superscriptii reverse transcriptase kit  (Thermo Fisher)


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    Thermo Fisher superscriptii reverse transcriptase kit
    Superscriptii Reverse Transcriptase Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscriptii reverse transcriptase kit/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    superscriptii reverse transcriptase kit - by Bioz Stars, 2020-04
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Functional endogenous LINE-1 retrotransposons are expressed and mobilized in rat chloroleukemia cells
    Article Snippet: .. Cloning of transcribed L1Rn sequences from RCL cells Total and poly(A)+ RNA were prepared from control and irradiated RCL cells with the RNeasy and Oligotex mRNA kits (Qiagen), respectively. cDNA was synthesized from poly(A)+ RNA employing SuperScript™ II reverse transcriptase (Invitrogen) and oligo(dT) primer. .. About 6 kb long L1Rn sequences encompassing ORF1 and ORF2 were amplified from cDNA with the Taq/Pwo Proof Mix (AGS-Hybaid) and primers Pr-L1Rn1 (5′-GGA AGA GAC CAC CAA CAC TGC TCA C-3′, pos.

    Amplification:

    Article Title: Parental haplotype-specific single-cell transcriptomics reveal incomplete epigenetic reprogramming in human female germ cells
    Article Snippet: .. Reverse transcription (RT) was performed by the addition of 5.6 µl Smart-Seq2 RT mix (0.5 µl SuperScriptII (Invitrogen Cat. 18064-014); 2 µl 5 × SuperScriptII buffer; 0.5 µl 100 mM DTT; 2 µl 5 M betaine; 0.1 µl 1 mM MgCl2 ; 0.25 µl Recombinant RNase inhibitor (TaKaRa 40U/µl Ref. 2313A); 0.1 µl 100 µM LNA strand switch primer; 0.15 µl water, per reaction) and incubation (90 min 42 °C; 10 “strand-switch” cycles of (2 min 50 °C; 2 min 70 °C); 4 °C) in a thermal cycler. cDNA amplification: cDNA amplification was performed by the addition of 15 µl of Smart-seq2 PCR mix [12.5 µl 2× KAPA HiFi HotStart ReadyMix (KAPA Biosystems Ref. KK2602); 0.25 µl 10 µM ISPCR primers; 2.25 µl water, per reaction] and incubation [3 min 98 °C; 19 cycles of (20 s (s) 98 °C; 15 s 67 °C; 6 min 72 °C); 5 min 70 °C; 4 °C] in a thermal cycler. .. The cDNA was purified using 0.7:1 volume of AMPure XP beads (Beckman Coulter, Ref. ) according to the manufacturer’s protocol (No. PT5163-1).

    Article Title: Apoptotic and Early Innate Immune Responses to PB1-F2 Protein of Influenza A Viruses Belonging to Different Subtypes in Human Lung Epithelial A549 Cells
    Article Snippet: .. To determine the mRNA expression of PB1-F2 gene in A549 cells, 500 ng of RNA was reverse transcribed using the Superscript II RT-PCR kit (Invitrogen, Carlsbad, CA, USA) and Uni12 primer and the PB1-F2 gene was amplified as mentioned above. .. The total cellular protein was extracted using radio-immunoprecipitation assay (RIPA) buffer (Sigma, St. Louis, Missouri, USA).

    Article Title: Time-course microarray analysis for identifying candidate genes involved in obesity-associated pathological changes in the mouse colon
    Article Snippet: Template RNA isolated from the colon tissue was reverse transcribed using Superscript™ II RT-PCR System (Invitrogen, Karlsruhe, Germany), according to the manufacturer’s instructions, for performing dT 20-primed complementary DNA (cDNA) synthesis. .. Amplifications were performed using the following protocol: initial template denaturation at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. All the samples were amplified in triplicate, and data were analyzed using Sequence Detector software (Applied Biosystems).

    Article Title: Gene Regulatory Networks for the Haploid-to-Diploid Transition of Chlamydomonas reinhardtii 1 1 [OPEN]
    Article Snippet: To analyze the relative abundance of transcripts using qRT-PCR, DNase-treated 5 μg of total RNA was reverse-transcribed with Superscript II RT-PCR kit (Invitrogen). .. Primers were designed to produce PCR amplicon lengths of 100–150 bp. qRT–PCR was performed in optical 96-well plates using Bio-Rad CFX96 Real-Time PCR systems.

    Article Title: Functional endogenous LINE-1 retrotransposons are expressed and mobilized in rat chloroleukemia cells
    Article Snippet: Cloning of transcribed L1Rn sequences from RCL cells Total and poly(A)+ RNA were prepared from control and irradiated RCL cells with the RNeasy and Oligotex mRNA kits (Qiagen), respectively. cDNA was synthesized from poly(A)+ RNA employing SuperScript™ II reverse transcriptase (Invitrogen) and oligo(dT) primer. .. About 6 kb long L1Rn sequences encompassing ORF1 and ORF2 were amplified from cDNA with the Taq/Pwo Proof Mix (AGS-Hybaid) and primers Pr-L1Rn1 (5′-GGA AGA GAC CAC CAA CAC TGC TCA C-3′, pos.

    Article Title: Corky, a gypsy-like retrotransposon is differentially transcribed in Quercus suber tissues
    Article Snippet: .. RNA samples were treated with RQ1 RNase-Free DNase (Promega, Madison, WI). cDNA was synthesized from 2 μg of total RNA using random hexamers and Superscript II RNase H- reverse transcriptase (Invitrogen®, Carlsbad, CA), according to the manufacturer’s recommendations followed by PCR amplification using specific primers for the RVT and a region between Integrase and the chromodomain of Corky (Figure ). .. As expected, amplification products were not obtained in RNA samples not yielded to reverse transcription prior to PCR. cDNA was stored at −20°C.

    Article Title: Distinct Signature of Altered Homeostasis in Aging Rod Photoreceptors: Implications for Retinal Diseases
    Article Snippet: .. Amplified photoreceptor cDNA was derived using WT-Ovation Pico System (NuGEN), and whole retina cDNA was derived using Superscript II RT-PCR system (Invitrogen). cDNA was used for real-time qRT-PCR using SYBR(R) Green PCR Mastermix (Applied Biosystems, Foster City, CA) on the 7900HT Fast Real-Time PCR System (Applied Biosystems). .. Candidate genes were selected for analysis based on microarray predictions of fold-change > 2, pattern of change, and gene function.

    Article Title: Epstein-Barr Virus Latent Nuclear Antigens Can Induce Metastasis in a Nude Mouse Model ▿
    Article Snippet: RT was carried out with SuperScript II RNase H-reverse transcriptase (Life Technologies, Gaithersburg, MD). .. The Nm23-H1-Myc transcript was amplified for 30 cycles (30 s at 94°C, 1 min at 50°C, and 1 min at 72°C) using the forward primer 5′-GATTACACGAGCTGTGCTCA-3′ and the reverse primer 5′-TTCGCTAGCCAAGTCTTCTT-3′. designed to amplify junction sequence between Nm23-H1 and Myc tag.

    Article Title: Characterization of Moraxella (Branhamella) catarrhalis lbpB, lbpA, and Lactoferrin Receptor orf3 Isogenic Mutants
    Article Snippet: The RT-PCR experiments were performed with SuperScript II RNase H− reverse transcriptase (Gibco BRL) according to the manufacturer’s instructions. .. Total RNA was isolated with the Qiagen RNeasy mini kit (Qiagen, Inc.) and subjected to amplification-grade DNase I (Gibco BRL) treatment in the presence of RNasin (Promega Corporation). cDNA was generated by using oligonucleotide 728 (Fig. ) and treated with RNase H (Gibco BRL), prior to being used as a template for subsequent PCR.

    Article Title: Mef2c is an essential regulatory element required for unique expression of the cardiac-specific CARK gene
    Article Snippet: Total RNAs were isolated using Trizol reagent (Invitrogen) and treated with DNase I (Ambion) according to the manufacturer's instructions. cDNAs were generated using oligo(dT)12-18 as a primer and SuperScript II RT-PCR kit (Invitrogen).cDNA was added to 25 μl of reaction volume containing 12.5 μl of 2× SYBR Green Premix EX Taq (TaKaRa, Otsu, Japan), and 400 nmol/L CARK gene-specific primers (forward: 5′-GACAAAGCAGCCAGGGAA; reverse: 5′-AGCAGGCAATCGGAAGAA). .. To confirm amplification specificity, the PCR products were subjected to a melting curve analysis.

    Synthesized:

    Article Title: Functional endogenous LINE-1 retrotransposons are expressed and mobilized in rat chloroleukemia cells
    Article Snippet: .. Cloning of transcribed L1Rn sequences from RCL cells Total and poly(A)+ RNA were prepared from control and irradiated RCL cells with the RNeasy and Oligotex mRNA kits (Qiagen), respectively. cDNA was synthesized from poly(A)+ RNA employing SuperScript™ II reverse transcriptase (Invitrogen) and oligo(dT) primer. .. About 6 kb long L1Rn sequences encompassing ORF1 and ORF2 were amplified from cDNA with the Taq/Pwo Proof Mix (AGS-Hybaid) and primers Pr-L1Rn1 (5′-GGA AGA GAC CAC CAA CAC TGC TCA C-3′, pos.

    Article Title: Annexin A1 down-regulation in head and neck squamous cell carcinoma is mediated via transcriptional control with direct involvement of miR-196a/b
    Article Snippet: .. RNA extraction and real-time RT-PCR Total RNA was extracted from HNSCC cells using Trizol reagent (Invitrogen Life Technologies), and cDNA synthesized with Superscript II RT-PCR System (Invitrogen Life Technologies), according to manufacturer’s protocols. .. Gene expression was analyzed by Real-time PCR using the StepOnePlus Real-Time PCR System (Applied Biosystems) following Applied Biosystems’ SYBR Green Master Mix protocol.

    Article Title: Corky, a gypsy-like retrotransposon is differentially transcribed in Quercus suber tissues
    Article Snippet: .. RNA samples were treated with RQ1 RNase-Free DNase (Promega, Madison, WI). cDNA was synthesized from 2 μg of total RNA using random hexamers and Superscript II RNase H- reverse transcriptase (Invitrogen®, Carlsbad, CA), according to the manufacturer’s recommendations followed by PCR amplification using specific primers for the RVT and a region between Integrase and the chromodomain of Corky (Figure ). .. As expected, amplification products were not obtained in RNA samples not yielded to reverse transcription prior to PCR. cDNA was stored at −20°C.

    Quantitative RT-PCR:

    Article Title: Gene Regulatory Networks for the Haploid-to-Diploid Transition of Chlamydomonas reinhardtii 1 1 [OPEN]
    Article Snippet: .. To analyze the relative abundance of transcripts using qRT-PCR, DNase-treated 5 μg of total RNA was reverse-transcribed with Superscript II RT-PCR kit (Invitrogen). .. Primers were designed to produce PCR amplicon lengths of 100–150 bp. qRT–PCR was performed in optical 96-well plates using Bio-Rad CFX96 Real-Time PCR systems.

