ng/ml IL-1β, 50 ng/ml IL-6, 50 ng/ml IL-17, and 33.3 μM RA. B . LNCaP cells were treated with different dosages (1 to 50 ng/ml) of TNFα for 48 h. C . LNCaP cells were also treated with 20 ng/ml TNFα for different time periods (8 to 48 h). 20 μl of each medium supernatant was subjected to Western blot analysis of midkine expression using rabbit anti-midkine antibodies, horseradish peroxidase-conjungated secondary antibodies and enhanced chemiluminescence reagents. D . Total RNA was extracted from LNCaP cells not treated or treated with 20 ng/ml TNFα, using RNeasy Mini Kit; cDNA was made from total RNA using Superscript™ First-Strand Synthesis System with oligo dT primers; real-time quantitative PCR was done in triplicates with Sybr-Green reagents; results were normalized to GAPDH levels as described in Methods; the data (mean ± standard deviation of three independent experiments) were presented as fold change of midkine mRNA compared to the LNCaP cells without treatment for 8 h, where fold = 2 ΔΔCt ; solid bar, TNFα treated; open bar, control; * P

    Techniques Used: Expressing, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, SYBR Green Assay, Standard Deviation

    2) Product Images from "In Vitro Selection of Highly Darunavir-Resistant and Replication-Competent HIV-1 Variants by Using a Mixture of Clinical HIV-1 Isolates Resistant to Multiple Conventional Protease Inhibitors ▿ Selection of Highly Darunavir-Resistant and Replication-Competent HIV-1 Variants by Using a Mixture of Clinical HIV-1 Isolates Resistant to Multiple Conventional Protease Inhibitors ▿ †"

    Article Title: In Vitro Selection of Highly Darunavir-Resistant and Replication-Competent HIV-1 Variants by Using a Mixture of Clinical HIV-1 Isolates Resistant to Multiple Conventional Protease Inhibitors ▿ Selection of Highly Darunavir-Resistant and Replication-Competent HIV-1 Variants by Using a Mixture of Clinical HIV-1 Isolates Resistant to Multiple Conventional Protease Inhibitors ▿ †

    Journal: Journal of Virology

    doi: 10.1128/JVI.00967-10

    Sequence analysis of the protease-encoding regions in the mixture of 8 HIV MDR isolates. Viral RNA was purified from each indicated supernatant using the QIAamp viral RNA minikit (Qiagen Inc., Valencia, CA), and RT-PCR was carried out using the Superscript First-Strand synthesis system for RT-PCR (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The amino acid sequences of protease deduced from nucleotide sequences of the protease-encoding region of HIV-1 clones determined are shown. The fraction of clones examined is indicated on the right. The amino acid sequence of protease of a wild-type pNL4-3 clone is shown as a reference. Identity with this sequence at individual amino acid positions is indicated (dots).
    Figure Legend Snippet: Sequence analysis of the protease-encoding regions in the mixture of 8 HIV MDR isolates. Viral RNA was purified from each indicated supernatant using the QIAamp viral RNA minikit (Qiagen Inc., Valencia, CA), and RT-PCR was carried out using the Superscript First-Strand synthesis system for RT-PCR (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The amino acid sequences of protease deduced from nucleotide sequences of the protease-encoding region of HIV-1 clones determined are shown. The fraction of clones examined is indicated on the right. The amino acid sequence of protease of a wild-type pNL4-3 clone is shown as a reference. Identity with this sequence at individual amino acid positions is indicated (dots).

    Techniques Used: Sequencing, Purification, Reverse Transcription Polymerase Chain Reaction, Clone Assay

    3) Product Images from "NNK, a Tobacco-Specific Carcinogen, Inhibits the Expression of Lysyl Oxidase, a Tumor Suppressor"

    Article Title: NNK, a Tobacco-Specific Carcinogen, Inhibits the Expression of Lysyl Oxidase, a Tumor Suppressor

    Journal: International Journal of Environmental Research and Public Health

    doi: 10.3390/ijerph120100064

    NNK inhibition of LOX steady–state mRNA levels in treated cells as revealed by reverse transcription (RT)-PCR and agarose gel electrophoresis ( A ) and quantitative real-time-PCR ( B ). ( A ) Total RNA (1 µg) was extracted from growth-arrested control and treated cells using Trizol reagent. Reverse-transcription cDNA was produced using the SuperScript first-strand synthesis system. LOX and GAPDH (an internal control) cDNA fragments were amplified by PCR and analyzed on a 2.2% agarose gel. Densities of PCR-amplified gene fragments on the gel as described here and below were measured with the 1D Scan software. ( B ) The real-time PCR was performed by the GeneAmpR 5700 Sequence Detection System (SDS) using reverse-transcription DNA as a template. PCR products were monitored by fluorescence from the TaqMan probes for LOX and GAPDH (an internal control) and analyzed using the GeneAmp 5700 SDS software. Data shown are the mean ± SD ( n = 3). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 compared with the control.
    Figure Legend Snippet: NNK inhibition of LOX steady–state mRNA levels in treated cells as revealed by reverse transcription (RT)-PCR and agarose gel electrophoresis ( A ) and quantitative real-time-PCR ( B ). ( A ) Total RNA (1 µg) was extracted from growth-arrested control and treated cells using Trizol reagent. Reverse-transcription cDNA was produced using the SuperScript first-strand synthesis system. LOX and GAPDH (an internal control) cDNA fragments were amplified by PCR and analyzed on a 2.2% agarose gel. Densities of PCR-amplified gene fragments on the gel as described here and below were measured with the 1D Scan software. ( B ) The real-time PCR was performed by the GeneAmpR 5700 Sequence Detection System (SDS) using reverse-transcription DNA as a template. PCR products were monitored by fluorescence from the TaqMan probes for LOX and GAPDH (an internal control) and analyzed using the GeneAmp 5700 SDS software. Data shown are the mean ± SD ( n = 3). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 compared with the control.

    Techniques Used: Inhibition, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Produced, Amplification, Polymerase Chain Reaction, Software, Sequencing, Fluorescence

    4) Product Images from "The Friend leukaemia virus integration 1 (Fli-1) transcription factor affects lupus nephritis development by regulating inflammatory cell infiltration into the kidney"

    Article Title: The Friend leukaemia virus integration 1 (Fli-1) transcription factor affects lupus nephritis development by regulating inflammatory cell infiltration into the kidney

    Journal: Clinical and Experimental Immunology

    doi: 10.1111/cei.12310

    Increased expression of inflammatory chemokines in kidneys of wild-type mice compared to Friend leukaemia virus integration 1 (Fli-1 +/− ) Murphy Roths large (MRL)/ lpr mice. Total RNA was prepared from kidneys at the ages of 22–24 weeks (wild-type MRL/ lpr , n = 11, Fli-1 +/− MRL/ lpr mice, n = 6). Total RNA was converted to cDNA with the SuperScript First-Strand Synthesis System. Real-time polymerase chain reaction was performed in triplicate with the appropriate primers. A single asterisk indicates P
    Figure Legend Snippet: Increased expression of inflammatory chemokines in kidneys of wild-type mice compared to Friend leukaemia virus integration 1 (Fli-1 +/− ) Murphy Roths large (MRL)/ lpr mice. Total RNA was prepared from kidneys at the ages of 22–24 weeks (wild-type MRL/ lpr , n = 11, Fli-1 +/− MRL/ lpr mice, n = 6). Total RNA was converted to cDNA with the SuperScript First-Strand Synthesis System. Real-time polymerase chain reaction was performed in triplicate with the appropriate primers. A single asterisk indicates P

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

    5) Product Images from "Vibrio vulnificus rtxE Is Important for Virulence, and Its Expression Is Induced by Exposure to Host Cells "

    Article Title: Vibrio vulnificus rtxE Is Important for Virulence, and Its Expression Is Induced by Exposure to Host Cells

    Journal: Infection and Immunity

    doi: 10.1128/IAI.01503-07

    Analysis of the rtxBDE transcript by RT-PCR. Shown is an RT-PCR analysis of RNA isolated from a mid-exponential-phase culture of M06-24/O(pMW0504). The RNA was treated with DNase I (Sigma, St. Louis, MO) and used for the synthesis of cDNA by reverse transcription (SuperScript first-strand synthesis system for RT-PCR; Invitrogen, Carlsbad, CA). The cDNA (lane 1), DNase I-treated RNA (negative control; lane 2), and wild-type genomic DNA (positive control; lane 3) were used as templates for PCR. Molecular size markers (1 kb plus ladder; Invitrogen) and a PCR product are indicated.
    Figure Legend Snippet: Analysis of the rtxBDE transcript by RT-PCR. Shown is an RT-PCR analysis of RNA isolated from a mid-exponential-phase culture of M06-24/O(pMW0504). The RNA was treated with DNase I (Sigma, St. Louis, MO) and used for the synthesis of cDNA by reverse transcription (SuperScript first-strand synthesis system for RT-PCR; Invitrogen, Carlsbad, CA). The cDNA (lane 1), DNase I-treated RNA (negative control; lane 2), and wild-type genomic DNA (positive control; lane 3) were used as templates for PCR. Molecular size markers (1 kb plus ladder; Invitrogen) and a PCR product are indicated.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation, Negative Control, Positive Control, Polymerase Chain Reaction

    Related Articles

    Amplification:

    Article Title: Metatranscriptomic and functional metagenomic analysis of methylphosphonate utilization by marine bacteria
    Article Snippet: .. In brief, roughly 10–20 ng of total RNA were amplified using MessageAmp II Bacteria kit (Ambion) and T7Bpm IdT16 VN, an oligo(dT) primer containing a promoter sequence for T7 RNA polymerase and a Bpm I recognition site. cDNA was synthesized from the amplified RNA using Superscript III (Invitrogen) and random hexamers for first-strand complementary DNA synthesis, and the SuperScript Double Stranded cDNA Synthesis kit (Invitrogen) for second-strand synthesis. .. The resulting cDNA was purified using the Qiaquick PCR purification kit (Qiagen), digested with Bpm I to remove poly(A) tails and used directly for pyrosequencing. gDNA and cDNA single-stranded libraries were prepared and sequenced using standard GS FLX protocols (454 Life Sciences, Roche) in a Roche Genome Sequencer FLX (Roche Applied Science, Indianapolis, IN).

    DNA Synthesis:

    Article Title: Metatranscriptomic and functional metagenomic analysis of methylphosphonate utilization by marine bacteria
    Article Snippet: .. In brief, roughly 10–20 ng of total RNA were amplified using MessageAmp II Bacteria kit (Ambion) and T7Bpm IdT16 VN, an oligo(dT) primer containing a promoter sequence for T7 RNA polymerase and a Bpm I recognition site. cDNA was synthesized from the amplified RNA using Superscript III (Invitrogen) and random hexamers for first-strand complementary DNA synthesis, and the SuperScript Double Stranded cDNA Synthesis kit (Invitrogen) for second-strand synthesis. .. The resulting cDNA was purified using the Qiaquick PCR purification kit (Qiagen), digested with Bpm I to remove poly(A) tails and used directly for pyrosequencing. gDNA and cDNA single-stranded libraries were prepared and sequenced using standard GS FLX protocols (454 Life Sciences, Roche) in a Roche Genome Sequencer FLX (Roche Applied Science, Indianapolis, IN).

    Synthesized:

    Article Title: M-CSF Induces Vascular Endothelial Growth Factor Production and Angiogenic Activity From Human Monocytes
    Article Snippet: .. RNA integrity was verified on a 1.5% agarose gel to detect 18S and 28S ribosomal RNA bands at 0.7 and 1.5 kDa, respectively. cDNA was synthesized from monocyte total cellular RNA using the SuperScript First-Strand Synthesis System for RT-PCR Kit (Life Technologies). .. The VEGF forward primer, VEGF reverse primer, and the VEGF probe with TAMRA quencher (PE Applied Biosystems) for VEGF mRNA were designed using Primer Express version 1.0 software (ABI PRISM, PerkinElmer, Branchburg, NJ) based on the human VEGF sequence obtained from PubMed (accession no. NM 003376).

    Article Title: Metatranscriptomic and functional metagenomic analysis of methylphosphonate utilization by marine bacteria
    Article Snippet: .. In brief, roughly 10–20 ng of total RNA were amplified using MessageAmp II Bacteria kit (Ambion) and T7Bpm IdT16 VN, an oligo(dT) primer containing a promoter sequence for T7 RNA polymerase and a Bpm I recognition site. cDNA was synthesized from the amplified RNA using Superscript III (Invitrogen) and random hexamers for first-strand complementary DNA synthesis, and the SuperScript Double Stranded cDNA Synthesis kit (Invitrogen) for second-strand synthesis. .. The resulting cDNA was purified using the Qiaquick PCR purification kit (Qiagen), digested with Bpm I to remove poly(A) tails and used directly for pyrosequencing. gDNA and cDNA single-stranded libraries were prepared and sequenced using standard GS FLX protocols (454 Life Sciences, Roche) in a Roche Genome Sequencer FLX (Roche Applied Science, Indianapolis, IN).

    Isolation:

    Article Title: Reduction of lung metastasis, cell invasion, and adhesion in mouse melanoma by statin-induced blockade of the Rho/Rho-associated coiled-coil-containing protein kinase pathway
    Article Snippet: .. Reverse transcription-polymerase chain reaction Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and a 1-μg aliquot of purified total RNA was subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis using a SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). .. The resulting cDNAs were used as a template for PCR amplification to generate products corresponding to the mRNAs encoding various gene products.

    Article Title: Presenilins regulate ?? T cell development by modulating TCR signaling
    Article Snippet: .. RNA was isolated using Trizol (Invitrogen) according to the manufacturer's instructions. cDNA was generated using a SuperScript III First-strand Synthesis System for RT-PCR kit (Invitrogen) according to manufacturer's instructions. ..

    Purification:

    Article Title: Reduction of lung metastasis, cell invasion, and adhesion in mouse melanoma by statin-induced blockade of the Rho/Rho-associated coiled-coil-containing protein kinase pathway
    Article Snippet: .. Reverse transcription-polymerase chain reaction Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and a 1-μg aliquot of purified total RNA was subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis using a SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). .. The resulting cDNAs were used as a template for PCR amplification to generate products corresponding to the mRNAs encoding various gene products.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: M-CSF Induces Vascular Endothelial Growth Factor Production and Angiogenic Activity From Human Monocytes
    Article Snippet: .. RNA integrity was verified on a 1.5% agarose gel to detect 18S and 28S ribosomal RNA bands at 0.7 and 1.5 kDa, respectively. cDNA was synthesized from monocyte total cellular RNA using the SuperScript First-Strand Synthesis System for RT-PCR Kit (Life Technologies). .. The VEGF forward primer, VEGF reverse primer, and the VEGF probe with TAMRA quencher (PE Applied Biosystems) for VEGF mRNA were designed using Primer Express version 1.0 software (ABI PRISM, PerkinElmer, Branchburg, NJ) based on the human VEGF sequence obtained from PubMed (accession no. NM 003376).

    Article Title: Tropomyosin 3.5 protects the F-actin networks required for tissue biomechanical properties
    Article Snippet: .. Reverse transcription was performed using the SuperscriptTM III First-Strand Synthesis System for RT-PCR kit (Thermo Fisher Scientific) from an equal amount of total RNA for each sample. .. PCR was performed with the same amount of cDNA from each sample in a 20 µl volume reaction.

    Article Title: Identification and characterization of the Plasmodium vivax thrombospondin-related apical merozoite protein
    Article Snippet: .. A 20 μL aliquot of RNA was used a template for RT-PCR cDNA synthesis using the SuperScript III enzyme (Invitrogen, California, USA), according to manufacturer's recommendations. .. Briefly, cDNA was synthesized for 60 min at 50°C and then amplified by PCR with Platinum Pfx DNA polymerase (Invitrogen, California, USA) using pvtramp specific forward (5'-atgTACATTTTGCAGTTGCTCC-3') and reverse (5'-TTCATACATAAATCTGCCAGC-3') primers, for 35 cycles at the following temperatures: 94°C for 15 s, 54°C for 30 s, 68°C for 60 s and a final 5 min extension at 68°C.

    Article Title: Molecular breeding of water lily: engineering cold stress tolerance into tropical water lily
    Article Snippet: .. First-strand complementary DNA (cDNA) synthesis was performed using SuperScript III First-Strand Synthesis System for reverse transcription PCR (RT-PCR) (Invitrogen, Carlsbad, CA, USA). ..

    Article Title: Reduction of lung metastasis, cell invasion, and adhesion in mouse melanoma by statin-induced blockade of the Rho/Rho-associated coiled-coil-containing protein kinase pathway
    Article Snippet: .. Reverse transcription-polymerase chain reaction Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and a 1-μg aliquot of purified total RNA was subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis using a SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). .. The resulting cDNAs were used as a template for PCR amplification to generate products corresponding to the mRNAs encoding various gene products.

    Article Title: Presenilins regulate ?? T cell development by modulating TCR signaling
    Article Snippet: .. RNA was isolated using Trizol (Invitrogen) according to the manufacturer's instructions. cDNA was generated using a SuperScript III First-strand Synthesis System for RT-PCR kit (Invitrogen) according to manufacturer's instructions. ..

    Article Title: Tumor protein D52 represents a negative regulator of ATM protein levels
    Article Snippet: .. Total RNA was extracted from 3T3 cell lines using TRIzol LS reagent (Life Technologies) and 1 μg RNA was subjected to cDNA synthesis using the SuperScript III First-Strand Synthesis System for RT-PCR kit (Life Technologies) according to the manufacturer’s instructions. .. The resulting cDNA was amplified using Taq DNA polymerase (QIAGEN) and 2 sets of Atm primers: Atm-2, sense 5′-TGCTAGATCTTCTGAGAGCG-3′ and antisense 5′-TATTGTTGAGGGCAGTCAGC-3′; Atm-3, sense 5′-CTGTATCTACAGCAGAGACC-3′ and antisense 5′-TCACACCCAAGCTTTCCATC-3′, which amplified mouse Atm (GenBank ) mRNA sequence fragments at nt 5040–5318 and nt 9071–9340, respectively.

