5x first strand buffer  (Thermo Fisher)


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    Structured Review

    Thermo Fisher 5x first strand buffer
    5x First Strand Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/5x first strand buffer/product/Thermo Fisher
    Average 99 stars, based on 22 article reviews
    Price from $9.99 to $1999.99
    5x first strand buffer - by Bioz Stars, 2020-02
    99/100 stars

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    Related Articles

    Centrifugation:

    Article Title: Protective Effect of Heme Oxygenase-1 on High Glucose-Induced Pancreatic ?-Cell Injury
    Article Snippet: RNA was extracted with BCP (1-bromo-3-chloropropane; Molecular Research Center, Cincinnati, OH, USA) followed by centrifugation to separate the sample into aqueous and organic phases. .. After quantification by spectrophotometer (UV/VIS Spectrophotometer ND-1000; Nanodrop, Silmington, DE, USA), 2 µg of RNA was subjected to 85℃ for 3 minutes prior to the addition of 5X First-Strand buffer (Invitrogen), 0.1 M dTT (Invitrogen), 50 µg/µL Random hexamer (Invitrogen), 10 mM dNTP (Invitrogen), RNaseOUT™ (40 U/µL, Invitorogen), and SuperScrip™ II Reverse Transcriptase (200 U/µL, Invitrogen).

    Amplification:

    Article Title: Optimized amplification and single-cell analysis identify GnRH-mediated activation of Rap1b in primary rat gonadotropes
    Article Snippet: Paragraph title: C. RNA amplification using two-cycle T7 RNA polymerase ... Briefly, 100 ng total RNA was incubated in a 10-μl volume containing 2 μl T7-Oligo(dT) primer, 2 μl 5X first strand buffer, 1 μl 0.1 M DTT, 0.5 μl RNase inhibitor, 0.5 μl 10 mM dNTP, and 1 μl SuperScript II (Invitrogen) at 42 ºC for 1 h. Second strand cDNA synthesis was carried out by incubating 10 μl first strand cDNA products with 4.8 μl water, 4 μl 17.5 mM MgCl2 , 0.4 μl 10 mM dNTP, 0.6 μl E. coli DNA Polymerase I, and 0.2 μl RNase H at 16ºC for 2 h. In vitro transcription was performed using the MegaScript T7 Kit (Ambion).

    Synthesized:

    Article Title: Identification of Appropriate Reference Genes for qRT-PCR Analysis of Heat-Stressed Mammary Epithelial Cells in Riverine Buffaloes (Bubalus bubalis)
    Article Snippet: All the extracted RNA samples were stored at −80°C and utilized within one month. cDNA was synthesized using 100 ng RNA, 1 μ L dT12–18 (Invitrogen Corp. CA), 1 μ L 10 mmol/L dNTP mix (Invitrogen Corp., CA), 1 μ L random primers (Invitrogen Corp., CA), and 10 μ L DNase-/RNase-free water. .. The mixture was incubated at 65°C for 5 min and kept on ice for 3 min. A total of 6 μ L of master mix composed of 4.5 μ L 5X First-Strand Buffer, 1 μ L 0.1 M DTT, 0.25 μ L (50 U) of SuperScript III RT (Invitrogen Corp., CA), and 0.25 μ L of RNase inhibitor (10 U, Promega, WI) was added.

    Quantitative RT-PCR:

    Article Title: Pair-wise comparison analysis of differential expression of mRNAs in early and advanced stage primary colorectal adenocarcinomas
    Article Snippet: Paragraph title: RT-qPCR analysis ... The reaction mixture was then incubated at 80°C for 3 min, followed by chilling on ice for another 2 min. Next, 4 µL of 5X first-strand buffer (Invitrogen), 5 µL of 2 mM dNTP (Fermentas), 0.5 µL of 40 U/µL RNaseOUT recombinant RNase inhibitor (Invitrogen) and 1 µL of 200 U/µL M-MLV reverse transcriptase (Invitrogen) were added to the mixture.

    Article Title: Individual co-variation between viral RNA load and gene expression reveals novel host factors during early dengue virus infection of the Aedes aegypti midgut
    Article Snippet: Paragraph title: Gene expression quantification by RT-qPCR ... After incubation at 65°C for 10 min, samples were chilled on ice for 5 min. For each reaction, 4 μL of 5X First-Strand buffer, 1 μL of 0.1 mM Dithiothreitol, 40 units of RNase-OUT and 100 units of MML-V reverse transcriptase (Life Technologies) were added to a final volume of 20 μL.

    Article Title: Transcription activated p73-modulated cyclin D1 expression leads to doxorubicin resistance in gastric cancer
    Article Snippet: Paragraph title: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) ... For cDNA synthesis, 1 µg of RNA was mixed with Oligo(dT) primers, dNTP mix, 5X first-strand buffer, Ditiotreitol, RNAseOUT and SuperScript II RT, all according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.).

    Real-time Polymerase Chain Reaction:

    Article Title: Withania somnifera as a potential candidate to ameliorate high fat diet-induced anxiety and neuroinflammation
    Article Snippet: Paragraph title: mRNA expression analysis by quantitative real-time PCR ... 4 µL of 5X first-strand buffer, 2 μL of 0.1 M DTT, and 1 μL of recombinant ribonuclease inhibitor (40 units/μL) (Life Technologies, Carlsbad, CA, USA) were added and incubated at 37 °C for 2 min. Then, 1 μL (200 units) of M-MLV reverse transcriptase (Life Technologies, Carlsbad, CA, USA) was added for 20 μL reaction volume.

    Article Title: Individual co-variation between viral RNA load and gene expression reveals novel host factors during early dengue virus infection of the Aedes aegypti midgut
    Article Snippet: After incubation at 65°C for 10 min, samples were chilled on ice for 5 min. For each reaction, 4 μL of 5X First-Strand buffer, 1 μL of 0.1 mM Dithiothreitol, 40 units of RNase-OUT and 100 units of MML-V reverse transcriptase (Life Technologies) were added to a final volume of 20 μL. .. Gene expression was assayed by relative quantitative PCR (qPCR) using a LightCycler96 machine (Roche).

    Article Title: Prediction of relapse and prognosis by expression levels of long noncoding RNA PEG10 in glioma patients
    Article Snippet: Paragraph title: RNA extraction and reverse transcription quantitative polymerase chain reaction assay ... Synthesis of cDNA was performed using 2 μg of total RNA, lncRNA PEG10 reverse transcription primer (Invitrogen; Thermo Fisher Scientific, Inc.), 5X first-strand buffer (Thermo Fisher Scientific, Inc.), 0.1 mol/L DTT (Thermo Fisher Scientific, Inc.), a dNTP mixture (Takara, Bio, Inc., Otsu, Japan), Moloney Murine Leukemia Virus reverse transcriptase (Thermo Fisher Scientific, Inc.) and recombinant RNasin RNase inhibitor (Promega Corporation, Madison, WI).

    Article Title: In Situ Mechanotransduction via Vinculin Regulates Stem Cell Differentiation
    Article Snippet: .. Samples containing 2 µg of cDNA were added to a mastermix containing of 10 mM dNTP (Roche), 100 mM DTT (Invitrogen), 5X First Strand Buffer (Invitrogen), 50 mM Random Hexamers (Qiagen), 200U/µL Reverse Transcriptase (Invitrogen), and DEPC H2 O. qPCR was then performed using an 7900HT Fast Real-Time PCR machine (Applied Biosystems). .. Primers coding for Pax3, Pax7, MyoD, Mrf4, Myf5, GAPDH, and the vinculin constructs H, T and FL are listed in .