    Article Title: Annexin A1 down-regulation in head and neck squamous cell carcinoma is mediated via transcriptional control with direct involvement of miR-196a/b
    Article Snippet: .. RNA extraction and real-time RT-PCR Total RNA was extracted from HNSCC cells using Trizol reagent (Invitrogen Life Technologies), and cDNA synthesized with Superscript II RT-PCR System (Invitrogen Life Technologies), according to manufacturer’s protocols. .. Gene expression was analyzed by Real-time PCR using the StepOnePlus Real-Time PCR System (Applied Biosystems) following Applied Biosystems’ SYBR Green Master Mix protocol.

    Article Title: Receptor Tyrosine Kinase-like Orphan Receptor 2 (Ror2) Expression Creates a Poised State of Wnt Signaling in Renal Cancer *
    Article Snippet: Paragraph title: Quantitative RT-PCR and Human Wnt Signaling RT2 Profiler PCR Array ... Total RNA was extracted from cells using a Qiagen RNeasy mini kit (Valencia, CA). cDNA was made from 500 ng of total RNA using random primers (Invitrogen) and Superscript II RT-PCR reagents (Invitrogen) and analyzed using the ABI 7900HT fast real-time PCR system with the following proprietary FAM-labeled primers: Ror2, Axin2, Fzd1, Jun, 18S, and β-actin (Applied Biosystems, Foster City, CA).

    Article Title: Corky, a gypsy-like retrotransposon is differentially transcribed in Quercus suber tissues
    Article Snippet: Paragraph title: RNA isolation and cDNA preparation for RT-qPCR ... RNA samples were treated with RQ1 RNase-Free DNase (Promega, Madison, WI). cDNA was synthesized from 2 μg of total RNA using random hexamers and Superscript II RNase H- reverse transcriptase (Invitrogen®, Carlsbad, CA), according to the manufacturer’s recommendations followed by PCR amplification using specific primers for the RVT and a region between Integrase and the chromodomain of Corky (Figure ).

    Article Title: Distinct Signature of Altered Homeostasis in Aging Rod Photoreceptors: Implications for Retinal Diseases
    Article Snippet: .. Amplified photoreceptor cDNA was derived using WT-Ovation Pico System (NuGEN), and whole retina cDNA was derived using Superscript II RT-PCR system (Invitrogen). cDNA was used for real-time qRT-PCR using SYBR(R) Green PCR Mastermix (Applied Biosystems, Foster City, CA) on the 7900HT Fast Real-Time PCR System (Applied Biosystems). .. Candidate genes were selected for analysis based on microarray predictions of fold-change > 2, pattern of change, and gene function.

    Article Title: Mef2c is an essential regulatory element required for unique expression of the cardiac-specific CARK gene
    Article Snippet: Paragraph title: Quantitative real-time RT-PCR analysis ... Total RNAs were isolated using Trizol reagent (Invitrogen) and treated with DNase I (Ambion) according to the manufacturer's instructions. cDNAs were generated using oligo(dT)12-18 as a primer and SuperScript II RT-PCR kit (Invitrogen).cDNA was added to 25 μl of reaction volume containing 12.5 μl of 2× SYBR Green Premix EX Taq (TaKaRa, Otsu, Japan), and 400 nmol/L CARK gene-specific primers (forward: 5′-GACAAAGCAGCCAGGGAA; reverse: 5′-AGCAGGCAATCGGAAGAA).

    Article Title: Functional Characterization of the Infection-Inducible Peptide Edin in Drosophila melanogaster
    Article Snippet: .. Semi-quantitative and quantitative RT-PCR Semi-quantitative RT-PCR reactions for edin , Attacin A and Act5C were performed using Super-Script™ II One-Step RT-PCR with Platinum Taq kit (Invitrogen/Life Technologies, Carlsbad, CA, USA). ..

    SYBR Green Assay:

    Article Title: Gene Regulatory Networks for the Haploid-to-Diploid Transition of Chlamydomonas reinhardtii 1 1 [OPEN]
    Article Snippet: To analyze the relative abundance of transcripts using qRT-PCR, DNase-treated 5 μg of total RNA was reverse-transcribed with Superscript II RT-PCR kit (Invitrogen). .. Reactions were performed in a final volume of 20 μL containing 10 μL of 23 SYBR Green Master Mix, 0.5 μ m each primer, and 10 ng of cDNA.

    Article Title: Annexin A1 down-regulation in head and neck squamous cell carcinoma is mediated via transcriptional control with direct involvement of miR-196a/b
    Article Snippet: RNA extraction and real-time RT-PCR Total RNA was extracted from HNSCC cells using Trizol reagent (Invitrogen Life Technologies), and cDNA synthesized with Superscript II RT-PCR System (Invitrogen Life Technologies), according to manufacturer’s protocols. .. Gene expression was analyzed by Real-time PCR using the StepOnePlus Real-Time PCR System (Applied Biosystems) following Applied Biosystems’ SYBR Green Master Mix protocol.

    Article Title: Mef2c is an essential regulatory element required for unique expression of the cardiac-specific CARK gene
    Article Snippet: .. Total RNAs were isolated using Trizol reagent (Invitrogen) and treated with DNase I (Ambion) according to the manufacturer's instructions. cDNAs were generated using oligo(dT)12-18 as a primer and SuperScript II RT-PCR kit (Invitrogen).cDNA was added to 25 μl of reaction volume containing 12.5 μl of 2× SYBR Green Premix EX Taq (TaKaRa, Otsu, Japan), and 400 nmol/L CARK gene-specific primers (forward: 5′-GACAAAGCAGCCAGGGAA; reverse: 5′-AGCAGGCAATCGGAAGAA). .. To confirm amplification specificity, the PCR products were subjected to a melting curve analysis.

    Article Title: Functional Characterization of the Infection-Inducible Peptide Edin in Drosophila melanogaster
    Article Snippet: Semi-quantitative and quantitative RT-PCR Semi-quantitative RT-PCR reactions for edin , Attacin A and Act5C were performed using Super-Script™ II One-Step RT-PCR with Platinum Taq kit (Invitrogen/Life Technologies, Carlsbad, CA, USA). .. Quantitative RT-PCR was carried out using the QuantiTect SYBR Green RT-PCR kit (Qiagen) and an ABI7000 (Applied Biosystems) instrument according to the manufacturer's instructions.

    Microarray:

    Article Title: Time-course microarray analysis for identifying candidate genes involved in obesity-associated pathological changes in the mouse colon
    Article Snippet: Real-time quantitative polymerase chain reaction analysis Real-time quantitative polymerase chain reaction (qPCR) was conducted to validate microarray data of several differentially expressed genes that were selected based on the clustering and network analyses and that were associated with the biological function of interest, including CRC and obesity. .. Template RNA isolated from the colon tissue was reverse transcribed using Superscript™ II RT-PCR System (Invitrogen, Karlsruhe, Germany), according to the manufacturer’s instructions, for performing dT 20-primed complementary DNA (cDNA) synthesis.

    Article Title: Distinct Signature of Altered Homeostasis in Aging Rod Photoreceptors: Implications for Retinal Diseases
    Article Snippet: Amplified photoreceptor cDNA was derived using WT-Ovation Pico System (NuGEN), and whole retina cDNA was derived using Superscript II RT-PCR system (Invitrogen). cDNA was used for real-time qRT-PCR using SYBR(R) Green PCR Mastermix (Applied Biosystems, Foster City, CA) on the 7900HT Fast Real-Time PCR System (Applied Biosystems). .. Candidate genes were selected for analysis based on microarray predictions of fold-change > 2, pattern of change, and gene function.

    Incubation:

    Article Title: Parental haplotype-specific single-cell transcriptomics reveal incomplete epigenetic reprogramming in human female germ cells
    Article Snippet: .. Reverse transcription (RT) was performed by the addition of 5.6 µl Smart-Seq2 RT mix (0.5 µl SuperScriptII (Invitrogen Cat. 18064-014); 2 µl 5 × SuperScriptII buffer; 0.5 µl 100 mM DTT; 2 µl 5 M betaine; 0.1 µl 1 mM MgCl2 ; 0.25 µl Recombinant RNase inhibitor (TaKaRa 40U/µl Ref. 2313A); 0.1 µl 100 µM LNA strand switch primer; 0.15 µl water, per reaction) and incubation (90 min 42 °C; 10 “strand-switch” cycles of (2 min 50 °C; 2 min 70 °C); 4 °C) in a thermal cycler. cDNA amplification: cDNA amplification was performed by the addition of 15 µl of Smart-seq2 PCR mix [12.5 µl 2× KAPA HiFi HotStart ReadyMix (KAPA Biosystems Ref. KK2602); 0.25 µl 10 µM ISPCR primers; 2.25 µl water, per reaction] and incubation [3 min 98 °C; 19 cycles of (20 s (s) 98 °C; 15 s 67 °C; 6 min 72 °C); 5 min 70 °C; 4 °C] in a thermal cycler. .. The cDNA was purified using 0.7:1 volume of AMPure XP beads (Beckman Coulter, Ref. ) according to the manufacturer’s protocol (No. PT5163-1).

    Expressing:

    Article Title: Apoptotic and Early Innate Immune Responses to PB1-F2 Protein of Influenza A Viruses Belonging to Different Subtypes in Human Lung Epithelial A549 Cells
    Article Snippet: .. To determine the mRNA expression of PB1-F2 gene in A549 cells, 500 ng of RNA was reverse transcribed using the Superscript II RT-PCR kit (Invitrogen, Carlsbad, CA, USA) and Uni12 primer and the PB1-F2 gene was amplified as mentioned above. .. The total cellular protein was extracted using radio-immunoprecipitation assay (RIPA) buffer (Sigma, St. Louis, Missouri, USA).

    Article Title: Annexin A1 down-regulation in head and neck squamous cell carcinoma is mediated via transcriptional control with direct involvement of miR-196a/b
    Article Snippet: RNA extraction and real-time RT-PCR Total RNA was extracted from HNSCC cells using Trizol reagent (Invitrogen Life Technologies), and cDNA synthesized with Superscript II RT-PCR System (Invitrogen Life Technologies), according to manufacturer’s protocols. .. Gene expression was analyzed by Real-time PCR using the StepOnePlus Real-Time PCR System (Applied Biosystems) following Applied Biosystems’ SYBR Green Master Mix protocol.

    Article Title: Receptor Tyrosine Kinase-like Orphan Receptor 2 (Ror2) Expression Creates a Poised State of Wnt Signaling in Renal Cancer *
    Article Snippet: Total RNA was extracted from cells using a Qiagen RNeasy mini kit (Valencia, CA). cDNA was made from 500 ng of total RNA using random primers (Invitrogen) and Superscript II RT-PCR reagents (Invitrogen) and analyzed using the ABI 7900HT fast real-time PCR system with the following proprietary FAM-labeled primers: Ror2, Axin2, Fzd1, Jun, 18S, and β-actin (Applied Biosystems, Foster City, CA). .. Wnt-related gene expression was examined with the human Wnt signaling RT2 Profiler PCR array (SABiosciences, Frederick, MD) using cDNA from 786-0 +TAP-hRor2, 786-0, and 786-0 shRor2.2 cells with the ABI 7500 real-time PCR system.

