dnasei treated total rna  (Thermo Fisher)


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    DNase I Solution 1 unit µL
    Description:
    Thermo Scientific DNase I removes unwanted DNA from cell lysates to improve protein extraction efficiency Features of RNase free DNase I • Degrades and removes unwanted DNA from samples • Cleaves both single stranded and double stranded DNA • Compatible with Thermo Scientific Pierce Cell Lysis Reagents • Reduces viscosity of bacterial lysates protein extracts to facilitate pipetting Deoxyribonuclease I DNase I is a single glycosylated polypeptide that degrades unwanted single and double stranded DNA The enzyme works by cleaving DNA into 5 phosphodinucleotide and small oligonucleotide fragments DNase I is commonly added to cell lysis reagents to remove the viscosity caused by the DNA content in bacterial cell lysates or to remove the DNA templates from RNAs produced by in vitro transcription RNase free DNase is useful for any application requiring the digestion of DNA in which it is crucial to avoid damage to RNA General information about the use of DNase I • Calcium ions are required for activity of DNase I Trace amounts of Ca may be present at high enough concentration for DNase I to be active however use of EGTA or calcium free buffers can reduce DNase I activity to undetectable levels • High levels i e 100 mM of monovalent ions such as Na and K will decrease DNase I activity • DNase I is inactivated by heating to 65°C for 10 minutes • Kunitz unit 1 Kunitz unit is the amount of enzyme required to cause an increase of 0 001 A260nm min mL at 25°C in 0 1M NaOAc pH 5 0 due to degradation of highly polymerized DNA • Degradation assay units 1 unit is defined as the amount of enzyme required to completely degrade 1 µg of plasmid DNA in the 10 minutes at 37°C in 10 mM Tris HCl pH 7 5 50 mM MgCl2 13 mM CaCl2 Specifications • Quantity 1000 units 1 mL • Concentration 1 unit µL 20 • Unit definition 1 unit completely degrades 1 µg of plasmid DNA in 10 minutes at 37°C One DNase I unit is equivalent to 0 3 Kunitz units • RNase Contamination No ribonuclease activity detectable based on incubation with RNA transcript • Source E coli containing cloned gene encoding bovine DNase I • Molecular Weight Approx 29 000 • Formulation Bovine DNase I in 50 mM Tris HCl pH 7 5 10 mM CaCl2 50 glycerol • Supplied with 1 mL of 10X Reaction Buffer 100 mM Tris HCl pH 7 5 25 mM MgCl2 1 mM CaCl2 50 mM EDTA Related Products DNase I Solution 2500 U mL
    Catalog Number:
    89836
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    Category:
    Proteins Enzymes Peptides
    Applications:
    Cell Lysis|Cell Lysis & Fractionation|Protein Biology|Protein Purification & Isolation
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    Structured Review

    Thermo Fisher dnasei treated total rna
    Derepressed genes are transcribed and enriched for active chromatin marks in WT TSCs. a Inactive-X profiles of H3K27me3 in WT TSCs of derepressed and non-derepressed genes in Eed –/– TSCs and of X-inactivation escapees in WT TSCs. b Inactive-X profiles for <t>DNAseI</t> hypersensitivity (DNAseI HS), <t>RNA</t> PolII occupancy, H3K27ac, H3K4me2, and H3K36me3. c Correlation between percent paternal-X expression in WT and Eed –/– TSCs. d Paternal-X contribution for all expressed X-linked genes in WT TSCs. The x-axis indicates the rank order of each gene from least to most paternal-X expression in WT TSCs. Gray dots indicate non-derepressed genes and red dots mark genes that are derepressed in Eed –/– TSCs. The y-axis depicts percent paternal-X expression for each gene. The expressed paternal X-linked genes can be divided into five quintiles. The derepressed genes are over-represented in quintile 4. e Table of number of genes and their percent paternal-X expression within the quintiles described in ( d ). f Left , boxplots of absolute paternal-X expression (as RNA-seq reads per kilobase of gene per million reads [RPKM]) for non-derepressed and derepressed genes in quintile 4 (3–6% paternal-X:total X-chromosomal expression). Derepressed genes exhibit a significantly higher median expression level than non-derepressed genes ( p
    Thermo Scientific DNase I removes unwanted DNA from cell lysates to improve protein extraction efficiency Features of RNase free DNase I • Degrades and removes unwanted DNA from samples • Cleaves both single stranded and double stranded DNA • Compatible with Thermo Scientific Pierce Cell Lysis Reagents • Reduces viscosity of bacterial lysates protein extracts to facilitate pipetting Deoxyribonuclease I DNase I is a single glycosylated polypeptide that degrades unwanted single and double stranded DNA The enzyme works by cleaving DNA into 5 phosphodinucleotide and small oligonucleotide fragments DNase I is commonly added to cell lysis reagents to remove the viscosity caused by the DNA content in bacterial cell lysates or to remove the DNA templates from RNAs produced by in vitro transcription RNase free DNase is useful for any application requiring the digestion of DNA in which it is crucial to avoid damage to RNA General information about the use of DNase I • Calcium ions are required for activity of DNase I Trace amounts of Ca may be present at high enough concentration for DNase I to be active however use of EGTA or calcium free buffers can reduce DNase I activity to undetectable levels • High levels i e 100 mM of monovalent ions such as Na and K will decrease DNase I activity • DNase I is inactivated by heating to 65°C for 10 minutes • Kunitz unit 1 Kunitz unit is the amount of enzyme required to cause an increase of 0 001 A260nm min mL at 25°C in 0 1M NaOAc pH 5 0 due to degradation of highly polymerized DNA • Degradation assay units 1 unit is defined as the amount of enzyme required to completely degrade 1 µg of plasmid DNA in the 10 minutes at 37°C in 10 mM Tris HCl pH 7 5 50 mM MgCl2 13 mM CaCl2 Specifications • Quantity 1000 units 1 mL • Concentration 1 unit µL 20 • Unit definition 1 unit completely degrades 1 µg of plasmid DNA in 10 minutes at 37°C One DNase I unit is equivalent to 0 3 Kunitz units • RNase Contamination No ribonuclease activity detectable based on incubation with RNA transcript • Source E coli containing cloned gene encoding bovine DNase I • Molecular Weight Approx 29 000 • Formulation Bovine DNase I in 50 mM Tris HCl pH 7 5 10 mM CaCl2 50 glycerol • Supplied with 1 mL of 10X Reaction Buffer 100 mM Tris HCl pH 7 5 25 mM MgCl2 1 mM CaCl2 50 mM EDTA Related Products DNase I Solution 2500 U mL
    https://www.bioz.com/result/dnasei treated total rna/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dnasei treated total rna - by Bioz Stars, 2021-04
    97/100 stars

    Images

    1) Product Images from "PRC2 represses transcribed genes on the imprinted inactive X chromosome in mice"

    Article Title: PRC2 represses transcribed genes on the imprinted inactive X chromosome in mice

    Journal: Genome Biology

    doi: 10.1186/s13059-017-1211-5

    Derepressed genes are transcribed and enriched for active chromatin marks in WT TSCs. a Inactive-X profiles of H3K27me3 in WT TSCs of derepressed and non-derepressed genes in Eed –/– TSCs and of X-inactivation escapees in WT TSCs. b Inactive-X profiles for DNAseI hypersensitivity (DNAseI HS), RNA PolII occupancy, H3K27ac, H3K4me2, and H3K36me3. c Correlation between percent paternal-X expression in WT and Eed –/– TSCs. d Paternal-X contribution for all expressed X-linked genes in WT TSCs. The x-axis indicates the rank order of each gene from least to most paternal-X expression in WT TSCs. Gray dots indicate non-derepressed genes and red dots mark genes that are derepressed in Eed –/– TSCs. The y-axis depicts percent paternal-X expression for each gene. The expressed paternal X-linked genes can be divided into five quintiles. The derepressed genes are over-represented in quintile 4. e Table of number of genes and their percent paternal-X expression within the quintiles described in ( d ). f Left , boxplots of absolute paternal-X expression (as RNA-seq reads per kilobase of gene per million reads [RPKM]) for non-derepressed and derepressed genes in quintile 4 (3–6% paternal-X:total X-chromosomal expression). Derepressed genes exhibit a significantly higher median expression level than non-derepressed genes ( p
    Figure Legend Snippet: Derepressed genes are transcribed and enriched for active chromatin marks in WT TSCs. a Inactive-X profiles of H3K27me3 in WT TSCs of derepressed and non-derepressed genes in Eed –/– TSCs and of X-inactivation escapees in WT TSCs. b Inactive-X profiles for DNAseI hypersensitivity (DNAseI HS), RNA PolII occupancy, H3K27ac, H3K4me2, and H3K36me3. c Correlation between percent paternal-X expression in WT and Eed –/– TSCs. d Paternal-X contribution for all expressed X-linked genes in WT TSCs. The x-axis indicates the rank order of each gene from least to most paternal-X expression in WT TSCs. Gray dots indicate non-derepressed genes and red dots mark genes that are derepressed in Eed –/– TSCs. The y-axis depicts percent paternal-X expression for each gene. The expressed paternal X-linked genes can be divided into five quintiles. The derepressed genes are over-represented in quintile 4. e Table of number of genes and their percent paternal-X expression within the quintiles described in ( d ). f Left , boxplots of absolute paternal-X expression (as RNA-seq reads per kilobase of gene per million reads [RPKM]) for non-derepressed and derepressed genes in quintile 4 (3–6% paternal-X:total X-chromosomal expression). Derepressed genes exhibit a significantly higher median expression level than non-derepressed genes ( p

    Techniques Used: Expressing, RNA Sequencing Assay

    2) Product Images from "Transcriptional profiling of Rickettsia prowazekii coding and non-coding transcripts during in vitro host-pathogen and vector-pathogen interactions"

    Article Title: Transcriptional profiling of Rickettsia prowazekii coding and non-coding transcripts during in vitro host-pathogen and vector-pathogen interactions

    Journal: Ticks and tick-borne diseases

    doi: 10.1016/j.ttbdis.2017.06.008

    qPCR of novel differentially expressed Rp _sRs Confluent monolayers of human microvascular endothelial cells (HMECs) cultured at 37°C or 34°C or Amblyomma americanum cells (AAE2) cultured at 34°C were infected with R. prowazekii for 0.5h, 3h, and 24h. Total RNA was extracted using Trizol, DNaseI treated, and reverse transcribed for RT-PCR (n≥3). AAE2 and HMEC expression were baselined to 0.5h and normalized to 16S rRNA. The data is presented as mean±SEM for four trans-acting Rp _sRs, namely, Rp _sR76, Rp_ sR83, Rp_ sR86, and Rp_ sR159. Asterisks indicate *p
    Figure Legend Snippet: qPCR of novel differentially expressed Rp _sRs Confluent monolayers of human microvascular endothelial cells (HMECs) cultured at 37°C or 34°C or Amblyomma americanum cells (AAE2) cultured at 34°C were infected with R. prowazekii for 0.5h, 3h, and 24h. Total RNA was extracted using Trizol, DNaseI treated, and reverse transcribed for RT-PCR (n≥3). AAE2 and HMEC expression were baselined to 0.5h and normalized to 16S rRNA. The data is presented as mean±SEM for four trans-acting Rp _sRs, namely, Rp _sR76, Rp_ sR83, Rp_ sR86, and Rp_ sR159. Asterisks indicate *p

    Techniques Used: Real-time Polymerase Chain Reaction, Cell Culture, