superscriptii reverse transcriptase  (Thermo Fisher)


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    Structured Review

    Thermo Fisher superscriptii reverse transcriptase
    Superscriptii Reverse Transcriptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 92 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscriptii reverse transcriptase/product/Thermo Fisher
    Average 99 stars, based on 92 article reviews
    Price from $9.99 to $1999.99
    superscriptii reverse transcriptase - 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: 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: 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: 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: 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.

    Expressing:

    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: 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: 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.

    Real-time Polymerase Chain Reaction:

    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).

    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.

    Reverse Transcription Polymerase Chain Reaction:

    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: 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: 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.

    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: 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).

    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.

    Isolation:

    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).

    Purification:

    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).

    Sequencing:

    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.

    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: 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.

    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.

    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: 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: 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.

    Lysis:

    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 rt
    Construction and characterization of synZIKV-R2A reporter virus genomes. ( A ) Schematic representation of the synZIKV-R2A reporter virus genomes. For both strains the R2A reporter cassette (light red) was inserted into the wild-type pFK-synZIKV plasmids via MLuI/KpnI restriction sites. The NotI/NruI sites flanking the RLuc gene allow for the exchange of the reporter gene. ( B ) Immunofluorescence analysis of VeroE6 cells transfected with synZIKV-R2A in vitro transcripts. Cells were grown on coverslips, fixed 72 h and 96 h after transfection and stained with E-specific antibody (green). Nuclear DNA was counterstained with DAPI (grey). Scale bar = 15 μm. ( C ) Replication kinetics of the synZIKV-R2A reporter viruses in VeroE6 cells. After electroporation (EPO) cells were harvested at given time points and RLuc activity was determined. Values were normalized to the 4 h-value reflecting transfection efficiency. Mean ± SEM of <t>three</t> independent experiments is shown. Replication deficient mutants containing two mutations affecting the active site of the <t>RNA-dependent-RNA</t> polymerase in NS5 (GAA) served as negative controls. ( D ) VeroE6 cells were transfected with synZIKV-R2A RNAs, cell culture supernatants were collected 72 h post- transfection (P0) and passaged three times by infection of VeroE6 cells (P1-P3) in 72 h intervals. Culture supernatants obtained from each passage were used to inoculate Huh7 cells. In the case of supernatant obtained directly from transfected VeroE6 cells (P0), Huh7 cells were inoculated with undiluted (undil) or 1:10 diluted supernatant. After 72 h cells were harvested and RLuc activity in cell lysates was determined. Mean ± SEM from two independent experiments is shown. ( E ) Virus titres as determined by plaque assay for each synZIKV-R2A passage; values are mean ± SEM of two independent experiments. ( F ) Stability of the reporter gene. SynZIKV-R2A viruses released into culture supernatants were harvested after each passage as described in panel D, RNA was isolated and the region encompassing the RLuc coding sequence was amplified by using random hexamer primers for reverse transcription and specific primers for subsequent PCR. The ~1350 bp long DNA fragment in the P0 virus sample corresponds to the reporter gene, while the ~250 bp long fragment corresponds to the WT sequence. ( G ) Antiviral assay using synZIKV-R2A viruses. VeroE6 cells were inoculated with a 1:10 dilution of a P0 stock and one hour later the medium was replaced with DMEM containing the indicated amount of 2′CMC. RLuc activity was measured in cell lysates 72 h post-infection. Mean ± SEM from two independent experiments is shown.
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    Construction and characterization of synZIKV-R2A reporter virus genomes. ( A ) Schematic representation of the synZIKV-R2A reporter virus genomes. For both strains the R2A reporter cassette (light red) was inserted into the wild-type pFK-synZIKV plasmids via MLuI/KpnI restriction sites. The NotI/NruI sites flanking the RLuc gene allow for the exchange of the reporter gene. ( B ) Immunofluorescence analysis of VeroE6 cells transfected with synZIKV-R2A in vitro transcripts. Cells were grown on coverslips, fixed 72 h and 96 h after transfection and stained with E-specific antibody (green). Nuclear DNA was counterstained with DAPI (grey). Scale bar = 15 μm. ( C ) Replication kinetics of the synZIKV-R2A reporter viruses in VeroE6 cells. After electroporation (EPO) cells were harvested at given time points and RLuc activity was determined. Values were normalized to the 4 h-value reflecting transfection efficiency. Mean ± SEM of three independent experiments is shown. Replication deficient mutants containing two mutations affecting the active site of the RNA-dependent-RNA polymerase in NS5 (GAA) served as negative controls. ( D ) VeroE6 cells were transfected with synZIKV-R2A RNAs, cell culture supernatants were collected 72 h post- transfection (P0) and passaged three times by infection of VeroE6 cells (P1-P3) in 72 h intervals. Culture supernatants obtained from each passage were used to inoculate Huh7 cells. In the case of supernatant obtained directly from transfected VeroE6 cells (P0), Huh7 cells were inoculated with undiluted (undil) or 1:10 diluted supernatant. After 72 h cells were harvested and RLuc activity in cell lysates was determined. Mean ± SEM from two independent experiments is shown. ( E ) Virus titres as determined by plaque assay for each synZIKV-R2A passage; values are mean ± SEM of two independent experiments. ( F ) Stability of the reporter gene. SynZIKV-R2A viruses released into culture supernatants were harvested after each passage as described in panel D, RNA was isolated and the region encompassing the RLuc coding sequence was amplified by using random hexamer primers for reverse transcription and specific primers for subsequent PCR. The ~1350 bp long DNA fragment in the P0 virus sample corresponds to the reporter gene, while the ~250 bp long fragment corresponds to the WT sequence. ( G ) Antiviral assay using synZIKV-R2A viruses. VeroE6 cells were inoculated with a 1:10 dilution of a P0 stock and one hour later the medium was replaced with DMEM containing the indicated amount of 2′CMC. RLuc activity was measured in cell lysates 72 h post-infection. Mean ± SEM from two independent experiments is shown.