    Article Title: Transcription activated p73-modulated cyclin D1 expression leads to doxorubicin resistance in gastric cancer
    Article Snippet: For cDNA synthesis, 1 µg of RNA was mixed with Oligo(dT) primers, dNTP mix, 5X first-strand buffer, Ditiotreitol, RNAseOUT and SuperScript II RT, all according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.). .. The thermocycling conditions were 10 min of initial denaturation at 95°C, followed by 45 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 30 sec and extension at 72°C for 30 sec with a single fluorescence acquisition step at the end of extension, followed by final cooling at 40°C for 30 sec. PCR analysis was then conducted using the Mx3000P qPCR system (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, USA).

    Microarray:

    Article Title: Mutual independence of alkaline‐ and calcium‐mediated signalling in Aspergillus fumigatus refutes the existence of a conserved druggable signalling nexus
    Article Snippet: Paragraph title: DNA microarray methods ... The tubes were briefly chilled to 4°C for 5 min and mixed with 5 μl of 5X first‐strand buffer (Invitrogen), 2 μl of DTT (100 mM, Invitrogen), 2 μl of 25 mM dNTP, 2 μl of SuperScript RT (Clontech) and 1 μl of Cy3‐dUTP (25 nmol) or Cy5‐dUTP (25 nmol).

    Random Hexamer Labeling:

    Article Title: Protective Effect of Heme Oxygenase-1 on High Glucose-Induced Pancreatic ?-Cell Injury
    Article Snippet: .. After quantification by spectrophotometer (UV/VIS Spectrophotometer ND-1000; Nanodrop, Silmington, DE, USA), 2 µg of RNA was subjected to 85℃ for 3 minutes prior to the addition of 5X First-Strand buffer (Invitrogen), 0.1 M dTT (Invitrogen), 50 µg/µL Random hexamer (Invitrogen), 10 mM dNTP (Invitrogen), RNaseOUT™ (40 U/µL, Invitorogen), and SuperScrip™ II Reverse Transcriptase (200 U/µL, Invitrogen). ..

    Article Title: Human and entomologic investigations of chikungunya outbreak in Mandera, Northeastern Kenya, 2016
    Article Snippet: In a 200 μl PCR tube, 10 μl of the extracted sample RNA was mixed with 2 μl of 50 ng/μl random hexamer primer and 1 μl of 10 mM deoxynucleotide solution (dNTPs), and incubated in a thermocycler for 5 minutes at 65°C and immediately chilled for 1 minute at 4°C. .. The following components were then added to the PCR tube: 4 μl of 5X First Strand Buffer (Invitrogen), 1 μl of 0.1 M DTT, 1 μl of RNAse OUT(40 U/μl) and 1 μl of Superscript III Reverse transcriptase (200 U/μl).

    Expressing:

    Article Title: Pair-wise comparison analysis of differential expression of mRNAs in early and advanced stage primary colorectal adenocarcinomas
    Article Snippet: The reaction mixture was then incubated at 80°C for 3 min, followed by chilling on ice for another 2 min. Next, 4 µL of 5X first-strand buffer (Invitrogen), 5 µL of 2 mM dNTP (Fermentas), 0.5 µL of 40 U/µL RNaseOUT recombinant RNase inhibitor (Invitrogen) and 1 µL of 200 U/µL M-MLV reverse transcriptase (Invitrogen) were added to the mixture. .. ΔΔCT analysis The relative expression of identified DEGs in all paired colorectal tumours and control samples was determined via ΔΔCT method.

    Article Title: Withania somnifera as a potential candidate to ameliorate high fat diet-induced anxiety and neuroinflammation
    Article Snippet: Paragraph title: mRNA expression analysis by quantitative real-time PCR ... 4 µL of 5X first-strand buffer, 2 μL of 0.1 M DTT, and 1 μL of recombinant ribonuclease inhibitor (40 units/μL) (Life Technologies, Carlsbad, CA, USA) were added and incubated at 37 °C for 2 min. Then, 1 μL (200 units) of M-MLV reverse transcriptase (Life Technologies, Carlsbad, CA, USA) was added for 20 μL reaction volume.

    Article Title: Individual co-variation between viral RNA load and gene expression reveals novel host factors during early dengue virus infection of the Aedes aegypti midgut
    Article Snippet: Paragraph title: Gene expression quantification by RT-qPCR ... After incubation at 65°C for 10 min, samples were chilled on ice for 5 min. For each reaction, 4 μL of 5X First-Strand buffer, 1 μL of 0.1 mM Dithiothreitol, 40 units of RNase-OUT and 100 units of MML-V reverse transcriptase (Life Technologies) were added to a final volume of 20 μL.

    Modification:

    Article Title: Transcriptome of the Female Synganglion of the Black-Legged Tick Ixodes scapularis (Acari: Ixodidae) with Comparison between Illumina and 454 Systems
    Article Snippet: Purified mRNA was ethanol precipitated, rehydrated in 2 µl of RNase-free water and combined with 10 pmol of modified 3′ reverse transcription primer (5′-ATTCTAGAGACCGAGGCGGCCGACATGT(4) GT(9) CT(10) VN-3′) and 10 pmol SMART IV oligo ( 5′-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3′ ) . .. The resulting 4 µl were incubated at 72°C for 2 min and then combined with the following reagents on ice: 1 µl RNase Out (40 U/µl, 2 µl 5X first strand buffer, 1 µl 20 mM DTT, 1 µl dNTP mix (10 mM each) and 1 µl Superscript II reverse transcriptase) (Invitrogen, Carlsbad, CA).

    Hybridization:

    Article Title: Mutual independence of alkaline‐ and calcium‐mediated signalling in Aspergillus fumigatus refutes the existence of a conserved druggable signalling nexus
    Article Snippet: DNA microarray methods We used the A . fumigatus oligonucleotide slides version 3 for microarray hybridizations (for details see http://pfgrc.tigr.org/slide_html/microarray_descriptions.shtml ) using a common reference hybridization scheme whereby temporal alterations (5, 15 30, 45 and 60 min) in transcript abundance relative to a common time zero (unshifted) sample, were quantified, including technical replications for each time‐point and condition via a dye swapped cohybridisation. .. The tubes were briefly chilled to 4°C for 5 min and mixed with 5 μl of 5X first‐strand buffer (Invitrogen), 2 μl of DTT (100 mM, Invitrogen), 2 μl of 25 mM dNTP, 2 μl of SuperScript RT (Clontech) and 1 μl of Cy3‐dUTP (25 nmol) or Cy5‐dUTP (25 nmol).