    Article Title: Characterization of Moraxella (Branhamella) catarrhalis lbpB, lbpA, and Lactoferrin Receptor orf3 Isogenic Mutants
    Article Snippet: Neither TbpA expression (solid box) nor TbpB expression (solid circle) was altered by any of the kan insertions. .. The RT-PCR experiments were performed with SuperScript II RNase H− reverse transcriptase (Gibco BRL) according to the manufacturer’s instructions.

    Article Title: Developmental arrest of T cells in RpL22-deficient mice is dependent upon multiple p53 effectors 1
    Article Snippet: Purified mRNA was converted to cDNA using Superscript II reverse transcription PCR with oligo dT(12–18) primers (Invitrogen, Carlsbad, CA). .. Isolated RNA was converted to cDNA by the Multiscribe RT-PCR kit (Applied Biosystems). miR expression was determined by the Taqman Micro RNA assay for miR-34a and sno-202.

    Modification:

    Article Title: Parental haplotype-specific single-cell transcriptomics reveal incomplete epigenetic reprogramming in human female germ cells
    Article Snippet: Single cell isolation from human organs: The tissue culture dishes to use for the single cell picking were first coated for a couple of minutes (min) at room temperature with 0.5% bovine serum albumin (BSA, Life Technologies, Carlsbad, USA) in PBS and then filled with Dulbecco’s Modified Eagle Medium/F12 Nutrient mixture (DMEM/F12, Life Technologies, Paisley, UK). .. Reverse transcription (RT) was performed by the addition of 5.6 µl Smart-Seq2 RT mix (0.5 µl SuperScriptII (Invitrogen Cat. 18064-014); 2 µl 5 × SuperScriptII buffer; 0.5 µl 100 mM DTT; 2 µl 5 M betaine; 0.1 µl 1 mM MgCl2 ; 0.25 µl Recombinant RNase inhibitor (TaKaRa 40U/µl Ref. 2313A); 0.1 µl 100 µM LNA strand switch primer; 0.15 µl water, per reaction) and incubation (90 min 42 °C; 10 “strand-switch” cycles of (2 min 50 °C; 2 min 70 °C); 4 °C) in a thermal cycler. cDNA amplification: cDNA amplification was performed by the addition of 15 µl of Smart-seq2 PCR mix [12.5 µl 2× KAPA HiFi HotStart ReadyMix (KAPA Biosystems Ref. KK2602); 0.25 µl 10 µM ISPCR primers; 2.25 µl water, per reaction] and incubation [3 min 98 °C; 19 cycles of (20 s (s) 98 °C; 15 s 67 °C; 6 min 72 °C); 5 min 70 °C; 4 °C] in a thermal cycler.

    Derivative Assay:

    Article Title: Distinct Signature of Altered Homeostasis in Aging Rod Photoreceptors: Implications for Retinal Diseases
    Article Snippet: .. Amplified photoreceptor cDNA was derived using WT-Ovation Pico System (NuGEN), and whole retina cDNA was derived using Superscript II RT-PCR system (Invitrogen). cDNA was used for real-time qRT-PCR using SYBR(R) Green PCR Mastermix (Applied Biosystems, Foster City, CA) on the 7900HT Fast Real-Time PCR System (Applied Biosystems). .. Candidate genes were selected for analysis based on microarray predictions of fold-change > 2, pattern of change, and gene function.

    Transfection:

    Article Title: Apoptotic and Early Innate Immune Responses to PB1-F2 Protein of Influenza A Viruses Belonging to Different Subtypes in Human Lung Epithelial A549 Cells
    Article Snippet: Paragraph title: 2.5. PB1-F2 mRNA and Protein Expression in Transfected Cells ... To determine the mRNA expression of PB1-F2 gene in A549 cells, 500 ng of RNA was reverse transcribed using the Superscript II RT-PCR kit (Invitrogen, Carlsbad, CA, USA) and Uni12 primer and the PB1-F2 gene was amplified as mentioned above.

    Article Title: Mef2c is an essential regulatory element required for unique expression of the cardiac-specific CARK gene
    Article Snippet: Quantitative real-time RT-PCR analysis SYBR green-based quantitative real-time RT-PCR was used to examine changes in levels of CARK mRNAs in RNAs prepared from primary rat cardiomyocytes transfected with MEF2C morpholino antisense or sense oligonucleotide as described above. .. Total RNAs were isolated using Trizol reagent (Invitrogen) and treated with DNase I (Ambion) according to the manufacturer's instructions. cDNAs were generated using oligo(dT)12-18 as a primer and SuperScript II RT-PCR kit (Invitrogen).cDNA was added to 25 μl of reaction volume containing 12.5 μl of 2× SYBR Green Premix EX Taq (TaKaRa, Otsu, Japan), and 400 nmol/L CARK gene-specific primers (forward: 5′-GACAAAGCAGCCAGGGAA; reverse: 5′-AGCAGGCAATCGGAAGAA).

    Protease Inhibitor:

    Article Title: Apoptotic and Early Innate Immune Responses to PB1-F2 Protein of Influenza A Viruses Belonging to Different Subtypes in Human Lung Epithelial A549 Cells
    Article Snippet: To determine the mRNA expression of PB1-F2 gene in A549 cells, 500 ng of RNA was reverse transcribed using the Superscript II RT-PCR kit (Invitrogen, Carlsbad, CA, USA) and Uni12 primer and the PB1-F2 gene was amplified as mentioned above. .. Protease inhibitor and Phosphatase inhibitor cocktails (Calbiochem, USA) were added to RIPA lysis buffer.

    Generated:

    Article Title: Characterization of Moraxella (Branhamella) catarrhalis lbpB, lbpA, and Lactoferrin Receptor orf3 Isogenic Mutants
    Article Snippet: The RT-PCR experiments were performed with SuperScript II RNase H− reverse transcriptase (Gibco BRL) according to the manufacturer’s instructions. .. Total RNA was isolated with the Qiagen RNeasy mini kit (Qiagen, Inc.) and subjected to amplification-grade DNase I (Gibco BRL) treatment in the presence of RNasin (Promega Corporation). cDNA was generated by using oligonucleotide 728 (Fig. ) and treated with RNase H (Gibco BRL), prior to being used as a template for subsequent PCR.

    Article Title: Mef2c is an essential regulatory element required for unique expression of the cardiac-specific CARK gene
    Article Snippet: .. Total RNAs were isolated using Trizol reagent (Invitrogen) and treated with DNase I (Ambion) according to the manufacturer's instructions. cDNAs were generated using oligo(dT)12-18 as a primer and SuperScript II RT-PCR kit (Invitrogen).cDNA was added to 25 μl of reaction volume containing 12.5 μl of 2× SYBR Green Premix EX Taq (TaKaRa, Otsu, Japan), and 400 nmol/L CARK gene-specific primers (forward: 5′-GACAAAGCAGCCAGGGAA; reverse: 5′-AGCAGGCAATCGGAAGAA). .. To confirm amplification specificity, the PCR products were subjected to a melting curve analysis.

    Article Title: Developmental arrest of T cells in RpL22-deficient mice is dependent upon multiple p53 effectors 1
    Article Snippet: Purified mRNA was converted to cDNA using Superscript II reverse transcription PCR with oligo dT(12–18) primers (Invitrogen, Carlsbad, CA). .. P21waf / Cdkn1a and Bax primer/probe sets were custom generated by the Fox Chase Cancer Center Genomics Core Facility. miR RNA was isolated using the miR Easy system in conjunction with the RNeasy MinElute Cleanup Kit (Qiagen, Valencia, CA).

    Polymerase Chain Reaction:

    Article Title: Parental haplotype-specific single-cell transcriptomics reveal incomplete epigenetic reprogramming in human female germ cells
    Article Snippet: .. Reverse transcription (RT) was performed by the addition of 5.6 µl Smart-Seq2 RT mix (0.5 µl SuperScriptII (Invitrogen Cat. 18064-014); 2 µl 5 × SuperScriptII buffer; 0.5 µl 100 mM DTT; 2 µl 5 M betaine; 0.1 µl 1 mM MgCl2 ; 0.25 µl Recombinant RNase inhibitor (TaKaRa 40U/µl Ref. 2313A); 0.1 µl 100 µM LNA strand switch primer; 0.15 µl water, per reaction) and incubation (90 min 42 °C; 10 “strand-switch” cycles of (2 min 50 °C; 2 min 70 °C); 4 °C) in a thermal cycler. cDNA amplification: cDNA amplification was performed by the addition of 15 µl of Smart-seq2 PCR mix [12.5 µl 2× KAPA HiFi HotStart ReadyMix (KAPA Biosystems Ref. KK2602); 0.25 µl 10 µM ISPCR primers; 2.25 µl water, per reaction] and incubation [3 min 98 °C; 19 cycles of (20 s (s) 98 °C; 15 s 67 °C; 6 min 72 °C); 5 min 70 °C; 4 °C] in a thermal cycler. .. The cDNA was purified using 0.7:1 volume of AMPure XP beads (Beckman Coulter, Ref. ) according to the manufacturer’s protocol (No. PT5163-1).

    Article Title: Gene Regulatory Networks for the Haploid-to-Diploid Transition of Chlamydomonas reinhardtii 1 1 [OPEN]
    Article Snippet: To analyze the relative abundance of transcripts using qRT-PCR, DNase-treated 5 μg of total RNA was reverse-transcribed with Superscript II RT-PCR kit (Invitrogen). .. Primers were designed to produce PCR amplicon lengths of 100–150 bp. qRT–PCR was performed in optical 96-well plates using Bio-Rad CFX96 Real-Time PCR systems.

    Article Title: Receptor Tyrosine Kinase-like Orphan Receptor 2 (Ror2) Expression Creates a Poised State of Wnt Signaling in Renal Cancer *
    Article Snippet: Paragraph title: Quantitative RT-PCR and Human Wnt Signaling RT2 Profiler PCR Array ... Total RNA was extracted from cells using a Qiagen RNeasy mini kit (Valencia, CA). cDNA was made from 500 ng of total RNA using random primers (Invitrogen) and Superscript II RT-PCR reagents (Invitrogen) and analyzed using the ABI 7900HT fast real-time PCR system with the following proprietary FAM-labeled primers: Ror2, Axin2, Fzd1, Jun, 18S, and β-actin (Applied Biosystems, Foster City, CA).

    Article Title: Corky, a gypsy-like retrotransposon is differentially transcribed in Quercus suber tissues
    Article Snippet: .. RNA samples were treated with RQ1 RNase-Free DNase (Promega, Madison, WI). cDNA was synthesized from 2 μg of total RNA using random hexamers and Superscript II RNase H- reverse transcriptase (Invitrogen®, Carlsbad, CA), according to the manufacturer’s recommendations followed by PCR amplification using specific primers for the RVT and a region between Integrase and the chromodomain of Corky (Figure ). .. As expected, amplification products were not obtained in RNA samples not yielded to reverse transcription prior to PCR. cDNA was stored at −20°C.