    Polymerase Chain Reaction:

    Article Title: Molecular breeding of water lily: engineering cold stress tolerance into tropical water lily
    Article Snippet: .. First-strand complementary DNA (cDNA) synthesis was performed using SuperScript III First-Strand Synthesis System for reverse transcription PCR (RT-PCR) (Invitrogen, Carlsbad, CA, USA). ..

    Generated:

    Article Title: Presenilins regulate ?? T cell development by modulating TCR signaling
    Article Snippet: .. RNA was isolated using Trizol (Invitrogen) according to the manufacturer's instructions. cDNA was generated using a SuperScript III First-strand Synthesis System for RT-PCR kit (Invitrogen) according to manufacturer's instructions. ..

    Sequencing:

    Article Title: Metatranscriptomic and functional metagenomic analysis of methylphosphonate utilization by marine bacteria
    Article Snippet: .. In brief, roughly 10–20 ng of total RNA were amplified using MessageAmp II Bacteria kit (Ambion) and T7Bpm IdT16 VN, an oligo(dT) primer containing a promoter sequence for T7 RNA polymerase and a Bpm I recognition site. cDNA was synthesized from the amplified RNA using Superscript III (Invitrogen) and random hexamers for first-strand complementary DNA synthesis, and the SuperScript Double Stranded cDNA Synthesis kit (Invitrogen) for second-strand synthesis. .. The resulting cDNA was purified using the Qiaquick PCR purification kit (Qiagen), digested with Bpm I to remove poly(A) tails and used directly for pyrosequencing. gDNA and cDNA single-stranded libraries were prepared and sequenced using standard GS FLX protocols (454 Life Sciences, Roche) in a Roche Genome Sequencer FLX (Roche Applied Science, Indianapolis, IN).

    Agarose Gel Electrophoresis:

    Article Title: M-CSF Induces Vascular Endothelial Growth Factor Production and Angiogenic Activity From Human Monocytes
    Article Snippet: .. RNA integrity was verified on a 1.5% agarose gel to detect 18S and 28S ribosomal RNA bands at 0.7 and 1.5 kDa, respectively. cDNA was synthesized from monocyte total cellular RNA using the SuperScript First-Strand Synthesis System for RT-PCR Kit (Life Technologies). .. The VEGF forward primer, VEGF reverse primer, and the VEGF probe with TAMRA quencher (PE Applied Biosystems) for VEGF mRNA were designed using Primer Express version 1.0 software (ABI PRISM, PerkinElmer, Branchburg, NJ) based on the human VEGF sequence obtained from PubMed (accession no. NM 003376).

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    • superscript iii  (Thermo Fisher)
    99
    Thermo Fisher superscript iii
    Spatial relationship between snRNA biogenesis hub and histone locus bodies. (A) Immunofluorescence imaging of classical Cajal Body (Coilin) and nuclear gem (SMN) markers in mouse ES cells and HEK293T cells. Top: Mouse ES cells do not contain visible Coilin foci for any of the <t>three</t> anti-Coilin antibodies tested. Bottom: HEK293T cells show visible Coilin foci. SMN foci, which are markers for nuclear Gemini of Cajal bodies (“gems”) are present in both mouse ES cells and HEK293T cells. (B) Z-section of mouse ES cell co-stained for SMN protein and scaRNAs (pooled scaRNA2 and scaRNA17 probes) within the nucleus (DAPI). Inset shows an example of scaRNA localization near SMN foci (arrow). (C) Z-section of mouse ES cell with <t>RNA</t> FISH staining for U7 and scaRNAs (pooled scaRNA2 and scaRNA17 probes) within the nucleus (DAPI). Inset shows an example of scaRNA localization near U7 (arrow). (D) RNA-RNA contact frequency between scaRNA2 and all RNAs. Top hits include annotated scaRNAs and identify two previously unannotated scaRNAs (see Supplemental Methods ). (E) Weighted DNA-DNA contacts for all SPRITE clusters (top) and for SPRITE clusters containing scaRNAs (bottom) occurring within a region on chromosome 11 with snRNA gene clusters. scaRNA occupancy is demarcated with solid red boxes. (F) Weighted DNA-DNA contacts for all SPRITE clusters (top) and for SPRITE clusters containing the U7 ncRNA (bottom) occurring within a region on chromosome 13 containing the two Hist1 gene clusters. U7 and Hist1 RNA occupancy is demarcated with teal boxes. ( G ) Weighted DNA-DNA contacts shown for SPRITE clusters containing both scaRNAs and snRNAs simultaneously. (H) Weighted DNA-DNA contacts for SPRITE clusters containing the scaRNAs on chromosome 13.
    Superscript Iii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, 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 99 stars, based on 254 article reviews
    Price from $9.99 to $1999.99
    superscript iii - by Bioz Stars, 2021-01
    99/100 stars
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    superscript iii first strand synthesis system  (Thermo Fisher)
    99
    Thermo Fisher superscript iii first strand synthesis system
    Characterization of zebrafish Padi2. (A) Citrullination activity of bacterially expressed zebrafish Padi2 201a and 202 splice variants in total lysates with and without calcium. Absorbance of light was measured and expressed as mean (± SEM) relative light units (RLU), normalized for protein level. Data represent 3 independent replicates. (B) Citrullination activity of Padi2 201a and individual point mutations in calcium binding and catalytic amino acids. Fold change of enzymatic activity is shown relative to wild-type Padi2 201a. Data represent 2 independent replicates and wild-type values are also represented in A. (C) Schematic of padi2 gene with exon 7 gRNA sequence highlighted for CRISPR/Cas9 mutagenesis. gRNA sequence in blue, PAM site in red. (D) Sequence alignment of wild-type and padi2 −/− 20 bp mutation in exon 7. MwoI restriction site for genotyping highlighted in pink, predicted early stop codon highlighted in red. (E) <t>RT-qPCR</t> of padi2 exon5/6 on individual larvae from a padi2 +/- incross. Data are from <t>three</t> pooled independent replicates with the means and SEM reported and a one-sample t test performed. (F) Representative western blot for zebrafish Padi2 and Actin from pooled larvae (representative of 4 experiments). (G) Citrullination activity of pooled zebrafish lysates expressed as relative light units (RLU). Data are from 3 independent replicates with the means and SEM reported and an ANOVA performed. (H) Citrullination activity of pooled embryo lysates during development. Fold change of enzymatic activity is shown as a ratio of calcium-treated to no calcium for each condition. Data are from 3 independent replicates. (I) Representative western blot for zebrafish Padi2 and Actin from pooled zebrafish through stages of development (representative of 3 and 2 experiments).
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    Spatial relationship between snRNA biogenesis hub and histone locus bodies. (A) Immunofluorescence imaging of classical Cajal Body (Coilin) and nuclear gem (SMN) markers in mouse ES cells and HEK293T cells. Top: Mouse ES cells do not contain visible Coilin foci for any of the three anti-Coilin antibodies tested. Bottom: HEK293T cells show visible Coilin foci. SMN foci, which are markers for nuclear Gemini of Cajal bodies (“gems”) are present in both mouse ES cells and HEK293T cells. (B) Z-section of mouse ES cell co-stained for SMN protein and scaRNAs (pooled scaRNA2 and scaRNA17 probes) within the nucleus (DAPI). Inset shows an example of scaRNA localization near SMN foci (arrow). (C) Z-section of mouse ES cell with RNA FISH staining for U7 and scaRNAs (pooled scaRNA2 and scaRNA17 probes) within the nucleus (DAPI). Inset shows an example of scaRNA localization near U7 (arrow). (D) RNA-RNA contact frequency between scaRNA2 and all RNAs. Top hits include annotated scaRNAs and identify two previously unannotated scaRNAs (see Supplemental Methods ). (E) Weighted DNA-DNA contacts for all SPRITE clusters (top) and for SPRITE clusters containing scaRNAs (bottom) occurring within a region on chromosome 11 with snRNA gene clusters. scaRNA occupancy is demarcated with solid red boxes. (F) Weighted DNA-DNA contacts for all SPRITE clusters (top) and for SPRITE clusters containing the U7 ncRNA (bottom) occurring within a region on chromosome 13 containing the two Hist1 gene clusters. U7 and Hist1 RNA occupancy is demarcated with teal boxes. ( G ) Weighted DNA-DNA contacts shown for SPRITE clusters containing both scaRNAs and snRNAs simultaneously. (H) Weighted DNA-DNA contacts for SPRITE clusters containing the scaRNAs on chromosome 13.

    Journal: bioRxiv

    Article Title: RNA promotes the formation of spatial compartments in the nucleus

    doi: 10.1101/2020.08.25.267435

    Figure Lengend Snippet: Spatial relationship between snRNA biogenesis hub and histone locus bodies. (A) Immunofluorescence imaging of classical Cajal Body (Coilin) and nuclear gem (SMN) markers in mouse ES cells and HEK293T cells. Top: Mouse ES cells do not contain visible Coilin foci for any of the three anti-Coilin antibodies tested. Bottom: HEK293T cells show visible Coilin foci. SMN foci, which are markers for nuclear Gemini of Cajal bodies (“gems”) are present in both mouse ES cells and HEK293T cells. (B) Z-section of mouse ES cell co-stained for SMN protein and scaRNAs (pooled scaRNA2 and scaRNA17 probes) within the nucleus (DAPI). Inset shows an example of scaRNA localization near SMN foci (arrow). (C) Z-section of mouse ES cell with RNA FISH staining for U7 and scaRNAs (pooled scaRNA2 and scaRNA17 probes) within the nucleus (DAPI). Inset shows an example of scaRNA localization near U7 (arrow). (D) RNA-RNA contact frequency between scaRNA2 and all RNAs. Top hits include annotated scaRNAs and identify two previously unannotated scaRNAs (see Supplemental Methods ). (E) Weighted DNA-DNA contacts for all SPRITE clusters (top) and for SPRITE clusters containing scaRNAs (bottom) occurring within a region on chromosome 11 with snRNA gene clusters. scaRNA occupancy is demarcated with solid red boxes. (F) Weighted DNA-DNA contacts for all SPRITE clusters (top) and for SPRITE clusters containing the U7 ncRNA (bottom) occurring within a region on chromosome 13 containing the two Hist1 gene clusters. U7 and Hist1 RNA occupancy is demarcated with teal boxes. ( G ) Weighted DNA-DNA contacts shown for SPRITE clusters containing both scaRNAs and snRNAs simultaneously. (H) Weighted DNA-DNA contacts for SPRITE clusters containing the scaRNAs on chromosome 13.

    Article Snippet: After RPM ligation, RNA was converted to cDNA using Superscript III at 42°C for 1 hour using the “RPM bottom” RT primer that contains an internal biotin to facilitate on-bead library construction (as above) and a 5’end sticky end to ligate tags during SPRITE.

    Techniques: Immunofluorescence, Imaging, Staining, Fluorescence In Situ Hybridization

    Detection of host biomarkers of inflammation. a Analysis of clinical stool samples. Four clinical stool samples were processed for total RNA and analyzed by our paper-based platform and RT-qPCR. Data represent mean values. Paper-based error bars represent s.d. from nine replicates (three biological replicates (NASBA reactions) × three technical replicates (paper-based reactions)). RT-qPCR error bars represent s.d. from six replicates (two biological replicates (RT reactions) × three technical replicates (qPCR reactions)). b Correlation of clinical sample results. Non-zero paper-based concentrations from a were compared to RT-qPCR determined values