    High Performance Liquid Chromatography:

    Article Title: Strand-specific RNA-seq applied to malaria samples
    Article Snippet: .. Parasite cultures grown in complete RPMI medium at 5 % hematocrit ( see ) TRIzol® LS Reagent (Invitrogen™) pre-warmed at 37°C Chloroform Isopropanol pre-chilled on ice Nuclease-free non-DEPC water DNAse I RNAse-free (Ambion®) Deionized formamide Formaldehyde 37 % 10X MOPS EDTA buffer pH 7.0 Glycerol 50 % Bromophenol blue powder Ethidium bromide 20 mg/mL GenElute™ mRNA Miniprep Kit (Sigma-Aldrich) 5X RNA storage solution (Ambion) HPLC-purified random hexamers and Anchored OligodT(20) SuperScript® VILO™ cDNA synthesis kit (Invitrogen™) DNA Clean & Concentrator™ (Zymo Research) 5X First Strand Buffer (Invitrogen™): 250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2 5X Second Strand Buffer (Invitrogen™): 100 mM Tris-HCl (pH 6.9), 450 mM KCl, 23 mM MgCl2 , 0.75 mM β-NAD+ , 50 mM (NH4 )2 SO4 Set of dATP, dGTP, dCTP, and dUTP E. coli DNA Polymerase I 10 U/μL (Invitrogen™) E. coli DNA Ligase 10 U/μL (Invitrogen™) E. coli DNA RNase H 2 U/μL (Invitrogen™) 0.1 M DTT dsDNA Shearase™ (Zymo Research) Encore™ NGS Library System I (NuGEN®) Same-day 70 % ethanol in nuclease-free water USER™ Enzyme (New England Biolabs®) 1X TE buffer pH 8.0 ..

    Protease Inhibitor:

    Article Title: In situ RT-PCR Optimized for Electron Microscopy Allows Description of Subcellular Morphology of Target mRNA-Expressing Cells in the Brain
    Article Snippet: .. Afterwards, we added 5X first strand buffer (#Y02321, Invitrogen), 0.1 M DTT (#Y00122, Invitrogen) and RNase OUT, a protease inhibitor (40 U/μL) (#10777-019, Invitrogen), and reaction temperature was set at 25°C for 2 min. ..

    Generated:

    Article Title: Mutual independence of alkaline‐ and calcium‐mediated signalling in Aspergillus fumigatus refutes the existence of a conserved druggable signalling nexus
    Article Snippet: Thus, a total of 20 cohybridisation datasets were generated for the analysis. .. The tubes were briefly chilled to 4°C for 5 min and mixed with 5 μl of 5X first‐strand buffer (Invitrogen), 2 μl of DTT (100 mM, Invitrogen), 2 μl of 25 mM dNTP, 2 μl of SuperScript RT (Clontech) and 1 μl of Cy3‐dUTP (25 nmol) or Cy5‐dUTP (25 nmol).

    Article Title: Optimized amplification and single-cell analysis identify GnRH-mediated activation of Rap1b in primary rat gonadotropes
    Article Snippet: Briefly, 100 ng total RNA was incubated in a 10-μl volume containing 2 μl T7-Oligo(dT) primer, 2 μl 5X first strand buffer, 1 μl 0.1 M DTT, 0.5 μl RNase inhibitor, 0.5 μl 10 mM dNTP, and 1 μl SuperScript II (Invitrogen) at 42 ºC for 1 h. Second strand cDNA synthesis was carried out by incubating 10 μl first strand cDNA products with 4.8 μl water, 4 μl 17.5 mM MgCl2 , 0.4 μl 10 mM dNTP, 0.6 μl E. coli DNA Polymerase I, and 0.2 μl RNase H at 16ºC for 2 h. In vitro transcription was performed using the MegaScript T7 Kit (Ambion). .. Second strand cDNA products (20 μl) were incubated with 5 μl 10X Reaction buffer, 5 μl each of ATP, CTP, UTP and GTP solutions, and 5 μl Enzyme Mix at 37ºC for 16 h. The cRNA generated was purified using an RNeasy kit (Qiagen) and the manufacturer’s RNA cleanup protocol. cRNA was subsequently subjected to a second cycle of T7 amplification.

    Polymerase Chain Reaction:

    Article Title: Protective Effect of Heme Oxygenase-1 on High Glucose-Induced Pancreatic ?-Cell Injury
    Article Snippet: After quantification by spectrophotometer (UV/VIS Spectrophotometer ND-1000; Nanodrop, Silmington, DE, USA), 2 µg of RNA was subjected to 85℃ for 3 minutes prior to the addition of 5X First-Strand buffer (Invitrogen), 0.1 M dTT (Invitrogen), 50 µg/µL Random hexamer (Invitrogen), 10 mM dNTP (Invitrogen), RNaseOUT™ (40 U/µL, Invitorogen), and SuperScrip™ II Reverse Transcriptase (200 U/µL, Invitrogen). .. The PCR reaction that was designed to amplify the cDNA fragments (Promega, Madison, WI, USA) contained 1 µL of each forward and reverse primer (10 pM), 6 µL of DEPC water, and 2 µL of cDNA template.

    Article Title: Individual co-variation between viral RNA load and gene expression reveals novel host factors during early dengue virus infection of the Aedes aegypti midgut
    Article Snippet: After incubation at 65°C for 10 min, samples were chilled on ice for 5 min. For each reaction, 4 μL of 5X First-Strand buffer, 1 μL of 0.1 mM Dithiothreitol, 40 units of RNase-OUT and 100 units of MML-V reverse transcriptase (Life Technologies) were added to a final volume of 20 μL. .. The qPCR mix contained 200 nM of each primer, 10 μL of 2X SYBR-green I Master Mix (Roche) and PCR grade water to 18 μL, with 2 μL of cDNA template to a final volume of 20 μL.

    Article Title: Human and entomologic investigations of chikungunya outbreak in Mandera, Northeastern Kenya, 2016
    Article Snippet: .. The following components were then added to the PCR tube: 4 μl of 5X First Strand Buffer (Invitrogen), 1 μl of 0.1 M DTT, 1 μl of RNAse OUT(40 U/μl) and 1 μl of Superscript III Reverse transcriptase (200 U/μl). .. The mixture was then incubated in a thermocycler for 5 minutes at 25°C, 50 minutes at 50°C and 15 minutes at 70°C.

    Article Title: Prediction of relapse and prognosis by expression levels of long noncoding RNA PEG10 in glioma patients
    Article Snippet: Synthesis of cDNA was performed using 2 μg of total RNA, lncRNA PEG10 reverse transcription primer (Invitrogen; Thermo Fisher Scientific, Inc.), 5X first-strand buffer (Thermo Fisher Scientific, Inc.), 0.1 mol/L DTT (Thermo Fisher Scientific, Inc.), a dNTP mixture (Takara, Bio, Inc., Otsu, Japan), Moloney Murine Leukemia Virus reverse transcriptase (Thermo Fisher Scientific, Inc.) and recombinant RNasin RNase inhibitor (Promega Corporation, Madison, WI). .. After first-strand synthesis, quantitative PCR (qPCR) was performed by a 7900 HT Fast RealTime PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

    Article Title: Transcriptome of the Female Synganglion of the Black-Legged Tick Ixodes scapularis (Acari: Ixodidae) with Comparison between Illumina and 454 Systems
    Article Snippet: The resulting 4 µl were incubated at 72°C for 2 min and then combined with the following reagents on ice: 1 µl RNase Out (40 U/µl, 2 µl 5X first strand buffer, 1 µl 20 mM DTT, 1 µl dNTP mix (10 mM each) and 1 µl Superscript II reverse transcriptase) (Invitrogen, Carlsbad, CA). .. To synthesize second strand cDNA, 5 µl of first-strand cDNA was mixed with 10 pmol of modified 3′ PCR primer (5′-ATTCTAGAGGCCGAGGCGGCCGACATGT(4) GTCT(4) GTTCTGT(3) CT(4) VN-3′) , 10 pmol of 5′ PCR primer ( 5′-AAGCAGTGGTATCAACGCAGAGT-3′ ) , 5 µl 10X reaction buffer, 1 µl dNTP mix, 2 µl MgSO4 , 0.4 µl Platinum HiFi Taq Polymerase and 34.6 µl H2 O (Invitrogen).