    Journal: Viruses

    Article Title: A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy

    doi: 10.3390/v10070368

    Figure Lengend Snippet: Construction and characterization of synZIKV-R2A reporter virus genomes. ( A ) Schematic representation of the synZIKV-R2A reporter virus genomes. For both strains the R2A reporter cassette (light red) was inserted into the wild-type pFK-synZIKV plasmids via MLuI/KpnI restriction sites. The NotI/NruI sites flanking the RLuc gene allow for the exchange of the reporter gene. ( B ) Immunofluorescence analysis of VeroE6 cells transfected with synZIKV-R2A in vitro transcripts. Cells were grown on coverslips, fixed 72 h and 96 h after transfection and stained with E-specific antibody (green). Nuclear DNA was counterstained with DAPI (grey). Scale bar = 15 μm. ( C ) Replication kinetics of the synZIKV-R2A reporter viruses in VeroE6 cells. After electroporation (EPO) cells were harvested at given time points and RLuc activity was determined. Values were normalized to the 4 h-value reflecting transfection efficiency. Mean ± SEM of three independent experiments is shown. Replication deficient mutants containing two mutations affecting the active site of the RNA-dependent-RNA polymerase in NS5 (GAA) served as negative controls. ( D ) VeroE6 cells were transfected with synZIKV-R2A RNAs, cell culture supernatants were collected 72 h post- transfection (P0) and passaged three times by infection of VeroE6 cells (P1-P3) in 72 h intervals. Culture supernatants obtained from each passage were used to inoculate Huh7 cells. In the case of supernatant obtained directly from transfected VeroE6 cells (P0), Huh7 cells were inoculated with undiluted (undil) or 1:10 diluted supernatant. After 72 h cells were harvested and RLuc activity in cell lysates was determined. Mean ± SEM from two independent experiments is shown. ( E ) Virus titres as determined by plaque assay for each synZIKV-R2A passage; values are mean ± SEM of two independent experiments. ( F ) Stability of the reporter gene. SynZIKV-R2A viruses released into culture supernatants were harvested after each passage as described in panel D, RNA was isolated and the region encompassing the RLuc coding sequence was amplified by using random hexamer primers for reverse transcription and specific primers for subsequent PCR. The ~1350 bp long DNA fragment in the P0 virus sample corresponds to the reporter gene, while the ~250 bp long fragment corresponds to the WT sequence. ( G ) Antiviral assay using synZIKV-R2A viruses. VeroE6 cells were inoculated with a 1:10 dilution of a P0 stock and one hour later the medium was replaced with DMEM containing the indicated amount of 2′CMC. RLuc activity was measured in cell lysates 72 h post-infection. Mean ± SEM from two independent experiments is shown.