    Article Title: Distinct Signature of Altered Homeostasis in Aging Rod Photoreceptors: Implications for Retinal Diseases
    Article Snippet: .. Amplified photoreceptor cDNA was derived using WT-Ovation Pico System (NuGEN), and whole retina cDNA was derived using Superscript II RT-PCR system (Invitrogen). cDNA was used for real-time qRT-PCR using SYBR(R) Green PCR Mastermix (Applied Biosystems, Foster City, CA) on the 7900HT Fast Real-Time PCR System (Applied Biosystems). .. Candidate genes were selected for analysis based on microarray predictions of fold-change > 2, pattern of change, and gene function.

    Article Title: Characterization of Moraxella (Branhamella) catarrhalis lbpB, lbpA, and Lactoferrin Receptor orf3 Isogenic Mutants
    Article Snippet: The RT-PCR experiments were performed with SuperScript II RNase H− reverse transcriptase (Gibco BRL) according to the manufacturer’s instructions. .. Total RNA was isolated with the Qiagen RNeasy mini kit (Qiagen, Inc.) and subjected to amplification-grade DNase I (Gibco BRL) treatment in the presence of RNasin (Promega Corporation). cDNA was generated by using oligonucleotide 728 (Fig. ) and treated with RNase H (Gibco BRL), prior to being used as a template for subsequent PCR.

    Article Title: Mef2c is an essential regulatory element required for unique expression of the cardiac-specific CARK gene
    Article Snippet: Total RNAs were isolated using Trizol reagent (Invitrogen) and treated with DNase I (Ambion) according to the manufacturer's instructions. cDNAs were generated using oligo(dT)12-18 as a primer and SuperScript II RT-PCR kit (Invitrogen).cDNA was added to 25 μl of reaction volume containing 12.5 μl of 2× SYBR Green Premix EX Taq (TaKaRa, Otsu, Japan), and 400 nmol/L CARK gene-specific primers (forward: 5′-GACAAAGCAGCCAGGGAA; reverse: 5′-AGCAGGCAATCGGAAGAA). .. To confirm amplification specificity, the PCR products were subjected to a melting curve analysis.

    Article Title: Developmental arrest of T cells in RpL22-deficient mice is dependent upon multiple p53 effectors 1
    Article Snippet: .. Purified mRNA was converted to cDNA using Superscript II reverse transcription PCR with oligo dT(12–18) primers (Invitrogen, Carlsbad, CA). .. Quantitative real-time PCR was performed on the Prism 7700 thermocycler (Applied Biosystems, Carlsbad, CA) using Taqman Real-time PCR primer/probe sets specific to murine PUMA/ Bbc3 (Mm00519268_m1), Noxa/ Pmaip1 (Mm00451763_m1), and Bim/( Bcl2l-11 ) (Mm01333921_m1).

    Single-cell Isolation:

    Article Title: Parental haplotype-specific single-cell transcriptomics reveal incomplete epigenetic reprogramming in human female germ cells
    Article Snippet: Single cell isolation from human organs: The tissue culture dishes to use for the single cell picking were first coated for a couple of minutes (min) at room temperature with 0.5% bovine serum albumin (BSA, Life Technologies, Carlsbad, USA) in PBS and then filled with Dulbecco’s Modified Eagle Medium/F12 Nutrient mixture (DMEM/F12, Life Technologies, Paisley, UK). .. Reverse transcription (RT) was performed by the addition of 5.6 µl Smart-Seq2 RT mix (0.5 µl SuperScriptII (Invitrogen Cat. 18064-014); 2 µl 5 × SuperScriptII buffer; 0.5 µl 100 mM DTT; 2 µl 5 M betaine; 0.1 µl 1 mM MgCl2 ; 0.25 µl Recombinant RNase inhibitor (TaKaRa 40U/µl Ref. 2313A); 0.1 µl 100 µM LNA strand switch primer; 0.15 µl water, per reaction) and incubation (90 min 42 °C; 10 “strand-switch” cycles of (2 min 50 °C; 2 min 70 °C); 4 °C) in a thermal cycler. cDNA amplification: cDNA amplification was performed by the addition of 15 µl of Smart-seq2 PCR mix [12.5 µl 2× KAPA HiFi HotStart ReadyMix (KAPA Biosystems Ref. KK2602); 0.25 µl 10 µM ISPCR primers; 2.25 µl water, per reaction] and incubation [3 min 98 °C; 19 cycles of (20 s (s) 98 °C; 15 s 67 °C; 6 min 72 °C); 5 min 70 °C; 4 °C] in a thermal cycler.

    Sequencing:

    Article Title: Parental haplotype-specific single-cell transcriptomics reveal incomplete epigenetic reprogramming in human female germ cells
    Article Snippet: Reverse transcription (RT) was performed by the addition of 5.6 µl Smart-Seq2 RT mix (0.5 µl SuperScriptII (Invitrogen Cat. 18064-014); 2 µl 5 × SuperScriptII buffer; 0.5 µl 100 mM DTT; 2 µl 5 M betaine; 0.1 µl 1 mM MgCl2 ; 0.25 µl Recombinant RNase inhibitor (TaKaRa 40U/µl Ref. 2313A); 0.1 µl 100 µM LNA strand switch primer; 0.15 µl water, per reaction) and incubation (90 min 42 °C; 10 “strand-switch” cycles of (2 min 50 °C; 2 min 70 °C); 4 °C) in a thermal cycler. cDNA amplification: cDNA amplification was performed by the addition of 15 µl of Smart-seq2 PCR mix [12.5 µl 2× KAPA HiFi HotStart ReadyMix (KAPA Biosystems Ref. KK2602); 0.25 µl 10 µM ISPCR primers; 2.25 µl water, per reaction] and incubation [3 min 98 °C; 19 cycles of (20 s (s) 98 °C; 15 s 67 °C; 6 min 72 °C); 5 min 70 °C; 4 °C] in a thermal cycler. .. Tagmentation and sequencing: Successful cDNA libraries were tagmented using the transposase Tn5 .

    Article Title: Time-course microarray analysis for identifying candidate genes involved in obesity-associated pathological changes in the mouse colon
    Article Snippet: Template RNA isolated from the colon tissue was reverse transcribed using Superscript™ II RT-PCR System (Invitrogen, Karlsruhe, Germany), according to the manufacturer’s instructions, for performing dT 20-primed complementary DNA (cDNA) synthesis. .. Next, real-time qPCR was performed using an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) in 384-well microtiter plates containing a final reaction volume of 10 μl.

    Article Title: Epstein-Barr Virus Latent Nuclear Antigens Can Induce Metastasis in a Nude Mouse Model ▿
    Article Snippet: RT was carried out with SuperScript II RNase H-reverse transcriptase (Life Technologies, Gaithersburg, MD). .. The Nm23-H1-Myc transcript was amplified for 30 cycles (30 s at 94°C, 1 min at 50°C, and 1 min at 72°C) using the forward primer 5′-GATTACACGAGCTGTGCTCA-3′ and the reverse primer 5′-TTCGCTAGCCAAGTCTTCTT-3′. designed to amplify junction sequence between Nm23-H1 and Myc tag.

    Recombinant:

    Article Title: Parental haplotype-specific single-cell transcriptomics reveal incomplete epigenetic reprogramming in human female germ cells
    Article Snippet: .. Reverse transcription (RT) was performed by the addition of 5.6 µl Smart-Seq2 RT mix (0.5 µl SuperScriptII (Invitrogen Cat. 18064-014); 2 µl 5 × SuperScriptII buffer; 0.5 µl 100 mM DTT; 2 µl 5 M betaine; 0.1 µl 1 mM MgCl2 ; 0.25 µl Recombinant RNase inhibitor (TaKaRa 40U/µl Ref. 2313A); 0.1 µl 100 µM LNA strand switch primer; 0.15 µl water, per reaction) and incubation (90 min 42 °C; 10 “strand-switch” cycles of (2 min 50 °C; 2 min 70 °C); 4 °C) in a thermal cycler. cDNA amplification: cDNA amplification was performed by the addition of 15 µl of Smart-seq2 PCR mix [12.5 µl 2× KAPA HiFi HotStart ReadyMix (KAPA Biosystems Ref. KK2602); 0.25 µl 10 µM ISPCR primers; 2.25 µl water, per reaction] and incubation [3 min 98 °C; 19 cycles of (20 s (s) 98 °C; 15 s 67 °C; 6 min 72 °C); 5 min 70 °C; 4 °C] in a thermal cycler. .. The cDNA was purified using 0.7:1 volume of AMPure XP beads (Beckman Coulter, Ref. ) according to the manufacturer’s protocol (No. PT5163-1).

    Cellular Antioxidant Activity Assay:

    Article Title: Functional endogenous LINE-1 retrotransposons are expressed and mobilized in rat chloroleukemia cells
    Article Snippet: Cloning of transcribed L1Rn sequences from RCL cells Total and poly(A)+ RNA were prepared from control and irradiated RCL cells with the RNeasy and Oligotex mRNA kits (Qiagen), respectively. cDNA was synthesized from poly(A)+ RNA employing SuperScript™ II reverse transcriptase (Invitrogen) and oligo(dT) primer. .. About 6 kb long L1Rn sequences encompassing ORF1 and ORF2 were amplified from cDNA with the Taq/Pwo Proof Mix (AGS-Hybaid) and primers Pr-L1Rn1 (5′-GGA AGA GAC CAC CAA CAC TGC TCA C-3′, pos.

    RNA Sequencing Assay:

    Article Title: Parental haplotype-specific single-cell transcriptomics reveal incomplete epigenetic reprogramming in human female germ cells
    Article Snippet: Paragraph title: Single-cell RNA-sequencing ... Reverse transcription (RT) was performed by the addition of 5.6 µl Smart-Seq2 RT mix (0.5 µl SuperScriptII (Invitrogen Cat. 18064-014); 2 µl 5 × SuperScriptII buffer; 0.5 µl 100 mM DTT; 2 µl 5 M betaine; 0.1 µl 1 mM MgCl2 ; 0.25 µl Recombinant RNase inhibitor (TaKaRa 40U/µl Ref. 2313A); 0.1 µl 100 µM LNA strand switch primer; 0.15 µl water, per reaction) and incubation (90 min 42 °C; 10 “strand-switch” cycles of (2 min 50 °C; 2 min 70 °C); 4 °C) in a thermal cycler. cDNA amplification: cDNA amplification was performed by the addition of 15 µl of Smart-seq2 PCR mix [12.5 µl 2× KAPA HiFi HotStart ReadyMix (KAPA Biosystems Ref. KK2602); 0.25 µl 10 µM ISPCR primers; 2.25 µl water, per reaction] and incubation [3 min 98 °C; 19 cycles of (20 s (s) 98 °C; 15 s 67 °C; 6 min 72 °C); 5 min 70 °C; 4 °C] in a thermal cycler.