    Article Title: Transcription activated p73-modulated cyclin D1 expression leads to doxorubicin resistance in gastric cancer
    Article Snippet: Paragraph title: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) ... For cDNA synthesis, 1 µg of RNA was mixed with Oligo(dT) primers, dNTP mix, 5X first-strand buffer, Ditiotreitol, RNAseOUT and SuperScript II RT, all according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.).

    Article Title: Isolation of Sphere-Forming Stem Cells from the Mouse Inner Ear
    Article Snippet: SuperScript™ II reverse transcriptase, including 5X first-strand buffer and 0.1 M DTT solution (2,000 units, cat. no. 18064-022, Invitrogen). .. Thermal cycler (e.g., GeneAmp PCR system 9700, Applied Biosystems, or similar apparatus).

    Sequencing:

    Article Title: Transcriptome of the Female Synganglion of the Black-Legged Tick Ixodes scapularis (Acari: Ixodidae) with Comparison between Illumina and 454 Systems
    Article Snippet: The samples were evaluated using the Bioanalyzer 2100 as described above to insure that they met the minimum requirements for 454 sequencing. .. The resulting 4 µl were incubated at 72°C for 2 min and then combined with the following reagents on ice: 1 µl RNase Out (40 U/µl, 2 µl 5X first strand buffer, 1 µl 20 mM DTT, 1 µl dNTP mix (10 mM each) and 1 µl Superscript II reverse transcriptase) (Invitrogen, Carlsbad, CA).

    Recombinant:

    Article Title: Pair-wise comparison analysis of differential expression of mRNAs in early and advanced stage primary colorectal adenocarcinomas
    Article Snippet: .. The reaction mixture was then incubated at 80°C for 3 min, followed by chilling on ice for another 2 min. Next, 4 µL of 5X first-strand buffer (Invitrogen), 5 µL of 2 mM dNTP (Fermentas), 0.5 µL of 40 U/µL RNaseOUT recombinant RNase inhibitor (Invitrogen) and 1 µL of 200 U/µL M-MLV reverse transcriptase (Invitrogen) were added to the mixture. ..

    Article Title: Withania somnifera as a potential candidate to ameliorate high fat diet-induced anxiety and neuroinflammation
    Article Snippet: .. 4 µL of 5X first-strand buffer, 2 μL of 0.1 M DTT, and 1 μL of recombinant ribonuclease inhibitor (40 units/μL) (Life Technologies, Carlsbad, CA, USA) were added and incubated at 37 °C for 2 min. Then, 1 μL (200 units) of M-MLV reverse transcriptase (Life Technologies, Carlsbad, CA, USA) was added for 20 μL reaction volume. .. 50 µg of cDNA was then used to amplify gene of interest using Step One Plus Real Time PCR system (Applied Biosystems, Foster City, CA, USA).

    Article Title: Prediction of relapse and prognosis by expression levels of long noncoding RNA PEG10 in glioma patients
    Article Snippet: .. Synthesis of cDNA was performed using 2 μg of total RNA, lncRNA PEG10 reverse transcription primer (Invitrogen; Thermo Fisher Scientific, Inc.), 5X first-strand buffer (Thermo Fisher Scientific, Inc.), 0.1 mol/L DTT (Thermo Fisher Scientific, Inc.), a dNTP mixture (Takara, Bio, Inc., Otsu, Japan), Moloney Murine Leukemia Virus reverse transcriptase (Thermo Fisher Scientific, Inc.) and recombinant RNasin RNase inhibitor (Promega Corporation, Madison, WI). .. After first-strand synthesis, quantitative PCR (qPCR) was performed by a 7900 HT Fast RealTime PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

    Fluorescence:

    Article Title: Transcription activated p73-modulated cyclin D1 expression leads to doxorubicin resistance in gastric cancer
    Article Snippet: For cDNA synthesis, 1 µg of RNA was mixed with Oligo(dT) primers, dNTP mix, 5X first-strand buffer, Ditiotreitol, RNAseOUT and SuperScript II RT, all according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.). .. The thermocycling conditions were 10 min of initial denaturation at 95°C, followed by 45 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 30 sec and extension at 72°C for 30 sec with a single fluorescence acquisition step at the end of extension, followed by final cooling at 40°C for 30 sec. PCR analysis was then conducted using the Mx3000P qPCR system (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, USA).

    Isolation:

    Article Title: Pair-wise comparison analysis of differential expression of mRNAs in early and advanced stage primary colorectal adenocarcinomas
    Article Snippet: RT-qPCR analysis Reverse transcription The total RNA isolated from 27 paired samples was reverse transcribed to first-strand cDNA, with the following protocol: 3 µg of total RNA was added with 2 µL of 0.5 µg/µL oligo(dT)12–18 (Invitrogen) and RNase-free water to a final volume of 9.5 µL. .. The reaction mixture was then incubated at 80°C for 3 min, followed by chilling on ice for another 2 min. Next, 4 µL of 5X first-strand buffer (Invitrogen), 5 µL of 2 mM dNTP (Fermentas), 0.5 µL of 40 U/µL RNaseOUT recombinant RNase inhibitor (Invitrogen) and 1 µL of 200 U/µL M-MLV reverse transcriptase (Invitrogen) were added to the mixture.

    Article Title: Prediction of relapse and prognosis by expression levels of long noncoding RNA PEG10 in glioma patients
    Article Snippet: 2.3 RNA extraction and reverse transcription quantitative polymerase chain reaction assay Total RNA from all the 147 cases of glioma tissues and 23 cases of normal control brain tissues specimens was purified as recommended by the manufacturer using RecoverAll Total Nucleic Acid Isolation kit (Thermo Fisher Scientific, Inc., Waltham, MA). .. Synthesis of cDNA was performed using 2 μg of total RNA, lncRNA PEG10 reverse transcription primer (Invitrogen; Thermo Fisher Scientific, Inc.), 5X first-strand buffer (Thermo Fisher Scientific, Inc.), 0.1 mol/L DTT (Thermo Fisher Scientific, Inc.), a dNTP mixture (Takara, Bio, Inc., Otsu, Japan), Moloney Murine Leukemia Virus reverse transcriptase (Thermo Fisher Scientific, Inc.) and recombinant RNasin RNase inhibitor (Promega Corporation, Madison, WI).

    Article Title: Identification of Appropriate Reference Genes for qRT-PCR Analysis of Heat-Stressed Mammary Epithelial Cells in Riverine Buffaloes (Bubalus bubalis)
    Article Snippet: Paragraph title: 2.3. Isolation of Total RNA and cDNA Synthesis ... The mixture was incubated at 65°C for 5 min and kept on ice for 3 min. A total of 6 μ L of master mix composed of 4.5 μ L 5X First-Strand Buffer, 1 μ L 0.1 M DTT, 0.25 μ L (50 U) of SuperScript III RT (Invitrogen Corp., CA), and 0.25 μ L of RNase inhibitor (10 U, Promega, WI) was added.