    Article Snippet: Viral RNA was isolated from cell lysates using the NucleoSpin RNA II kit (Machery-Nagel, Düren, Germany) and reverse transcribed using SuperScript III RT (Thermo Fisher Scientific Waltham, MA, USA). cDNA was amplified by PCR and amplicons were sequenced by Sanger sequencing (GATC Biotech, Constance, Germany) using primers spanning the complete ZIKV genome.

    Techniques: Immunofluorescence, Transfection, In Vitro, Staining, Electroporation, Activity Assay, Cell Culture, Infection, Plaque Assay, Isolation, Sequencing, Amplification, Random Hexamer Labeling, Polymerase Chain Reaction, Antiviral Assay

    Quantification of transcript amounts of jhp0832 in H. pylori strains J99, J99-mut and the J99 mutants with point mutations within the GCGC motifs. qPCR results are represented in the right panel, three different biological replicates were performed. Statistics: One-way ANOVA, ** P

    Journal: Nucleic Acids Research

    Article Title: The core genome m5C methyltransferase JHP1050 (M.Hpy99III) plays an important role in orchestrating gene expression in Helicobacter pylori

    doi: 10.1093/nar/gky1307

    Figure Lengend Snippet: Quantification of transcript amounts of jhp0832 in H. pylori strains J99, J99-mut and the J99 mutants with point mutations within the GCGC motifs. qPCR results are represented in the right panel, three different biological replicates were performed. Statistics: One-way ANOVA, ** P

    Article Snippet: Quantitative PCR (qPCR) One μg of RNA was used for cDNA synthesis using the SuperScript™ III Reverse Transcriptase (Thermo Fisher Scientific, Darmstadt, Germany) as described before ( ). qPCR was performed with gene specific primers ( ) and SYBR Green Master Mix (Qiagen, Hilden, Germany).

    Techniques: Real-time Polymerase Chain Reaction

    SGMS1 is transcriptionally up-regulated in K562 cells. A , B ) SGMS1 ) were used to quantify hnRNA expression, normalized to β-actin and expressed as MNE. Primer locations are indicated on the SGMS1 locus (white boxes represent untranslated exons, black boxes represent translated exons, and lines represent introns). Negative control for cDNA synthesis (without superscript III) in K562 showed no amplification, thus verifying the absence of genomic DNA. Results from 3 independent experiments are shown. C ) K562 and HL-60 cells were treated with vehicle (H 2 O) or actinomycin D (5 μg/ml) over a 2-h time course. SGMS1 ) to quantify the percent of remaining mRNA. D ) K562 cells were treated with imatinib (1 μM) for 8 h. Cells were harvested and lysates were prepared for Western blot analysis. Total STAT5 and pSTAT5 levels were measured in control and treated cells. E ) In control and imatinib-treated cells, RNA was extracted to measure hnRNA levels of SGMS1. Intron VII and exon 8–specific RT-PCR primers were used. Results represent 3 independent experiments. ND, not detectable; SSIII, superscript III. * P

    Journal: The FASEB Journal

    Article Title: Bcr-Abl regulation of sphingomyelin synthase 1 reveals a novel oncogenic-driven mechanism of protein up-regulation

    doi: 10.1096/fj.201701016R

    Figure Lengend Snippet: SGMS1 is transcriptionally up-regulated in K562 cells. A , B ) SGMS1 ) were used to quantify hnRNA expression, normalized to β-actin and expressed as MNE. Primer locations are indicated on the SGMS1 locus (white boxes represent untranslated exons, black boxes represent translated exons, and lines represent introns). Negative control for cDNA synthesis (without superscript III) in K562 showed no amplification, thus verifying the absence of genomic DNA. Results from 3 independent experiments are shown. C ) K562 and HL-60 cells were treated with vehicle (H 2 O) or actinomycin D (5 μg/ml) over a 2-h time course. SGMS1 ) to quantify the percent of remaining mRNA. D ) K562 cells were treated with imatinib (1 μM) for 8 h. Cells were harvested and lysates were prepared for Western blot analysis. Total STAT5 and pSTAT5 levels were measured in control and treated cells. E ) In control and imatinib-treated cells, RNA was extracted to measure hnRNA levels of SGMS1. Intron VII and exon 8–specific RT-PCR primers were used. Results represent 3 independent experiments. ND, not detectable; SSIII, superscript III. * P