    Irradiation:

    Article Title: Functional endogenous LINE-1 retrotransposons are expressed and mobilized in rat chloroleukemia cells
    Article Snippet: .. Cloning of transcribed L1Rn sequences from RCL cells Total and poly(A)+ RNA were prepared from control and irradiated RCL cells with the RNeasy and Oligotex mRNA kits (Qiagen), respectively. cDNA was synthesized from poly(A)+ RNA employing SuperScript™ II reverse transcriptase (Invitrogen) and oligo(dT) primer. .. About 6 kb long L1Rn sequences encompassing ORF1 and ORF2 were amplified from cDNA with the Taq/Pwo Proof Mix (AGS-Hybaid) and primers Pr-L1Rn1 (5′-GGA AGA GAC CAC CAA CAC TGC TCA C-3′, pos.

    Isolation:

    Article Title: Time-course microarray analysis for identifying candidate genes involved in obesity-associated pathological changes in the mouse colon
    Article Snippet: .. Template RNA isolated from the colon tissue was reverse transcribed using Superscript™ II RT-PCR System (Invitrogen, Karlsruhe, Germany), according to the manufacturer’s instructions, for performing dT 20-primed complementary DNA (cDNA) synthesis. .. Next, real-time qPCR was performed using an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) in 384-well microtiter plates containing a final reaction volume of 10 μl.

    Article Title: Corky, a gypsy-like retrotransposon is differentially transcribed in Quercus suber tissues
    Article Snippet: Paragraph title: RNA isolation and cDNA preparation for RT-qPCR ... RNA samples were treated with RQ1 RNase-Free DNase (Promega, Madison, WI). cDNA was synthesized from 2 μg of total RNA using random hexamers and Superscript II RNase H- reverse transcriptase (Invitrogen®, Carlsbad, CA), according to the manufacturer’s recommendations followed by PCR amplification using specific primers for the RVT and a region between Integrase and the chromodomain of Corky (Figure ).

    Article Title: Epstein-Barr Virus Latent Nuclear Antigens Can Induce Metastasis in a Nude Mouse Model ▿
    Article Snippet: Tissues were homogenized by using a tissue tearer (Biospec Products, Inc., Bartlesville, OK) prior to processing for RNA isolation. .. RT was carried out with SuperScript II RNase H-reverse transcriptase (Life Technologies, Gaithersburg, MD).

    Article Title: Characterization of Moraxella (Branhamella) catarrhalis lbpB, lbpA, and Lactoferrin Receptor orf3 Isogenic Mutants
    Article Snippet: The RT-PCR experiments were performed with SuperScript II RNase H− reverse transcriptase (Gibco BRL) according to the manufacturer’s instructions. .. Total RNA was isolated with the Qiagen RNeasy mini kit (Qiagen, Inc.) and subjected to amplification-grade DNase I (Gibco BRL) treatment in the presence of RNasin (Promega Corporation). cDNA was generated by using oligonucleotide 728 (Fig. ) and treated with RNase H (Gibco BRL), prior to being used as a template for subsequent PCR.

    Article Title: Mef2c is an essential regulatory element required for unique expression of the cardiac-specific CARK gene
    Article Snippet: .. Total RNAs were isolated using Trizol reagent (Invitrogen) and treated with DNase I (Ambion) according to the manufacturer's instructions. cDNAs were generated using oligo(dT)12-18 as a primer and SuperScript II RT-PCR kit (Invitrogen).cDNA was added to 25 μl of reaction volume containing 12.5 μl of 2× SYBR Green Premix EX Taq (TaKaRa, Otsu, Japan), and 400 nmol/L CARK gene-specific primers (forward: 5′-GACAAAGCAGCCAGGGAA; reverse: 5′-AGCAGGCAATCGGAAGAA). .. To confirm amplification specificity, the PCR products were subjected to a melting curve analysis.

    Article Title: Developmental arrest of T cells in RpL22-deficient mice is dependent upon multiple p53 effectors 1
    Article Snippet: Total RNA was isolated from primary cells using the RNA-Easy system (Qiagen, Valencia, Ca). .. Purified mRNA was converted to cDNA using Superscript II reverse transcription PCR with oligo dT(12–18) primers (Invitrogen, Carlsbad, CA).

    Flow Cytometry:

    Article Title: Distinct Signature of Altered Homeostasis in Aging Rod Photoreceptors: Implications for Retinal Diseases
    Article Snippet: Quantitative RT-PCR Retinas were dissected from four independent biological replicates for each of the three timepoints (1.5, 5, and 12 months), enzymatically dissociated as described above, and rod photoreceptors were flow-sorted into RNAprotect (Qiagen). .. Amplified photoreceptor cDNA was derived using WT-Ovation Pico System (NuGEN), and whole retina cDNA was derived using Superscript II RT-PCR system (Invitrogen). cDNA was used for real-time qRT-PCR using SYBR(R) Green PCR Mastermix (Applied Biosystems, Foster City, CA) on the 7900HT Fast Real-Time PCR System (Applied Biosystems).

    Microscopy:

    Article Title: Parental haplotype-specific single-cell transcriptomics reveal incomplete epigenetic reprogramming in human female germ cells
    Article Snippet: Single cells were then manually picked under a stereo microscope (Zeiss, Sliedrecht, the Netherlands) using a pulled glass capillary in picking volumes of ±0.5 µl medium and transferred to ice-cold PCR tubes containing 2.0 µl lysis buffer (1.9 µl 0.2% TritonX-100 in water + 0.1 µl Recombinant RNase inhibitor (TaKaRa 40U/µl Ref. 2313A) per tube), snap-frozen on dry ice and stored at −80 °C. .. Reverse transcription (RT) was performed by the addition of 5.6 µl Smart-Seq2 RT mix (0.5 µl SuperScriptII (Invitrogen Cat. 18064-014); 2 µl 5 × SuperScriptII buffer; 0.5 µl 100 mM DTT; 2 µl 5 M betaine; 0.1 µl 1 mM MgCl2 ; 0.25 µl Recombinant RNase inhibitor (TaKaRa 40U/µl Ref. 2313A); 0.1 µl 100 µM LNA strand switch primer; 0.15 µl water, per reaction) and incubation (90 min 42 °C; 10 “strand-switch” cycles of (2 min 50 °C; 2 min 70 °C); 4 °C) in a thermal cycler. cDNA amplification: cDNA amplification was performed by the addition of 15 µl of Smart-seq2 PCR mix [12.5 µl 2× KAPA HiFi HotStart ReadyMix (KAPA Biosystems Ref. KK2602); 0.25 µl 10 µM ISPCR primers; 2.25 µl water, per reaction] and incubation [3 min 98 °C; 19 cycles of (20 s (s) 98 °C; 15 s 67 °C; 6 min 72 °C); 5 min 70 °C; 4 °C] in a thermal cycler.

    Purification:

    Article Title: Parental haplotype-specific single-cell transcriptomics reveal incomplete epigenetic reprogramming in human female germ cells
    Article Snippet: Reverse transcription (RT) was performed by the addition of 5.6 µl Smart-Seq2 RT mix (0.5 µl SuperScriptII (Invitrogen Cat. 18064-014); 2 µl 5 × SuperScriptII buffer; 0.5 µl 100 mM DTT; 2 µl 5 M betaine; 0.1 µl 1 mM MgCl2 ; 0.25 µl Recombinant RNase inhibitor (TaKaRa 40U/µl Ref. 2313A); 0.1 µl 100 µM LNA strand switch primer; 0.15 µl water, per reaction) and incubation (90 min 42 °C; 10 “strand-switch” cycles of (2 min 50 °C; 2 min 70 °C); 4 °C) in a thermal cycler. cDNA amplification: cDNA amplification was performed by the addition of 15 µl of Smart-seq2 PCR mix [12.5 µl 2× KAPA HiFi HotStart ReadyMix (KAPA Biosystems Ref. KK2602); 0.25 µl 10 µM ISPCR primers; 2.25 µl water, per reaction] and incubation [3 min 98 °C; 19 cycles of (20 s (s) 98 °C; 15 s 67 °C; 6 min 72 °C); 5 min 70 °C; 4 °C] in a thermal cycler. .. The cDNA was purified using 0.7:1 volume of AMPure XP beads (Beckman Coulter, Ref. ) according to the manufacturer’s protocol (No. PT5163-1).

    Article Title: Apoptotic and Early Innate Immune Responses to PB1-F2 Protein of Influenza A Viruses Belonging to Different Subtypes in Human Lung Epithelial A549 Cells
    Article Snippet: RNA was purified using RNeasy mini kit (Qiagen, Carlsbad, CA) and their yields were evaluated in a Nanodrop spectrophotometer (Nanodrop technologies, Wilmington, DE, USA) at 260 nm. .. To determine the mRNA expression of PB1-F2 gene in A549 cells, 500 ng of RNA was reverse transcribed using the Superscript II RT-PCR kit (Invitrogen, Carlsbad, CA, USA) and Uni12 primer and the PB1-F2 gene was amplified as mentioned above.

    Article Title: Developmental arrest of T cells in RpL22-deficient mice is dependent upon multiple p53 effectors 1
    Article Snippet: .. Purified mRNA was converted to cDNA using Superscript II reverse transcription PCR with oligo dT(12–18) primers (Invitrogen, Carlsbad, CA). .. Quantitative real-time PCR was performed on the Prism 7700 thermocycler (Applied Biosystems, Carlsbad, CA) using Taqman Real-time PCR primer/probe sets specific to murine PUMA/ Bbc3 (Mm00519268_m1), Noxa/ Pmaip1 (Mm00451763_m1), and Bim/( Bcl2l-11 ) (Mm01333921_m1).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Apoptotic and Early Innate Immune Responses to PB1-F2 Protein of Influenza A Viruses Belonging to Different Subtypes in Human Lung Epithelial A549 Cells
    Article Snippet: .. To determine the mRNA expression of PB1-F2 gene in A549 cells, 500 ng of RNA was reverse transcribed using the Superscript II RT-PCR kit (Invitrogen, Carlsbad, CA, USA) and Uni12 primer and the PB1-F2 gene was amplified as mentioned above. .. The total cellular protein was extracted using radio-immunoprecipitation assay (RIPA) buffer (Sigma, St. Louis, Missouri, USA).

    Article Title: Time-course microarray analysis for identifying candidate genes involved in obesity-associated pathological changes in the mouse colon
    Article Snippet: .. Template RNA isolated from the colon tissue was reverse transcribed using Superscript™ II RT-PCR System (Invitrogen, Karlsruhe, Germany), according to the manufacturer’s instructions, for performing dT 20-primed complementary DNA (cDNA) synthesis. .. Next, real-time qPCR was performed using an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) in 384-well microtiter plates containing a final reaction volume of 10 μl.

    Article Title: Gene Regulatory Networks for the Haploid-to-Diploid Transition of Chlamydomonas reinhardtii 1 1 [OPEN]
    Article Snippet: .. To analyze the relative abundance of transcripts using qRT-PCR, DNase-treated 5 μg of total RNA was reverse-transcribed with Superscript II RT-PCR kit (Invitrogen). .. Primers were designed to produce PCR amplicon lengths of 100–150 bp. qRT–PCR was performed in optical 96-well plates using Bio-Rad CFX96 Real-Time PCR systems.