    Article Title: Transcriptome of the Female Synganglion of the Black-Legged Tick Ixodes scapularis (Acari: Ixodidae) with Comparison between Illumina and 454 Systems
    Article Snippet: 454 pyrosequencing For 454 pyrosequencing, the total RNA was thawed and mRNA isolated from each of 3 female synganglion samples, using an Oligotex mRNA isolation kit according to the manufacturer's recommendations. .. The resulting 4 µl were incubated at 72°C for 2 min and then combined with the following reagents on ice: 1 µl RNase Out (40 U/µl, 2 µl 5X first strand buffer, 1 µl 20 mM DTT, 1 µl dNTP mix (10 mM each) and 1 µl Superscript II reverse transcriptase) (Invitrogen, Carlsbad, CA).

    Size-exclusion Chromatography:

    Article Title: Transcriptome of the Female Synganglion of the Black-Legged Tick Ixodes scapularis (Acari: Ixodidae) with Comparison between Illumina and 454 Systems
    Article Snippet: The resulting 4 µl were incubated at 72°C for 2 min and then combined with the following reagents on ice: 1 µl RNase Out (40 U/µl, 2 µl 5X first strand buffer, 1 µl 20 mM DTT, 1 µl dNTP mix (10 mM each) and 1 µl Superscript II reverse transcriptase) (Invitrogen, Carlsbad, CA). .. Thermal cycling conditions were 94°C for 2 min followed by 20 cycles of 94°C for 20 sec, 65°C for 20 sec and 68°C for 6 min. For optimization of the PCR reaction, 5 µl aliquots from cycles 18, 22 and 25 were analyzed on a 1% agarose gel.

    Article Title: Transcription activated p73-modulated cyclin D1 expression leads to doxorubicin resistance in gastric cancer
    Article Snippet: For cDNA synthesis, 1 µg of RNA was mixed with Oligo(dT) primers, dNTP mix, 5X first-strand buffer, Ditiotreitol, RNAseOUT and SuperScript II RT, all according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.). .. The thermocycling conditions were 10 min of initial denaturation at 95°C, followed by 45 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 30 sec and extension at 72°C for 30 sec with a single fluorescence acquisition step at the end of extension, followed by final cooling at 40°C for 30 sec. PCR analysis was then conducted using the Mx3000P qPCR system (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, USA).

    Labeling:

    Article Title: Optimized amplification and single-cell analysis identify GnRH-mediated activation of Rap1b in primary rat gonadotropes
    Article Snippet: Two-cycle target labeling was performed according to the manufacturer’s protocol (Affymetrix). .. Briefly, 100 ng total RNA was incubated in a 10-μl volume containing 2 μl T7-Oligo(dT) primer, 2 μl 5X first strand buffer, 1 μl 0.1 M DTT, 0.5 μl RNase inhibitor, 0.5 μl 10 mM dNTP, and 1 μl SuperScript II (Invitrogen) at 42 ºC for 1 h. Second strand cDNA synthesis was carried out by incubating 10 μl first strand cDNA products with 4.8 μl water, 4 μl 17.5 mM MgCl2 , 0.4 μl 10 mM dNTP, 0.6 μl E. coli DNA Polymerase I, and 0.2 μl RNase H at 16ºC for 2 h. In vitro transcription was performed using the MegaScript T7 Kit (Ambion).

    Purification:

    Article Title: Prediction of relapse and prognosis by expression levels of long noncoding RNA PEG10 in glioma patients
    Article Snippet: 2.3 RNA extraction and reverse transcription quantitative polymerase chain reaction assay Total RNA from all the 147 cases of glioma tissues and 23 cases of normal control brain tissues specimens was purified as recommended by the manufacturer using RecoverAll Total Nucleic Acid Isolation kit (Thermo Fisher Scientific, Inc., Waltham, MA). .. Synthesis of cDNA was performed using 2 μg of total RNA, lncRNA PEG10 reverse transcription primer (Invitrogen; Thermo Fisher Scientific, Inc.), 5X first-strand buffer (Thermo Fisher Scientific, Inc.), 0.1 mol/L DTT (Thermo Fisher Scientific, Inc.), a dNTP mixture (Takara, Bio, Inc., Otsu, Japan), Moloney Murine Leukemia Virus reverse transcriptase (Thermo Fisher Scientific, Inc.) and recombinant RNasin RNase inhibitor (Promega Corporation, Madison, WI).

    Article Title: Transcriptome of the Female Synganglion of the Black-Legged Tick Ixodes scapularis (Acari: Ixodidae) with Comparison between Illumina and 454 Systems
    Article Snippet: Purified mRNA was ethanol precipitated, rehydrated in 2 µl of RNase-free water and combined with 10 pmol of modified 3′ reverse transcription primer (5′-ATTCTAGAGACCGAGGCGGCCGACATGT(4) GT(9) CT(10) VN-3′) and 10 pmol SMART IV oligo ( 5′-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3′ ) . .. The resulting 4 µl were incubated at 72°C for 2 min and then combined with the following reagents on ice: 1 µl RNase Out (40 U/µl, 2 µl 5X first strand buffer, 1 µl 20 mM DTT, 1 µl dNTP mix (10 mM each) and 1 µl Superscript II reverse transcriptase) (Invitrogen, Carlsbad, CA).

    Article Title: Mutual independence of alkaline‐ and calcium‐mediated signalling in Aspergillus fumigatus refutes the existence of a conserved druggable signalling nexus
    Article Snippet: The tubes were briefly chilled to 4°C for 5 min and mixed with 5 μl of 5X first‐strand buffer (Invitrogen), 2 μl of DTT (100 mM, Invitrogen), 2 μl of 25 mM dNTP, 2 μl of SuperScript RT (Clontech) and 1 μl of Cy3‐dUTP (25 nmol) or Cy5‐dUTP (25 nmol). .. The resulting first strand cDNA was purified and concentrated using a MiniElute column (Qiagen) according to the manufacturer instructions.

    Article Title: Optimized amplification and single-cell analysis identify GnRH-mediated activation of Rap1b in primary rat gonadotropes
    Article Snippet: Briefly, 100 ng total RNA was incubated in a 10-μl volume containing 2 μl T7-Oligo(dT) primer, 2 μl 5X first strand buffer, 1 μl 0.1 M DTT, 0.5 μl RNase inhibitor, 0.5 μl 10 mM dNTP, and 1 μl SuperScript II (Invitrogen) at 42 ºC for 1 h. Second strand cDNA synthesis was carried out by incubating 10 μl first strand cDNA products with 4.8 μl water, 4 μl 17.5 mM MgCl2 , 0.4 μl 10 mM dNTP, 0.6 μl E. coli DNA Polymerase I, and 0.2 μl RNase H at 16ºC for 2 h. In vitro transcription was performed using the MegaScript T7 Kit (Ambion). .. Second strand cDNA products (20 μl) were incubated with 5 μl 10X Reaction buffer, 5 μl each of ATP, CTP, UTP and GTP solutions, and 5 μl Enzyme Mix at 37ºC for 16 h. The cRNA generated was purified using an RNeasy kit (Qiagen) and the manufacturer’s RNA cleanup protocol. cRNA was subsequently subjected to a second cycle of T7 amplification.