    Journal: Nature Communications

    Article Title: A low-cost paper-based synthetic biology platform for analyzing gut microbiota and host biomarkers

    doi: 10.1038/s41467-018-05864-4

    Figure Lengend Snippet: Detection of host biomarkers of inflammation. a Analysis of clinical stool samples. Four clinical stool samples were processed for total RNA and analyzed by our paper-based platform and RT-qPCR. Data represent mean values. Paper-based error bars represent s.d. from nine replicates (three biological replicates (NASBA reactions) × three technical replicates (paper-based reactions)). RT-qPCR error bars represent s.d. from six replicates (two biological replicates (RT reactions) × three technical replicates (qPCR reactions)). b Correlation of clinical sample results. Non-zero paper-based concentrations from a were compared to RT-qPCR determined values

    Article Snippet: In vitro transcribed RNA diluted in 150 ng/µl yeast tRNA (ThermoFisher, AM7119) was used to generate standards for absolute quantitation based on calculations designed to incorporate 1 to 107 RNA copies per reverse transcription reaction. cDNA synthesis from stool samples was performed with 300 ng input RNA per reaction using Superscript III (ThermoFisher, 18080-400), according to the manufacturer’s protocol, using gene-specific primers (reverse qPCR primers as indicated in Supplementary Table ) at a final concentration of 2 µM and total volume of 20 µl.

    Techniques: Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    Paper-based detection of C. difficile infection. a Schematic of RNA-based CDI detection using a toehold switch sensor to detect toxin B mRNA. b Toxin B mRNA detection in stool samples. Two C. difficile strains (630 and VPI 10463) were grown in two different media (M1—TYG plus cysteine, M2—TY). Cells from each culture were spiked into 150 mg of a commercial stool sample and processed for total RNA. Toxin B mRNA was measured by our paper-based platform and RT-qPCR. Data represent mean values. Paper-based error bars represent s.d. from nine replicates (three biological replicates (NASBA reactions) × three technical replicates (paper-based reactions)). RT-qPCR error bars represent s.d. from six replicates (two biological replicates (RT reactions) × three technical replicates (qPCR reactions)). Toxin B DNA was confirmed in each sample using qPCR (Cq values shown in Supplementary Table 11 )

    Journal: Nature Communications

    Article Title: A low-cost paper-based synthetic biology platform for analyzing gut microbiota and host biomarkers

    doi: 10.1038/s41467-018-05864-4

    Figure Lengend Snippet: Paper-based detection of C. difficile infection. a Schematic of RNA-based CDI detection using a toehold switch sensor to detect toxin B mRNA. b Toxin B mRNA detection in stool samples. Two C. difficile strains (630 and VPI 10463) were grown in two different media (M1—TYG plus cysteine, M2—TY). Cells from each culture were spiked into 150 mg of a commercial stool sample and processed for total RNA. Toxin B mRNA was measured by our paper-based platform and RT-qPCR. Data represent mean values. Paper-based error bars represent s.d. from nine replicates (three biological replicates (NASBA reactions) × three technical replicates (paper-based reactions)). RT-qPCR error bars represent s.d. from six replicates (two biological replicates (RT reactions) × three technical replicates (qPCR reactions)). Toxin B DNA was confirmed in each sample using qPCR (Cq values shown in Supplementary Table 11 )

    Article Snippet: In vitro transcribed RNA diluted in 150 ng/µl yeast tRNA (ThermoFisher, AM7119) was used to generate standards for absolute quantitation based on calculations designed to incorporate 1 to 107 RNA copies per reverse transcription reaction. cDNA synthesis from stool samples was performed with 300 ng input RNA per reaction using Superscript III (ThermoFisher, 18080-400), according to the manufacturer’s protocol, using gene-specific primers (reverse qPCR primers as indicated in Supplementary Table ) at a final concentration of 2 µM and total volume of 20 µl.

    Techniques: Infection, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    16S rRNA sensors. a Schematic of toehold switch sensor function. b Best performing toehold switch sensors targeting the V3 hypervariable region of 16S rRNA for each species. Data represent mean GFP production rates from paper-based reactions with sensor alone and sensor plus 36-nucleotide trigger RNA (2 μM). Error bars represent high and low values from three technical replicates. c Schematic of NASBA-mediated RNA amplification. d Evaluation of NASBA primers. NASBA reactions were performed on 1 ng of total RNA for 90 min. Outputs from NASBA reactions were used to activate toehold switch sensors in paper-based reactions. Data represent mean values of three technical replicates. Error bars represent high and low values of the three replicates. e Orthogonality of 16S sensors. Each sensor was challenged with 2 μM of NASBA trigger RNAs from each species representing what would be amplified in a NASBA reaction. GFP production rates for an individual sensor were normalized to the production rate of the sensor plus its cognate trigger (100%). Data represent mean values of six replicates (two biological replicates × three technical replicates). Full data and s.d. are shown in Supplementary Figure 3

    Journal: Nature Communications

    Article Title: A low-cost paper-based synthetic biology platform for analyzing gut microbiota and host biomarkers

    doi: 10.1038/s41467-018-05864-4

    Figure Lengend Snippet: 16S rRNA sensors. a Schematic of toehold switch sensor function. b Best performing toehold switch sensors targeting the V3 hypervariable region of 16S rRNA for each species. Data represent mean GFP production rates from paper-based reactions with sensor alone and sensor plus 36-nucleotide trigger RNA (2 μM). Error bars represent high and low values from three technical replicates. c Schematic of NASBA-mediated RNA amplification. d Evaluation of NASBA primers. NASBA reactions were performed on 1 ng of total RNA for 90 min. Outputs from NASBA reactions were used to activate toehold switch sensors in paper-based reactions. Data represent mean values of three technical replicates. Error bars represent high and low values of the three replicates. e Orthogonality of 16S sensors. Each sensor was challenged with 2 μM of NASBA trigger RNAs from each species representing what would be amplified in a NASBA reaction. GFP production rates for an individual sensor were normalized to the production rate of the sensor plus its cognate trigger (100%). Data represent mean values of six replicates (two biological replicates × three technical replicates). Full data and s.d. are shown in Supplementary Figure 3

    Article Snippet: In vitro transcribed RNA diluted in 150 ng/µl yeast tRNA (ThermoFisher, AM7119) was used to generate standards for absolute quantitation based on calculations designed to incorporate 1 to 107 RNA copies per reverse transcription reaction. cDNA synthesis from stool samples was performed with 300 ng input RNA per reaction using Superscript III (ThermoFisher, 18080-400), according to the manufacturer’s protocol, using gene-specific primers (reverse qPCR primers as indicated in Supplementary Table ) at a final concentration of 2 µM and total volume of 20 µl.