    Article Title: Annexin A1 down-regulation in head and neck squamous cell carcinoma is mediated via transcriptional control with direct involvement of miR-196a/b
    Article Snippet: .. RNA extraction and real-time RT-PCR Total RNA was extracted from HNSCC cells using Trizol reagent (Invitrogen Life Technologies), and cDNA synthesized with Superscript II RT-PCR System (Invitrogen Life Technologies), according to manufacturer’s protocols. .. Gene expression was analyzed by Real-time PCR using the StepOnePlus Real-Time PCR System (Applied Biosystems) following Applied Biosystems’ SYBR Green Master Mix protocol.

    Article Title: Receptor Tyrosine Kinase-like Orphan Receptor 2 (Ror2) Expression Creates a Poised State of Wnt Signaling in Renal Cancer *
    Article Snippet: .. Total RNA was extracted from cells using a Qiagen RNeasy mini kit (Valencia, CA). cDNA was made from 500 ng of total RNA using random primers (Invitrogen) and Superscript II RT-PCR reagents (Invitrogen) and analyzed using the ABI 7900HT fast real-time PCR system with the following proprietary FAM-labeled primers: Ror2, Axin2, Fzd1, Jun, 18S, and β-actin (Applied Biosystems, Foster City, CA). .. Wnt-related gene expression was examined with the human Wnt signaling RT2 Profiler PCR array (SABiosciences, Frederick, MD) using cDNA from 786-0 +TAP-hRor2, 786-0, and 786-0 shRor2.2 cells with the ABI 7500 real-time PCR system.

    Article Title: Distinct Signature of Altered Homeostasis in Aging Rod Photoreceptors: Implications for Retinal Diseases
    Article Snippet: .. Amplified photoreceptor cDNA was derived using WT-Ovation Pico System (NuGEN), and whole retina cDNA was derived using Superscript II RT-PCR system (Invitrogen). cDNA was used for real-time qRT-PCR using SYBR(R) Green PCR Mastermix (Applied Biosystems, Foster City, CA) on the 7900HT Fast Real-Time PCR System (Applied Biosystems). .. Candidate genes were selected for analysis based on microarray predictions of fold-change > 2, pattern of change, and gene function.

    Article Title: Epstein-Barr Virus Latent Nuclear Antigens Can Induce Metastasis in a Nude Mouse Model ▿
    Article Snippet: Paragraph title: RT-PCR analysis for levels of viral and cellular gene transcripts. ... RT was carried out with SuperScript II RNase H-reverse transcriptase (Life Technologies, Gaithersburg, MD).

    Article Title: Mef2c is an essential regulatory element required for unique expression of the cardiac-specific CARK gene
    Article Snippet: .. Total RNAs were isolated using Trizol reagent (Invitrogen) and treated with DNase I (Ambion) according to the manufacturer's instructions. cDNAs were generated using oligo(dT)12-18 as a primer and SuperScript II RT-PCR kit (Invitrogen).cDNA was added to 25 μl of reaction volume containing 12.5 μl of 2× SYBR Green Premix EX Taq (TaKaRa, Otsu, Japan), and 400 nmol/L CARK gene-specific primers (forward: 5′-GACAAAGCAGCCAGGGAA; reverse: 5′-AGCAGGCAATCGGAAGAA). .. To confirm amplification specificity, the PCR products were subjected to a melting curve analysis.

    Article Title: Functional Characterization of the Infection-Inducible Peptide Edin in Drosophila melanogaster
    Article Snippet: .. Semi-quantitative and quantitative RT-PCR Semi-quantitative RT-PCR reactions for edin , Attacin A and Act5C were performed using Super-Script™ II One-Step RT-PCR with Platinum Taq kit (Invitrogen/Life Technologies, Carlsbad, CA, USA). ..

    Article Title: Developmental arrest of T cells in RpL22-deficient mice is dependent upon multiple p53 effectors 1
    Article Snippet: Purified mRNA was converted to cDNA using Superscript II reverse transcription PCR with oligo dT(12–18) primers (Invitrogen, Carlsbad, CA). .. Isolated RNA was converted to cDNA by the Multiscribe RT-PCR kit (Applied Biosystems). miR expression was determined by the Taqman Micro RNA assay for miR-34a and sno-202.

    Nucleic Acid Concentration:

    Article Title: Corky, a gypsy-like retrotransposon is differentially transcribed in Quercus suber tissues
    Article Snippet: Nucleic acid concentration of each sample was quantified by spectrophotometry using the software Gen5 1.09 (Synergy HT, Bio-Tek Instruments, Winooski, USA). .. RNA samples were treated with RQ1 RNase-Free DNase (Promega, Madison, WI). cDNA was synthesized from 2 μg of total RNA using random hexamers and Superscript II RNase H- reverse transcriptase (Invitrogen®, Carlsbad, CA), according to the manufacturer’s recommendations followed by PCR amplification using specific primers for the RVT and a region between Integrase and the chromodomain of Corky (Figure ).

    Software:

    Article Title: Time-course microarray analysis for identifying candidate genes involved in obesity-associated pathological changes in the mouse colon
    Article Snippet: Template RNA isolated from the colon tissue was reverse transcribed using Superscript™ II RT-PCR System (Invitrogen, Karlsruhe, Germany), according to the manufacturer’s instructions, for performing dT 20-primed complementary DNA (cDNA) synthesis. .. Amplifications were performed using the following protocol: initial template denaturation at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. All the samples were amplified in triplicate, and data were analyzed using Sequence Detector software (Applied Biosystems).

    Article Title: Corky, a gypsy-like retrotransposon is differentially transcribed in Quercus suber tissues
    Article Snippet: Nucleic acid concentration of each sample was quantified by spectrophotometry using the software Gen5 1.09 (Synergy HT, Bio-Tek Instruments, Winooski, USA). .. RNA samples were treated with RQ1 RNase-Free DNase (Promega, Madison, WI). cDNA was synthesized from 2 μg of total RNA using random hexamers and Superscript II RNase H- reverse transcriptase (Invitrogen®, Carlsbad, CA), according to the manufacturer’s recommendations followed by PCR amplification using specific primers for the RVT and a region between Integrase and the chromodomain of Corky (Figure ).

    Article Title: Functional Characterization of the Infection-Inducible Peptide Edin in Drosophila melanogaster
    Article Snippet: Semi-quantitative and quantitative RT-PCR Semi-quantitative RT-PCR reactions for edin , Attacin A and Act5C were performed using Super-Script™ II One-Step RT-PCR with Platinum Taq kit (Invitrogen/Life Technologies, Carlsbad, CA, USA). .. Results were analyzed with the ABI 7000 System SDS software version 1.2.3.

    Real-time Polymerase Chain Reaction:

    Article Title: Time-course microarray analysis for identifying candidate genes involved in obesity-associated pathological changes in the mouse colon
    Article Snippet: Paragraph title: Real-time quantitative polymerase chain reaction analysis ... Template RNA isolated from the colon tissue was reverse transcribed using Superscript™ II RT-PCR System (Invitrogen, Karlsruhe, Germany), according to the manufacturer’s instructions, for performing dT 20-primed complementary DNA (cDNA) synthesis.

    Article Title: Gene Regulatory Networks for the Haploid-to-Diploid Transition of Chlamydomonas reinhardtii 1 1 [OPEN]
    Article Snippet: To analyze the relative abundance of transcripts using qRT-PCR, DNase-treated 5 μg of total RNA was reverse-transcribed with Superscript II RT-PCR kit (Invitrogen). .. Primers were designed to produce PCR amplicon lengths of 100–150 bp. qRT–PCR was performed in optical 96-well plates using Bio-Rad CFX96 Real-Time PCR systems.

    Article Title: Annexin A1 down-regulation in head and neck squamous cell carcinoma is mediated via transcriptional control with direct involvement of miR-196a/b
    Article Snippet: RNA extraction and real-time RT-PCR Total RNA was extracted from HNSCC cells using Trizol reagent (Invitrogen Life Technologies), and cDNA synthesized with Superscript II RT-PCR System (Invitrogen Life Technologies), according to manufacturer’s protocols. .. Gene expression was analyzed by Real-time PCR using the StepOnePlus Real-Time PCR System (Applied Biosystems) following Applied Biosystems’ SYBR Green Master Mix protocol.

    Article Title: Receptor Tyrosine Kinase-like Orphan Receptor 2 (Ror2) Expression Creates a Poised State of Wnt Signaling in Renal Cancer *
    Article Snippet: .. Total RNA was extracted from cells using a Qiagen RNeasy mini kit (Valencia, CA). cDNA was made from 500 ng of total RNA using random primers (Invitrogen) and Superscript II RT-PCR reagents (Invitrogen) and analyzed using the ABI 7900HT fast real-time PCR system with the following proprietary FAM-labeled primers: Ror2, Axin2, Fzd1, Jun, 18S, and β-actin (Applied Biosystems, Foster City, CA). .. Wnt-related gene expression was examined with the human Wnt signaling RT2 Profiler PCR array (SABiosciences, Frederick, MD) using cDNA from 786-0 +TAP-hRor2, 786-0, and 786-0 shRor2.2 cells with the ABI 7500 real-time PCR system.

    Article Title: Distinct Signature of Altered Homeostasis in Aging Rod Photoreceptors: Implications for Retinal Diseases
    Article Snippet: .. Amplified photoreceptor cDNA was derived using WT-Ovation Pico System (NuGEN), and whole retina cDNA was derived using Superscript II RT-PCR system (Invitrogen). cDNA was used for real-time qRT-PCR using SYBR(R) Green PCR Mastermix (Applied Biosystems, Foster City, CA) on the 7900HT Fast Real-Time PCR System (Applied Biosystems). .. Candidate genes were selected for analysis based on microarray predictions of fold-change > 2, pattern of change, and gene function.

    Article Title: Mef2c is an essential regulatory element required for unique expression of the cardiac-specific CARK gene
    Article Snippet: Total RNAs were isolated using Trizol reagent (Invitrogen) and treated with DNase I (Ambion) according to the manufacturer's instructions. cDNAs were generated using oligo(dT)12-18 as a primer and SuperScript II RT-PCR kit (Invitrogen).cDNA was added to 25 μl of reaction volume containing 12.5 μl of 2× SYBR Green Premix EX Taq (TaKaRa, Otsu, Japan), and 400 nmol/L CARK gene-specific primers (forward: 5′-GACAAAGCAGCCAGGGAA; reverse: 5′-AGCAGGCAATCGGAAGAA). .. Assays were performed in triplicate with a DNA Engine Opticon 2 Real Time PCR Detector (Bio-Rad, Richmond, CA, USA).

    Article Title: Developmental arrest of T cells in RpL22-deficient mice is dependent upon multiple p53 effectors 1
    Article Snippet: Paragraph title: Quantitative real-time PCR ... Purified mRNA was converted to cDNA using Superscript II reverse transcription PCR with oligo dT(12–18) primers (Invitrogen, Carlsbad, CA).