    Article Title: Transcription activated p73-modulated cyclin D1 expression leads to doxorubicin resistance in gastric cancer
    Article Snippet: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) The AxyPrep™ Purification kit (Axygen Scientific, Inc., Union City, CA, USA) was utilized to obtain the total RNA from SGC7901 cells. .. For cDNA synthesis, 1 µg of RNA was mixed with Oligo(dT) primers, dNTP mix, 5X first-strand buffer, Ditiotreitol, RNAseOUT and SuperScript II RT, all according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Protective Effect of Heme Oxygenase-1 on High Glucose-Induced Pancreatic ?-Cell Injury
    Article Snippet: Paragraph title: Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis ... After quantification by spectrophotometer (UV/VIS Spectrophotometer ND-1000; Nanodrop, Silmington, DE, USA), 2 µg of RNA was subjected to 85℃ for 3 minutes prior to the addition of 5X First-Strand buffer (Invitrogen), 0.1 M dTT (Invitrogen), 50 µg/µL Random hexamer (Invitrogen), 10 mM dNTP (Invitrogen), RNaseOUT™ (40 U/µL, Invitorogen), and SuperScrip™ II Reverse Transcriptase (200 U/µL, Invitrogen).

    Article Title: Isolation of Sphere-Forming Stem Cells from the Mouse Inner Ear
    Article Snippet: Paragraph title: 2.5. RT-PCR ... SuperScript™ II reverse transcriptase, including 5X first-strand buffer and 0.1 M DTT solution (2,000 units, cat. no. 18064-022, Invitrogen).

    Construct:

    Article Title: In Situ Mechanotransduction via Vinculin Regulates Stem Cell Differentiation
    Article Snippet: Samples containing 2 µg of cDNA were added to a mastermix containing of 10 mM dNTP (Roche), 100 mM DTT (Invitrogen), 5X First Strand Buffer (Invitrogen), 50 mM Random Hexamers (Qiagen), 200U/µL Reverse Transcriptase (Invitrogen), and DEPC H2 O. qPCR was then performed using an 7900HT Fast Real-Time PCR machine (Applied Biosystems). .. Primers coding for Pax3, Pax7, MyoD, Mrf4, Myf5, GAPDH, and the vinculin constructs H, T and FL are listed in .

    IA:

    Article Title: Withania somnifera as a potential candidate to ameliorate high fat diet-induced anxiety and neuroinflammation
    Article Snippet: 4 µL of 5X first-strand buffer, 2 μL of 0.1 M DTT, and 1 μL of recombinant ribonuclease inhibitor (40 units/μL) (Life Technologies, Carlsbad, CA, USA) were added and incubated at 37 °C for 2 min. Then, 1 μL (200 units) of M-MLV reverse transcriptase (Life Technologies, Carlsbad, CA, USA) was added for 20 μL reaction volume. .. Each 5 μL reaction mixture comprised of 2.5 μL of 2X Power-SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA), 1 μL of 20X pre-designed Primer mix (Integrated DNA Technologies, Coralville, IA, USA), 1 μL of water, and 0.5 μL (50 μg) cDNA.

    SYBR Green Assay:

    Article Title: Individual co-variation between viral RNA load and gene expression reveals novel host factors during early dengue virus infection of the Aedes aegypti midgut
    Article Snippet: After incubation at 65°C for 10 min, samples were chilled on ice for 5 min. For each reaction, 4 μL of 5X First-Strand buffer, 1 μL of 0.1 mM Dithiothreitol, 40 units of RNase-OUT and 100 units of MML-V reverse transcriptase (Life Technologies) were added to a final volume of 20 μL. .. The qPCR mix contained 200 nM of each primer, 10 μL of 2X SYBR-green I Master Mix (Roche) and PCR grade water to 18 μL, with 2 μL of cDNA template to a final volume of 20 μL.

    RNA Extraction:

    Article Title: Prediction of relapse and prognosis by expression levels of long noncoding RNA PEG10 in glioma patients
    Article Snippet: Paragraph title: RNA extraction and reverse transcription quantitative polymerase chain reaction assay ... Synthesis of cDNA was performed using 2 μg of total RNA, lncRNA PEG10 reverse transcription primer (Invitrogen; Thermo Fisher Scientific, Inc.), 5X first-strand buffer (Thermo Fisher Scientific, Inc.), 0.1 mol/L DTT (Thermo Fisher Scientific, Inc.), a dNTP mixture (Takara, Bio, Inc., Otsu, Japan), Moloney Murine Leukemia Virus reverse transcriptase (Thermo Fisher Scientific, Inc.) and recombinant RNasin RNase inhibitor (Promega Corporation, Madison, WI).

    Agarose Gel Electrophoresis:

    Article Title: Transcriptome of the Female Synganglion of the Black-Legged Tick Ixodes scapularis (Acari: Ixodidae) with Comparison between Illumina and 454 Systems
    Article Snippet: The resulting 4 µl were incubated at 72°C for 2 min and then combined with the following reagents on ice: 1 µl RNase Out (40 U/µl, 2 µl 5X first strand buffer, 1 µl 20 mM DTT, 1 µl dNTP mix (10 mM each) and 1 µl Superscript II reverse transcriptase) (Invitrogen, Carlsbad, CA). .. Thermal cycling conditions were 94°C for 2 min followed by 20 cycles of 94°C for 20 sec, 65°C for 20 sec and 68°C for 6 min. For optimization of the PCR reaction, 5 µl aliquots from cycles 18, 22 and 25 were analyzed on a 1% agarose gel.

    In Vitro:

    Article Title: Optimized amplification and single-cell analysis identify GnRH-mediated activation of Rap1b in primary rat gonadotropes
    Article Snippet: .. Briefly, 100 ng total RNA was incubated in a 10-μl volume containing 2 μl T7-Oligo(dT) primer, 2 μl 5X first strand buffer, 1 μl 0.1 M DTT, 0.5 μl RNase inhibitor, 0.5 μl 10 mM dNTP, and 1 μl SuperScript II (Invitrogen) at 42 ºC for 1 h. Second strand cDNA synthesis was carried out by incubating 10 μl first strand cDNA products with 4.8 μl water, 4 μl 17.5 mM MgCl2 , 0.4 μl 10 mM dNTP, 0.6 μl E. coli DNA Polymerase I, and 0.2 μl RNase H at 16ºC for 2 h. In vitro transcription was performed using the MegaScript T7 Kit (Ambion). .. Second strand cDNA products (20 μl) were incubated with 5 μl 10X Reaction buffer, 5 μl each of ATP, CTP, UTP and GTP solutions, and 5 μl Enzyme Mix at 37ºC for 16 h. The cRNA generated was purified using an RNeasy kit (Qiagen) and the manufacturer’s RNA cleanup protocol. cRNA was subsequently subjected to a second cycle of T7 amplification.

    Electrophoresis:

    Article Title: Identification of Appropriate Reference Genes for qRT-PCR Analysis of Heat-Stressed Mammary Epithelial Cells in Riverine Buffaloes (Bubalus bubalis)
    Article Snippet: The quality check for all samples was performed using experion automated electrophoresis System (Biorad). .. The mixture was incubated at 65°C for 5 min and kept on ice for 3 min. A total of 6 μ L of master mix composed of 4.5 μ L 5X First-Strand Buffer, 1 μ L 0.1 M DTT, 0.25 μ L (50 U) of SuperScript III RT (Invitrogen Corp., CA), and 0.25 μ L of RNase inhibitor (10 U, Promega, WI) was added.