    Article Snippet: Dephosphorylated and uncapped mRNA was added to the GeneRacer RNA Oligo (provided in the kit) and incubated with T4 RNA ligase at 37°C for 1 h. The uncapped, full-length mRNA that was ligated to the GeneRacer RNA oligo (provided in the kit) was used for reverse transcription of mRNA into cDNA using Superscript III reverse transcriptase and random primers according to the standard protocol (Thermo Fisher Scientific).

    Techniques: Expressing, Negative Control, Amplification, Western Blot, Reverse Transcription Polymerase Chain Reaction

    Overexpression of wild-type or inactive ClpP proteins has varied effects on Chlamydia species. C. trachomatis serovar L2 was transformed with aTc-inducible shuttle vectors encoding either wild-type or active-site mutants of each ClpP paralog with a 6×His tag at the C terminus. HEp2 cells were infected with each transformant, and expression was induced at 10 h postinfection (hpi). (A) ClpP1 wild-type and S92A mutant overexpression assay. Overexpression of inactive ClpP1 has a negative impact on the bacteria. (B) ClpP2 wild-type and S98A mutant overexpression assay. Parameters were the same as those described above. Overexpression does not appear to negatively affect Chlamydia species. Samples were stained for major outer membrane protein (MOMP; green), 6×His-tagged ClpP protein of interest (red), and DNA (blue). Representative images of three independent experiments are presented. Scale bars are equal to 10 μm. Images were acquired on a Zeiss LSM 800 laser scanning confocal microscope with a 60× objective and a 3× digital magnification. (C) Inclusion-forming unit (IFU) assay measuring the effect of increasing levels of ClpP protein induction on chlamydial growth. Values are averages of the results from three independent experiments and are reported as a percentage of the respective uninduced sample. *, P

    Journal: Journal of Bacteriology

    Article Title: Initial Characterization of the Two ClpP Paralogs of Chlamydia trachomatis Suggests Unique Functionality for Each

    doi: 10.1128/JB.00635-18

    Figure Lengend Snippet: Overexpression of wild-type or inactive ClpP proteins has varied effects on Chlamydia species. C. trachomatis serovar L2 was transformed with aTc-inducible shuttle vectors encoding either wild-type or active-site mutants of each ClpP paralog with a 6×His tag at the C terminus. HEp2 cells were infected with each transformant, and expression was induced at 10 h postinfection (hpi). (A) ClpP1 wild-type and S92A mutant overexpression assay. Overexpression of inactive ClpP1 has a negative impact on the bacteria. (B) ClpP2 wild-type and S98A mutant overexpression assay. Parameters were the same as those described above. Overexpression does not appear to negatively affect Chlamydia species. Samples were stained for major outer membrane protein (MOMP; green), 6×His-tagged ClpP protein of interest (red), and DNA (blue). Representative images of three independent experiments are presented. Scale bars are equal to 10 μm. Images were acquired on a Zeiss LSM 800 laser scanning confocal microscope with a 60× objective and a 3× digital magnification. (C) Inclusion-forming unit (IFU) assay measuring the effect of increasing levels of ClpP protein induction on chlamydial growth. Values are averages of the results from three independent experiments and are reported as a percentage of the respective uninduced sample. *, P

    Article Snippet: DNA was removed from total RNA by rigorous DNA-free treatment (Thermo Fisher) before 1 µg was reverse transcribed with Superscript III reverse transcriptase (RT; Thermo Fisher).

    Techniques: Over Expression, Transformation Assay, Infection, Expressing, Mutagenesis, Staining, Microscopy