    Techniques: Amplification

    Quantification of NASBA-mediated amplification using toehold switch sensors. a Run-to-run variation in mRNA standards amplified by NASBA and measured by toehold sensors. mRNA standards for the B. thetaiotaomicron species-specific sensor were run in NASBA reactions for 30 min. Outputs from NASBA reactions were used to activate toehold switch sensors in paper-based reactions. b Calibration curve for the B.t . species-specific mRNA. Values from each standard in the individual runs in a were normalized to the 300 fM standard for that specific run and averaged across runs. c Quantifying species-specific mRNAs in stool. E. coli or B. fragilis cells were spiked into 150 mg of a commercial stool sample and processed for total RNA. Species-specific mRNAs were quantified using our paper-based platform and RT-qPCR. d Analysis of clinical stool samples. Six clinical stool samples were processed for total RNA and analyzed by our paper-based platform and RT-qPCR. Data and s.d. are shown in Supplementary Figure 11 . e Correlation of clinical sample results. Non-zero paper-based concentrations from d were compared to RT-qPCR determined values. Data represent mean values. Paper-based error bars in a , c , and e represent s.d. from nine replicates (three biological replicates (NASBA reactions) × three technical replicates (paper-based reactions)). RT-qPCR error bars in c and e represent s.d. from six replicates (two biological replicates (RT reactions) × three technical replicates (qPCR reactions))

    Journal: Nature Communications

    Article Title: A low-cost paper-based synthetic biology platform for analyzing gut microbiota and host biomarkers

    doi: 10.1038/s41467-018-05864-4

    Figure Lengend Snippet: Quantification of NASBA-mediated amplification using toehold switch sensors. a Run-to-run variation in mRNA standards amplified by NASBA and measured by toehold sensors. mRNA standards for the B. thetaiotaomicron species-specific sensor were run in NASBA reactions for 30 min. Outputs from NASBA reactions were used to activate toehold switch sensors in paper-based reactions. b Calibration curve for the B.t . species-specific mRNA. Values from each standard in the individual runs in a were normalized to the 300 fM standard for that specific run and averaged across runs. c Quantifying species-specific mRNAs in stool. E. coli or B. fragilis cells were spiked into 150 mg of a commercial stool sample and processed for total RNA. Species-specific mRNAs were quantified using our paper-based platform and RT-qPCR. d Analysis of clinical stool samples. Six clinical stool samples were processed for total RNA and analyzed by our paper-based platform and RT-qPCR. Data and s.d. are shown in Supplementary Figure 11 . e Correlation of clinical sample results. Non-zero paper-based concentrations from d were compared to RT-qPCR determined values. Data represent mean values. Paper-based error bars in a , c , and e represent s.d. from nine replicates (three biological replicates (NASBA reactions) × three technical replicates (paper-based reactions)). RT-qPCR error bars in c and e represent s.d. from six replicates (two biological replicates (RT reactions) × three technical replicates (qPCR reactions))

    Article Snippet: In vitro transcribed RNA diluted in 150 ng/µl yeast tRNA (ThermoFisher, AM7119) was used to generate standards for absolute quantitation based on calculations designed to incorporate 1 to 107 RNA copies per reverse transcription reaction. cDNA synthesis from stool samples was performed with 300 ng input RNA per reaction using Superscript III (ThermoFisher, 18080-400), according to the manufacturer’s protocol, using gene-specific primers (reverse qPCR primers as indicated in Supplementary Table ) at a final concentration of 2 µM and total volume of 20 µl.

    Techniques: Amplification, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    Species-specific mRNA sensors. a Bioinformatic pipeline for identifying species-specific mRNAs. b Best performing NASBA primers and species-specific mRNA sensors for each species. NASBA reactions were performed on 10 ng of total RNA for 90 min. Outputs from NASBA reactions were used to activate toehold switch sensors in paper-based reactions. Data represent mean values of three technical replicates. Error bars represent high and low values of the three replicates. c Orthogonality of species-specific sensors. Each sensor was challenged with 2 μM of trigger RNAs from each species representing what would be amplified in a NASBA reaction. GFP production rates for an individual sensor were normalized to the production rate of the sensor plus its cognate trigger (100%). Data represent mean values of six replicates (two biological replicates × three technical replicates). Full data and s.d. are shown in Supplementary Figure 6 . d Orthogonality of NASBA primer sets. NASBA reactions were performed on 10 ng of total RNA for 90 min. Data represent mean ± s.d. of six replicates (two biological replicates (NASBA reactions) × three technical replicates (paper-based reactions))

    Journal: Nature Communications

    Article Title: A low-cost paper-based synthetic biology platform for analyzing gut microbiota and host biomarkers

    doi: 10.1038/s41467-018-05864-4

    Figure Lengend Snippet: Species-specific mRNA sensors. a Bioinformatic pipeline for identifying species-specific mRNAs. b Best performing NASBA primers and species-specific mRNA sensors for each species. NASBA reactions were performed on 10 ng of total RNA for 90 min. Outputs from NASBA reactions were used to activate toehold switch sensors in paper-based reactions. Data represent mean values of three technical replicates. Error bars represent high and low values of the three replicates. c Orthogonality of species-specific sensors. Each sensor was challenged with 2 μM of trigger RNAs from each species representing what would be amplified in a NASBA reaction. GFP production rates for an individual sensor were normalized to the production rate of the sensor plus its cognate trigger (100%). Data represent mean values of six replicates (two biological replicates × three technical replicates). Full data and s.d. are shown in Supplementary Figure 6 . d Orthogonality of NASBA primer sets. NASBA reactions were performed on 10 ng of total RNA for 90 min. Data represent mean ± s.d. of six replicates (two biological replicates (NASBA reactions) × three technical replicates (paper-based reactions))

    Article Snippet: In vitro transcribed RNA diluted in 150 ng/µl yeast tRNA (ThermoFisher, AM7119) was used to generate standards for absolute quantitation based on calculations designed to incorporate 1 to 107 RNA copies per reverse transcription reaction. cDNA synthesis from stool samples was performed with 300 ng input RNA per reaction using Superscript III (ThermoFisher, 18080-400), according to the manufacturer’s protocol, using gene-specific primers (reverse qPCR primers as indicated in Supplementary Table ) at a final concentration of 2 µM and total volume of 20 µl.