    Homogenization:

    Article Title: Strand-specific RNA-seq applied to malaria samples
    Article Snippet: After tissue homogenization, samples should always be kept on ice. .. Parasite cultures grown in complete RPMI medium at 5 % hematocrit ( see ) TRIzol® LS Reagent (Invitrogen™) pre-warmed at 37°C Chloroform Isopropanol pre-chilled on ice Nuclease-free non-DEPC water DNAse I RNAse-free (Ambion®) Deionized formamide Formaldehyde 37 % 10X MOPS EDTA buffer pH 7.0 Glycerol 50 % Bromophenol blue powder Ethidium bromide 20 mg/mL GenElute™ mRNA Miniprep Kit (Sigma-Aldrich) 5X RNA storage solution (Ambion) HPLC-purified random hexamers and Anchored OligodT(20) SuperScript® VILO™ cDNA synthesis kit (Invitrogen™) DNA Clean & Concentrator™ (Zymo Research) 5X First Strand Buffer (Invitrogen™): 250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2 5X Second Strand Buffer (Invitrogen™): 100 mM Tris-HCl (pH 6.9), 450 mM KCl, 23 mM MgCl2 , 0.75 mM β-NAD+ , 50 mM (NH4 )2 SO4 Set of dATP, dGTP, dCTP, and dUTP E. coli DNA Polymerase I 10 U/μL (Invitrogen™) E. coli DNA Ligase 10 U/μL (Invitrogen™) E. coli DNA RNase H 2 U/μL (Invitrogen™) 0.1 M DTT dsDNA Shearase™ (Zymo Research) Encore™ NGS Library System I (NuGEN®) Same-day 70 % ethanol in nuclease-free water USER™ Enzyme (New England Biolabs®) 1X TE buffer pH 8.0

    Next-Generation Sequencing:

    Article Title: Strand-specific RNA-seq applied to malaria samples
    Article Snippet: .. Parasite cultures grown in complete RPMI medium at 5 % hematocrit ( see ) TRIzol® LS Reagent (Invitrogen™) pre-warmed at 37°C Chloroform Isopropanol pre-chilled on ice Nuclease-free non-DEPC water DNAse I RNAse-free (Ambion®) Deionized formamide Formaldehyde 37 % 10X MOPS EDTA buffer pH 7.0 Glycerol 50 % Bromophenol blue powder Ethidium bromide 20 mg/mL GenElute™ mRNA Miniprep Kit (Sigma-Aldrich) 5X RNA storage solution (Ambion) HPLC-purified random hexamers and Anchored OligodT(20) SuperScript® VILO™ cDNA synthesis kit (Invitrogen™) DNA Clean & Concentrator™ (Zymo Research) 5X First Strand Buffer (Invitrogen™): 250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2 5X Second Strand Buffer (Invitrogen™): 100 mM Tris-HCl (pH 6.9), 450 mM KCl, 23 mM MgCl2 , 0.75 mM β-NAD+ , 50 mM (NH4 )2 SO4 Set of dATP, dGTP, dCTP, and dUTP E. coli DNA Polymerase I 10 U/μL (Invitrogen™) E. coli DNA Ligase 10 U/μL (Invitrogen™) E. coli DNA RNase H 2 U/μL (Invitrogen™) 0.1 M DTT dsDNA Shearase™ (Zymo Research) Encore™ NGS Library System I (NuGEN®) Same-day 70 % ethanol in nuclease-free water USER™ Enzyme (New England Biolabs®) 1X TE buffer pH 8.0 ..

    Incubation:

    Article Title: Pair-wise comparison analysis of differential expression of mRNAs in early and advanced stage primary colorectal adenocarcinomas
    Article Snippet: .. The reaction mixture was then incubated at 80°C for 3 min, followed by chilling on ice for another 2 min. Next, 4 µL of 5X first-strand buffer (Invitrogen), 5 µL of 2 mM dNTP (Fermentas), 0.5 µL of 40 U/µL RNaseOUT recombinant RNase inhibitor (Invitrogen) and 1 µL of 200 U/µL M-MLV reverse transcriptase (Invitrogen) were added to the mixture. ..

    Article Title: Withania somnifera as a potential candidate to ameliorate high fat diet-induced anxiety and neuroinflammation
    Article Snippet: .. 4 µL of 5X first-strand buffer, 2 μL of 0.1 M DTT, and 1 μL of recombinant ribonuclease inhibitor (40 units/μL) (Life Technologies, Carlsbad, CA, USA) were added and incubated at 37 °C for 2 min. Then, 1 μL (200 units) of M-MLV reverse transcriptase (Life Technologies, Carlsbad, CA, USA) was added for 20 μL reaction volume. .. 50 µg of cDNA was then used to amplify gene of interest using Step One Plus Real Time PCR system (Applied Biosystems, Foster City, CA, USA).

    Article Title: Individual co-variation between viral RNA load and gene expression reveals novel host factors during early dengue virus infection of the Aedes aegypti midgut
    Article Snippet: .. After incubation at 65°C for 10 min, samples were chilled on ice for 5 min. For each reaction, 4 μL of 5X First-Strand buffer, 1 μL of 0.1 mM Dithiothreitol, 40 units of RNase-OUT and 100 units of MML-V reverse transcriptase (Life Technologies) were added to a final volume of 20 μL. .. After 10 min at 25°C, cDNA synthesis was conducted at 37°C for 50 min and terminated at 70°C for 15 min. cDNA samples were stored at -20°C until use.

    Article Title: Human and entomologic investigations of chikungunya outbreak in Mandera, Northeastern Kenya, 2016
    Article Snippet: In a 200 μl PCR tube, 10 μl of the extracted sample RNA was mixed with 2 μl of 50 ng/μl random hexamer primer and 1 μl of 10 mM deoxynucleotide solution (dNTPs), and incubated in a thermocycler for 5 minutes at 65°C and immediately chilled for 1 minute at 4°C. .. The following components were then added to the PCR tube: 4 μl of 5X First Strand Buffer (Invitrogen), 1 μl of 0.1 M DTT, 1 μl of RNAse OUT(40 U/μl) and 1 μl of Superscript III Reverse transcriptase (200 U/μl).

    Article Title: Identification of Appropriate Reference Genes for qRT-PCR Analysis of Heat-Stressed Mammary Epithelial Cells in Riverine Buffaloes (Bubalus bubalis)
    Article Snippet: .. The mixture was incubated at 65°C for 5 min and kept on ice for 3 min. A total of 6 μ L of master mix composed of 4.5 μ L 5X First-Strand Buffer, 1 μ L 0.1 M DTT, 0.25 μ L (50 U) of SuperScript III RT (Invitrogen Corp., CA), and 0.25 μ L of RNase inhibitor (10 U, Promega, WI) was added. .. The reaction was performed in an Eppendorf Gradient cycler using the program: 25°C for 5 min, 50°C for 60 min and 70°C for 15 min. cDNA was then diluted 1 : 4 (v : v) with DNase-/RNase-free water.

    Article Title: Transcriptome of the Female Synganglion of the Black-Legged Tick Ixodes scapularis (Acari: Ixodidae) with Comparison between Illumina and 454 Systems
    Article Snippet: .. The resulting 4 µl were incubated at 72°C for 2 min and then combined with the following reagents on ice: 1 µl RNase Out (40 U/µl, 2 µl 5X first strand buffer, 1 µl 20 mM DTT, 1 µl dNTP mix (10 mM each) and 1 µl Superscript II reverse transcriptase) (Invitrogen, Carlsbad, CA). .. The reaction was incubated at 42°C for 90 min then diluted to 30 µl with TE buffer (10 mM Tris HCL pH 7.5, 1 mM EDTA) and stored at −20°C until further use.