    RNA Extraction:

    Article Title: Annexin A1 down-regulation in head and neck squamous cell carcinoma is mediated via transcriptional control with direct involvement of miR-196a/b
    Article Snippet: .. RNA extraction and real-time RT-PCR Total RNA was extracted from HNSCC cells using Trizol reagent (Invitrogen Life Technologies), and cDNA synthesized with Superscript II RT-PCR System (Invitrogen Life Technologies), according to manufacturer’s protocols. .. Gene expression was analyzed by Real-time PCR using the StepOnePlus Real-Time PCR System (Applied Biosystems) following Applied Biosystems’ SYBR Green Master Mix protocol.

    Agarose Gel Electrophoresis:

    Article Title: Corky, a gypsy-like retrotransposon is differentially transcribed in Quercus suber tissues
    Article Snippet: Total RNA integrity was tested through 1% agarose gel electrophoresis under denaturing conditions. .. RNA samples were treated with RQ1 RNase-Free DNase (Promega, Madison, WI). cDNA was synthesized from 2 μg of total RNA using random hexamers and Superscript II RNase H- reverse transcriptase (Invitrogen®, Carlsbad, CA), according to the manufacturer’s recommendations followed by PCR amplification using specific primers for the RVT and a region between Integrase and the chromodomain of Corky (Figure ).

    Radio Immunoprecipitation:

    Article Title: Apoptotic and Early Innate Immune Responses to PB1-F2 Protein of Influenza A Viruses Belonging to Different Subtypes in Human Lung Epithelial A549 Cells
    Article Snippet: To determine the mRNA expression of PB1-F2 gene in A549 cells, 500 ng of RNA was reverse transcribed using the Superscript II RT-PCR kit (Invitrogen, Carlsbad, CA, USA) and Uni12 primer and the PB1-F2 gene was amplified as mentioned above. .. The total cellular protein was extracted using radio-immunoprecipitation assay (RIPA) buffer (Sigma, St. Louis, Missouri, USA).

    Spectrophotometry:

    Article Title: Apoptotic and Early Innate Immune Responses to PB1-F2 Protein of Influenza A Viruses Belonging to Different Subtypes in Human Lung Epithelial A549 Cells
    Article Snippet: RNA was purified using RNeasy mini kit (Qiagen, Carlsbad, CA) and their yields were evaluated in a Nanodrop spectrophotometer (Nanodrop technologies, Wilmington, DE, USA) at 260 nm. .. To determine the mRNA expression of PB1-F2 gene in A549 cells, 500 ng of RNA was reverse transcribed using the Superscript II RT-PCR kit (Invitrogen, Carlsbad, CA, USA) and Uni12 primer and the PB1-F2 gene was amplified as mentioned above.

    Article Title: Corky, a gypsy-like retrotransposon is differentially transcribed in Quercus suber tissues
    Article Snippet: Nucleic acid concentration of each sample was quantified by spectrophotometry using the software Gen5 1.09 (Synergy HT, Bio-Tek Instruments, Winooski, USA). .. RNA samples were treated with RQ1 RNase-Free DNase (Promega, Madison, WI). cDNA was synthesized from 2 μg of total RNA using random hexamers and Superscript II RNase H- reverse transcriptase (Invitrogen®, Carlsbad, CA), according to the manufacturer’s recommendations followed by PCR amplification using specific primers for the RVT and a region between Integrase and the chromodomain of Corky (Figure ).

    Concentration Assay:

    Article Title: Parental haplotype-specific single-cell transcriptomics reveal incomplete epigenetic reprogramming in human female germ cells
    Article Snippet: Reverse transcription (RT) was performed by the addition of 5.6 µl Smart-Seq2 RT mix (0.5 µl SuperScriptII (Invitrogen Cat. 18064-014); 2 µl 5 × SuperScriptII buffer; 0.5 µl 100 mM DTT; 2 µl 5 M betaine; 0.1 µl 1 mM MgCl2 ; 0.25 µl Recombinant RNase inhibitor (TaKaRa 40U/µl Ref. 2313A); 0.1 µl 100 µM LNA strand switch primer; 0.15 µl water, per reaction) and incubation (90 min 42 °C; 10 “strand-switch” cycles of (2 min 50 °C; 2 min 70 °C); 4 °C) in a thermal cycler. cDNA amplification: cDNA amplification was performed by the addition of 15 µl of Smart-seq2 PCR mix [12.5 µl 2× KAPA HiFi HotStart ReadyMix (KAPA Biosystems Ref. KK2602); 0.25 µl 10 µM ISPCR primers; 2.25 µl water, per reaction] and incubation [3 min 98 °C; 19 cycles of (20 s (s) 98 °C; 15 s 67 °C; 6 min 72 °C); 5 min 70 °C; 4 °C] in a thermal cycler. .. The purified cDNA for each cell was inspected on an Agilent 2100 Bioanalyzer to determine cDNA concentration and size distribution, using Agilent High Sensitivity DNA chips (Ref. 5067-4626).

    Lysis:

    Article Title: Parental haplotype-specific single-cell transcriptomics reveal incomplete epigenetic reprogramming in human female germ cells
    Article Snippet: Single cells were then manually picked under a stereo microscope (Zeiss, Sliedrecht, the Netherlands) using a pulled glass capillary in picking volumes of ±0.5 µl medium and transferred to ice-cold PCR tubes containing 2.0 µl lysis buffer (1.9 µl 0.2% TritonX-100 in water + 0.1 µl Recombinant RNase inhibitor (TaKaRa 40U/µl Ref. 2313A) per tube), snap-frozen on dry ice and stored at −80 °C. .. Reverse transcription (RT) was performed by the addition of 5.6 µl Smart-Seq2 RT mix (0.5 µl SuperScriptII (Invitrogen Cat. 18064-014); 2 µl 5 × SuperScriptII buffer; 0.5 µl 100 mM DTT; 2 µl 5 M betaine; 0.1 µl 1 mM MgCl2 ; 0.25 µl Recombinant RNase inhibitor (TaKaRa 40U/µl Ref. 2313A); 0.1 µl 100 µM LNA strand switch primer; 0.15 µl water, per reaction) and incubation (90 min 42 °C; 10 “strand-switch” cycles of (2 min 50 °C; 2 min 70 °C); 4 °C) in a thermal cycler. cDNA amplification: cDNA amplification was performed by the addition of 15 µl of Smart-seq2 PCR mix [12.5 µl 2× KAPA HiFi HotStart ReadyMix (KAPA Biosystems Ref. KK2602); 0.25 µl 10 µM ISPCR primers; 2.25 µl water, per reaction] and incubation [3 min 98 °C; 19 cycles of (20 s (s) 98 °C; 15 s 67 °C; 6 min 72 °C); 5 min 70 °C; 4 °C] in a thermal cycler.

    Article Title: Apoptotic and Early Innate Immune Responses to PB1-F2 Protein of Influenza A Viruses Belonging to Different Subtypes in Human Lung Epithelial A549 Cells
    Article Snippet: To determine the mRNA expression of PB1-F2 gene in A549 cells, 500 ng of RNA was reverse transcribed using the Superscript II RT-PCR kit (Invitrogen, Carlsbad, CA, USA) and Uni12 primer and the PB1-F2 gene was amplified as mentioned above. .. Protease inhibitor and Phosphatase inhibitor cocktails (Calbiochem, USA) were added to RIPA lysis buffer.

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    Thermo Fisher superscript iii reverse transcriptase kit
    Impact of MyD88 reactivation on the expression of factors that govern MO/DC function in infection and inflammation. (A, B) WT, MyD OFF , CD11c-MyD ON and LysM-MyD ON mice were infected orally with C . rodentium . On day 4 p.i. leukocytes were isolated from the cLP and sorted for DC (lin − CD64 − MHC-II + CD26 + CD11c + ) and MO (lin − CD64 + F4/80 + MHC-II + ). As lineage marker, antibodies against CD3, CD19, B220 and NK1.1 were included (A) Heat map displaying expression of genes involved in MNP function in infection and inflammation analyzed by real-time PCR from sorted DC/MO fractions. Data presented as mean log2 value of relative <t>mRNA</t> expression (see color scale). (B) Bar graphs show mean expression of selected genes in sorted DC/MO fractions relative to the expression of 5 housekeeping genes ( Actb , B2m , Gapdh , Gusb , Hsp90ab1 ). Data were pooled from <t>three</t> individual experiments with n = 4–6 mice per group. Error bar represents +SEM. One-Way ANOVA with Bonferroni’s Multiple Comparison test; *p
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    Impact of MyD88 reactivation on the expression of factors that govern MO/DC function in infection and inflammation. (A, B) WT, MyD OFF , CD11c-MyD ON and LysM-MyD ON mice were infected orally with C . rodentium . On day 4 p.i. leukocytes were isolated from the cLP and sorted for DC (lin − CD64 − MHC-II + CD26 + CD11c + ) and MO (lin − CD64 + F4/80 + MHC-II + ). As lineage marker, antibodies against CD3, CD19, B220 and NK1.1 were included (A) Heat map displaying expression of genes involved in MNP function in infection and inflammation analyzed by real-time PCR from sorted DC/MO fractions. Data presented as mean log2 value of relative mRNA expression (see color scale). (B) Bar graphs show mean expression of selected genes in sorted DC/MO fractions relative to the expression of 5 housekeeping genes ( Actb , B2m , Gapdh , Gusb , Hsp90ab1 ). Data were pooled from three individual experiments with n = 4–6 mice per group. Error bar represents +SEM. One-Way ANOVA with Bonferroni’s Multiple Comparison test; *p

    Journal: PLoS Pathogens

    Article Title: MyD88 signaling in dendritic cells and the intestinal epithelium controls immunity against intestinal infection with C. rodentium

    doi: 10.1371/journal.ppat.1006357

    Figure Lengend Snippet: Impact of MyD88 reactivation on the expression of factors that govern MO/DC function in infection and inflammation. (A, B) WT, MyD OFF , CD11c-MyD ON and LysM-MyD ON mice were infected orally with C . rodentium . On day 4 p.i. leukocytes were isolated from the cLP and sorted for DC (lin − CD64 − MHC-II + CD26 + CD11c + ) and MO (lin − CD64 + F4/80 + MHC-II + ). As lineage marker, antibodies against CD3, CD19, B220 and NK1.1 were included (A) Heat map displaying expression of genes involved in MNP function in infection and inflammation analyzed by real-time PCR from sorted DC/MO fractions. Data presented as mean log2 value of relative mRNA expression (see color scale). (B) Bar graphs show mean expression of selected genes in sorted DC/MO fractions relative to the expression of 5 housekeeping genes ( Actb , B2m , Gapdh , Gusb , Hsp90ab1 ). Data were pooled from three individual experiments with n = 4–6 mice per group. Error bar represents +SEM. One-Way ANOVA with Bonferroni’s Multiple Comparison test; *p

    Article Snippet: 50–100 ng of amplified mRNA was transcribed into cDNA using SuperScript III Reverse Transcriptase Kit (Thermo Fisher Scientific).

    Techniques: Expressing, Infection, Mouse Assay, Isolation, Marker, Real-time Polymerase Chain Reaction

    Cxcl8/cxcr1 signaling in endothelial cells induces gene expression changes favoring HSPC colonization . (a) Kdrl:cxcr1;kdrl:mCherry zebrafish and kdrl:mCherry clutchmates (WT) were dissociated at 72 hpf, and mCherry + endothelial cells were FACS sorted. Quantitative PCR for cxcr1 is shown. The experiment was repeated three times with similar results. (b–d) Kdrl:cxcr1;Runx1:mCherry zebrafish were imaged at 72 hpf for HSPC colonization of the CHT (a and b). Bars, 20 µm. (d) Plot showing increased HSPC colonization in kdrl:cxcr1 animals (P = 0.001, Wilcoxon’s rank sum test; n = 35 for WT control; n = 28 for kdrl:cxcr1 ) The experiment was repeated twice with similar results; combined results are shown. (e) Mpx:GFP (WT) and kdrl:cxcr1;mpx:GFP zebrafish were imaged at 72 hpf, and neutrophil numbers in the CHT were quantified (p = NS, Student’s t test; n = 47 for WT control; n = 35 for kdrl:cxcr1 ). The experiment was repeated three times with similar results. Combined results are shown. (f–k) Kdrl:cxcr1 and WT clutchmates were fixed at 72 hpf and WISH was performed for cxcl12a (f–h) and cxcl12b (i–k). Bar, 100 µm. h and k show the results of blinded semiquantitative scoring of CHT staining for each probe ( cxcl12a: P = 0.03, Wilcoxon’s rank sum test, n = 26 for WT control and n = 34 for kdrl:cxcr1 ; cxcl12b : p = NS, Wilcoxon’s rank sum test, n = 19 for WT control and n = 22 for kdrl:cxcr1 ). The experiment was performed three times with similar results; combined results are shown. (l) HUVECs were serum starved for 12 h, and then treated with 10 ng/ml rhCXCL8 or vehicle control. Quantitative RT-PCR was performed for expression of CXCL12, CXCL8, and survivin and VEGFA. (m) RNA sequencing was performed on HUVEC RNA. IPA analysis identifying the top enriched molecular and cellular functions is shown. The HUVEC experiments were performed with biological duplicates.

    Journal: The Journal of Experimental Medicine

    Article Title: CXCR1 remodels the vascular niche to promote hematopoietic stem and progenitor cell engraftment

    doi: 10.1084/jem.20161616

    Figure Lengend Snippet: Cxcl8/cxcr1 signaling in endothelial cells induces gene expression changes favoring HSPC colonization . (a) Kdrl:cxcr1;kdrl:mCherry zebrafish and kdrl:mCherry clutchmates (WT) were dissociated at 72 hpf, and mCherry + endothelial cells were FACS sorted. Quantitative PCR for cxcr1 is shown. The experiment was repeated three times with similar results. (b–d) Kdrl:cxcr1;Runx1:mCherry zebrafish were imaged at 72 hpf for HSPC colonization of the CHT (a and b). Bars, 20 µm. (d) Plot showing increased HSPC colonization in kdrl:cxcr1 animals (P = 0.001, Wilcoxon’s rank sum test; n = 35 for WT control; n = 28 for kdrl:cxcr1 ) The experiment was repeated twice with similar results; combined results are shown. (e) Mpx:GFP (WT) and kdrl:cxcr1;mpx:GFP zebrafish were imaged at 72 hpf, and neutrophil numbers in the CHT were quantified (p = NS, Student’s t test; n = 47 for WT control; n = 35 for kdrl:cxcr1 ). The experiment was repeated three times with similar results. Combined results are shown. (f–k) Kdrl:cxcr1 and WT clutchmates were fixed at 72 hpf and WISH was performed for cxcl12a (f–h) and cxcl12b (i–k). Bar, 100 µm. h and k show the results of blinded semiquantitative scoring of CHT staining for each probe ( cxcl12a: P = 0.03, Wilcoxon’s rank sum test, n = 26 for WT control and n = 34 for kdrl:cxcr1 ; cxcl12b : p = NS, Wilcoxon’s rank sum test, n = 19 for WT control and n = 22 for kdrl:cxcr1 ). The experiment was performed three times with similar results; combined results are shown. (l) HUVECs were serum starved for 12 h, and then treated with 10 ng/ml rhCXCL8 or vehicle control. Quantitative RT-PCR was performed for expression of CXCL12, CXCL8, and survivin and VEGFA. (m) RNA sequencing was performed on HUVEC RNA. IPA analysis identifying the top enriched molecular and cellular functions is shown. The HUVEC experiments were performed with biological duplicates.

    Article Snippet: Transgenesis Candidate zebrafish coding sequences were amplified from kidney marrow total RNA using the Superscript III RT kit (Thermo Fisher Scientific) and gene-specific primers.

    Techniques: Expressing, FACS, Real-time Polymerase Chain Reaction, Staining, Quantitative RT-PCR, RNA Sequencing Assay, Indirect Immunoperoxidase Assay

    RNase R treatments of dsRNA samples, where three consecutive digests were carried out, with 10 μL samples saved at each step for RT-PCR screening ( lower branch ), and a parallel no-enzyme control series ( upper branch ) to validate the procedure. Denaturation at 95 °C for 8 min was carried out before each RNase R treatment.

    Journal: Viruses

    Article Title: Blackcurrant Leaf Chlorosis Associated Virus: Evidence of the Presence of Circular RNA during Infections

    doi: 10.3390/v10050260

    Figure Lengend Snippet: RNase R treatments of dsRNA samples, where three consecutive digests were carried out, with 10 μL samples saved at each step for RT-PCR screening ( lower branch ), and a parallel no-enzyme control series ( upper branch ) to validate the procedure. Denaturation at 95 °C for 8 min was carried out before each RNase R treatment.

    Article Snippet: The RT-PCR tests were performed as two-step reactions, with the cDNA synthesis steps using SuperScript III reverse transcriptase kits, and PCR steps using Platinum Hi-Fi Taq DNA polymerase (Thermo-Fisher, Waltham, MA, USA).

    Techniques: Reverse Transcription Polymerase Chain Reaction

    Heterogeneous expression pattern of YFP + and YFP - signals within the aneuploid population was not due to the karyotypic variations. ( A ) A schematic representation of the genetic manipulations used to generate isogenic diploid and triploid strains from the parental WT haploid strain, which has nuclear localization sequence-tagged YFP inserted into the HML locus. ( B ) FACS sorting of YFP - vs YFP + cells from the strain with 2x Chr III and 1x Chr X gain. Red and yellow line outlines the population of YFP - and YFP + cells sorted for the qPCR analysis in C and D, respectively. ( C–D ) qPCR karyotyping of YFP - ( C ) and YFP + ( D ) cell population sorted from the strain with 2x Chr III and 1x Chr X gain. Chromosome copy numbers of sixteen yeast chromosomes are plotted as mean and SD of three technical replicates. ( E–G ) Confocal images of YFP fluorescence, taken at the indicated time points during time-lapse imaging, show transitions between repression and derepression of the HML locus in proliferating lineages of aneuploid yeast cells with the following karyotypes: ( E ) Gain of III, III, X; ( F ) Loss of I, V, VII, VIII, XI (basal ploidy, 2N); and ( G ) Gain of I, X, XII, XIII. Arrows point to mother cells that switched from the silenced to the desilenced state. The circle outlines the boundary of the cell. Scale bar, 4 µm.

    Journal: eLife

    Article Title: Aneuploidy as a cause of impaired chromatin silencing and mating-type specification in budding yeast

    doi: 10.7554/eLife.27991

    Figure Lengend Snippet: Heterogeneous expression pattern of YFP + and YFP - signals within the aneuploid population was not due to the karyotypic variations. ( A ) A schematic representation of the genetic manipulations used to generate isogenic diploid and triploid strains from the parental WT haploid strain, which has nuclear localization sequence-tagged YFP inserted into the HML locus. ( B ) FACS sorting of YFP - vs YFP + cells from the strain with 2x Chr III and 1x Chr X gain. Red and yellow line outlines the population of YFP - and YFP + cells sorted for the qPCR analysis in C and D, respectively. ( C–D ) qPCR karyotyping of YFP - ( C ) and YFP + ( D ) cell population sorted from the strain with 2x Chr III and 1x Chr X gain. Chromosome copy numbers of sixteen yeast chromosomes are plotted as mean and SD of three technical replicates. ( E–G ) Confocal images of YFP fluorescence, taken at the indicated time points during time-lapse imaging, show transitions between repression and derepression of the HML locus in proliferating lineages of aneuploid yeast cells with the following karyotypes: ( E ) Gain of III, III, X; ( F ) Loss of I, V, VII, VIII, XI (basal ploidy, 2N); and ( G ) Gain of I, X, XII, XIII. Arrows point to mother cells that switched from the silenced to the desilenced state. The circle outlines the boundary of the cell. Scale bar, 4 µm.

    Article Snippet: Quantitative reverse transcriptase-PCR (qPCR) analysis RNA was extracted as described above and cDNA was prepared from 2 µg of the resulting total RNA using the Super-Script III reverse transcriptase kit (Thermo Fisher Scientific, Waltham, MA). qPCR was performed using SYBR Green real-time PCR master mix (Quanta Biosciences, Beverly, MA) and analyzed by standard procedures ( ).

    Techniques: Expressing, Sequencing, FACS, Real-time Polymerase Chain Reaction, Fluorescence, Imaging

    HM desilencing in disome X cells correlates with increased H4K16 acetylation and reduced Sir2 enrichment across HM loci. ( A–B ) Bottom: The plots show levels of H4K16 acetylation across the HML ( A ) and HMR ( B ) loci in disome X and Δsir1 strains relative to WT haploid cells, determined using anti-H4K16ac chromatin immunoprecipitation (ChIP) followed by quantitative RT-PCR (qPCR) analysis. Top: Schematics of the HM loci indicate the genomic positioning of primer sets A to F used for qPCR. Plots show the mean and SD from three biological replicates. *p

    Journal: eLife

    Article Title: Aneuploidy as a cause of impaired chromatin silencing and mating-type specification in budding yeast

    doi: 10.7554/eLife.27991

    Figure Lengend Snippet: HM desilencing in disome X cells correlates with increased H4K16 acetylation and reduced Sir2 enrichment across HM loci. ( A–B ) Bottom: The plots show levels of H4K16 acetylation across the HML ( A ) and HMR ( B ) loci in disome X and Δsir1 strains relative to WT haploid cells, determined using anti-H4K16ac chromatin immunoprecipitation (ChIP) followed by quantitative RT-PCR (qPCR) analysis. Top: Schematics of the HM loci indicate the genomic positioning of primer sets A to F used for qPCR. Plots show the mean and SD from three biological replicates. *p

    Article Snippet: Quantitative reverse transcriptase-PCR (qPCR) analysis RNA was extracted as described above and cDNA was prepared from 2 µg of the resulting total RNA using the Super-Script III reverse transcriptase kit (Thermo Fisher Scientific, Waltham, MA). qPCR was performed using SYBR Green real-time PCR master mix (Quanta Biosciences, Beverly, MA) and analyzed by standard procedures ( ).

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