    Techniques: Amplification

    Characterization of zebrafish Padi2. (A) Citrullination activity of bacterially expressed zebrafish Padi2 201a and 202 splice variants in total lysates with and without calcium. Absorbance of light was measured and expressed as mean (± SEM) relative light units (RLU), normalized for protein level. Data represent 3 independent replicates. (B) Citrullination activity of Padi2 201a and individual point mutations in calcium binding and catalytic amino acids. Fold change of enzymatic activity is shown relative to wild-type Padi2 201a. Data represent 2 independent replicates and wild-type values are also represented in A. (C) Schematic of padi2 gene with exon 7 gRNA sequence highlighted for CRISPR/Cas9 mutagenesis. gRNA sequence in blue, PAM site in red. (D) Sequence alignment of wild-type and padi2 −/− 20 bp mutation in exon 7. MwoI restriction site for genotyping highlighted in pink, predicted early stop codon highlighted in red. (E) RT-qPCR of padi2 exon5/6 on individual larvae from a padi2 +/- incross. Data are from three pooled independent replicates with the means and SEM reported and a one-sample t test performed. (F) Representative western blot for zebrafish Padi2 and Actin from pooled larvae (representative of 4 experiments). (G) Citrullination activity of pooled zebrafish lysates expressed as relative light units (RLU). Data are from 3 independent replicates with the means and SEM reported and an ANOVA performed. (H) Citrullination activity of pooled embryo lysates during development. Fold change of enzymatic activity is shown as a ratio of calcium-treated to no calcium for each condition. Data are from 3 independent replicates. (I) Representative western blot for zebrafish Padi2 and Actin from pooled zebrafish through stages of development (representative of 3 and 2 experiments).

    Journal: bioRxiv

    Article Title: Citrullination regulates wound responses and tissue regeneration in zebrafish

    doi: 10.1101/2019.12.27.889378

    Figure Lengend Snippet: Characterization of zebrafish Padi2. (A) Citrullination activity of bacterially expressed zebrafish Padi2 201a and 202 splice variants in total lysates with and without calcium. Absorbance of light was measured and expressed as mean (± SEM) relative light units (RLU), normalized for protein level. Data represent 3 independent replicates. (B) Citrullination activity of Padi2 201a and individual point mutations in calcium binding and catalytic amino acids. Fold change of enzymatic activity is shown relative to wild-type Padi2 201a. Data represent 2 independent replicates and wild-type values are also represented in A. (C) Schematic of padi2 gene with exon 7 gRNA sequence highlighted for CRISPR/Cas9 mutagenesis. gRNA sequence in blue, PAM site in red. (D) Sequence alignment of wild-type and padi2 −/− 20 bp mutation in exon 7. MwoI restriction site for genotyping highlighted in pink, predicted early stop codon highlighted in red. (E) RT-qPCR of padi2 exon5/6 on individual larvae from a padi2 +/- incross. Data are from three pooled independent replicates with the means and SEM reported and a one-sample t test performed. (F) Representative western blot for zebrafish Padi2 and Actin from pooled larvae (representative of 4 experiments). (G) Citrullination activity of pooled zebrafish lysates expressed as relative light units (RLU). Data are from 3 independent replicates with the means and SEM reported and an ANOVA performed. (H) Citrullination activity of pooled embryo lysates during development. Fold change of enzymatic activity is shown as a ratio of calcium-treated to no calcium for each condition. Data are from 3 independent replicates. (I) Representative western blot for zebrafish Padi2 and Actin from pooled zebrafish through stages of development (representative of 3 and 2 experiments).

    Article Snippet: Embryos were genotyped using GoTaq (Promega) as described above and 2-3 embryos of each genotype were used for cDNA production using Superscript III First Strand Synthesis System with Oligo(dT) (Thermo Fisher Scientific). qPCR was performed using FastStart Essential Green Master (Roche) and a LightCycler96 (Rocher).

    Techniques: Activity Assay, Binding Assay, Sequencing, CRISPR, Mutagenesis, Quantitative RT-PCR, Western Blot

    Inhibition of mTOR-related genes and mTORC1 downstream effectors by RAD001. Notes: qPCR detection of mTOR-related genes, including ( A ) mTOR complexes, ( B ) mTORC1-positive regulation, and ( C ) mTORC2-positive regulation in T24 cells treated with 1 or 5 µM RAD001. Cells were seeded 24 hours before RAD001 treatment. Total RNA samples were extracted at 24 hours posttreatment, reverse transcribed, and subjected to the detection of genes involved in mTOR signaling. The values are shown as the mean ± SD of three independent experiments; * P

    Journal: Drug Design, Development and Therapy

    Article Title: Autophagy inhibition enhances RAD001-induced cytotoxicity in human bladder cancer cells

    doi: 10.2147/DDDT.S95900

    Figure Lengend Snippet: Inhibition of mTOR-related genes and mTORC1 downstream effectors by RAD001. Notes: qPCR detection of mTOR-related genes, including ( A ) mTOR complexes, ( B ) mTORC1-positive regulation, and ( C ) mTORC2-positive regulation in T24 cells treated with 1 or 5 µM RAD001. Cells were seeded 24 hours before RAD001 treatment. Total RNA samples were extracted at 24 hours posttreatment, reverse transcribed, and subjected to the detection of genes involved in mTOR signaling. The values are shown as the mean ± SD of three independent experiments; * P

    Article Snippet: Two micrograms of total RNA was subjected to reverse transcription using SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific). qPCR was performed as previously described.

    Techniques: Inhibition, Real-time Polymerase Chain Reaction