    Article Title: Mutual independence of alkaline‐ and calcium‐mediated signalling in Aspergillus fumigatus refutes the existence of a conserved druggable signalling nexus
    Article Snippet: The tubes were briefly chilled to 4°C for 5 min and mixed with 5 μl of 5X first‐strand buffer (Invitrogen), 2 μl of DTT (100 mM, Invitrogen), 2 μl of 25 mM dNTP, 2 μl of SuperScript RT (Clontech) and 1 μl of Cy3‐dUTP (25 nmol) or Cy5‐dUTP (25 nmol). .. After 16 h of incubation at 42°C, RNA was hydrolysed by adding 2.5 μl EDTA (0.5 M, pH 8) and 5 μl NaOH (1 M) following incubation at 37°C for 40 min.

    Article Title: Optimized amplification and single-cell analysis identify GnRH-mediated activation of Rap1b in primary rat gonadotropes
    Article Snippet: .. Briefly, 100 ng total RNA was incubated in a 10-μl volume containing 2 μl T7-Oligo(dT) primer, 2 μl 5X first strand buffer, 1 μl 0.1 M DTT, 0.5 μl RNase inhibitor, 0.5 μl 10 mM dNTP, and 1 μl SuperScript II (Invitrogen) at 42 ºC for 1 h. Second strand cDNA synthesis was carried out by incubating 10 μl first strand cDNA products with 4.8 μl water, 4 μl 17.5 mM MgCl2 , 0.4 μl 10 mM dNTP, 0.6 μl E. coli DNA Polymerase I, and 0.2 μl RNase H at 16ºC for 2 h. In vitro transcription was performed using the MegaScript T7 Kit (Ambion). .. Second strand cDNA products (20 μl) were incubated with 5 μl 10X Reaction buffer, 5 μl each of ATP, CTP, UTP and GTP solutions, and 5 μl Enzyme Mix at 37ºC for 16 h. The cRNA generated was purified using an RNeasy kit (Qiagen) and the manufacturer’s RNA cleanup protocol. cRNA was subsequently subjected to a second cycle of T7 amplification.

    Spectrophotometry:

    Article Title: Protective Effect of Heme Oxygenase-1 on High Glucose-Induced Pancreatic ?-Cell Injury
    Article Snippet: .. After quantification by spectrophotometer (UV/VIS Spectrophotometer ND-1000; Nanodrop, Silmington, DE, USA), 2 µg of RNA was subjected to 85℃ for 3 minutes prior to the addition of 5X First-Strand buffer (Invitrogen), 0.1 M dTT (Invitrogen), 50 µg/µL Random hexamer (Invitrogen), 10 mM dNTP (Invitrogen), RNaseOUT™ (40 U/µL, Invitorogen), and SuperScrip™ II Reverse Transcriptase (200 U/µL, Invitrogen). ..

    Article Title: Identification of Appropriate Reference Genes for qRT-PCR Analysis of Heat-Stressed Mammary Epithelial Cells in Riverine Buffaloes (Bubalus bubalis)
    Article Snippet: Total RNA concentration and purity were measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). .. The mixture was incubated at 65°C for 5 min and kept on ice for 3 min. A total of 6 μ L of master mix composed of 4.5 μ L 5X First-Strand Buffer, 1 μ L 0.1 M DTT, 0.25 μ L (50 U) of SuperScript III RT (Invitrogen Corp., CA), and 0.25 μ L of RNase inhibitor (10 U, Promega, WI) was added.

    Article Title: Transcription activated p73-modulated cyclin D1 expression leads to doxorubicin resistance in gastric cancer
    Article Snippet: Next, the quality and quantity of total RNA were examined by a Nanodrop 2000 micro-volume spectrophotometer (Thermo Fisher Scientific, Inc.) through evaluation of absorbance. .. For cDNA synthesis, 1 µg of RNA was mixed with Oligo(dT) primers, dNTP mix, 5X first-strand buffer, Ditiotreitol, RNAseOUT and SuperScript II RT, all according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.).

    Article Title: Isolation of Sphere-Forming Stem Cells from the Mouse Inner Ear
    Article Snippet: UV spectrophotometer. g vial, 500 μg / mL). .. SuperScript™ II reverse transcriptase, including 5X first-strand buffer and 0.1 M DTT solution (2,000 units, cat. no. 18064-022, Invitrogen).

    Concentration Assay:

    Article Title: Identification of Appropriate Reference Genes for qRT-PCR Analysis of Heat-Stressed Mammary Epithelial Cells in Riverine Buffaloes (Bubalus bubalis)
    Article Snippet: Total RNA concentration and purity were measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). .. The mixture was incubated at 65°C for 5 min and kept on ice for 3 min. A total of 6 μ L of master mix composed of 4.5 μ L 5X First-Strand Buffer, 1 μ L 0.1 M DTT, 0.25 μ L (50 U) of SuperScript III RT (Invitrogen Corp., CA), and 0.25 μ L of RNase inhibitor (10 U, Promega, WI) was added.

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    Thermo Fisher superscript iii reverse transcriptase
    Microcapillary electrophoresis of total <t>RNA</t> from whole cells and projections. Microcapillary electrophoresis of total RNA (Bioanalyzer RNA pico assay) from whole cells and projections of <t>three</t> biological replicates. Only RNAs > ~150 nt were sequenced for this study. In projections, the band at ~100 nt was striking. By sequencing RNAs within 20–150 nt size range, we found that they were mostly full length and fragmented tRNAs (unpublished data).
    Superscript Iii Reverse Transcriptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 3158 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii reverse transcriptase/product/Thermo Fisher
    Average 90 stars, based on 3158 article reviews
    Price from $9.99 to $1999.99
    superscript iii reverse transcriptase - by Bioz Stars, 2020-02
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    Thermo Fisher first strand buffer
    Microcapillary electrophoresis of total <t>RNA</t> from whole cells and projections. Microcapillary electrophoresis of total RNA (Bioanalyzer RNA pico assay) from whole cells and projections of <t>three</t> biological replicates. Only RNAs > ~150 nt were sequenced for this study. In projections, the band at ~100 nt was striking. By sequencing RNAs within 20–150 nt size range, we found that they were mostly full length and fragmented tRNAs (unpublished data).
    First Strand Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 939 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Microcapillary electrophoresis of total RNA from whole cells and projections. Microcapillary electrophoresis of total RNA (Bioanalyzer RNA pico assay) from whole cells and projections of three biological replicates. Only RNAs > ~150 nt were sequenced for this study. In projections, the band at ~100 nt was striking. By sequencing RNAs within 20–150 nt size range, we found that they were mostly full length and fragmented tRNAs (unpublished data).

    Journal: eLife

    Article Title: Free circular introns with an unusual branchpoint in neuronal projections

    doi: 10.7554/eLife.47809

    Figure Lengend Snippet: Microcapillary electrophoresis of total RNA from whole cells and projections. Microcapillary electrophoresis of total RNA (Bioanalyzer RNA pico assay) from whole cells and projections of three biological replicates. Only RNAs > ~150 nt were sequenced for this study. In projections, the band at ~100 nt was striking. By sequencing RNAs within 20–150 nt size range, we found that they were mostly full length and fragmented tRNAs (unpublished data).

    Article Snippet: RNA was hydrolyzed using 5X first strand buffer (provided with Superscript III reverse transcriptase, ThermoFisher #18080044) at 94°C for 4 min and 50 s and immediately moved to ice.

    Techniques: Electrophoresis, Sequencing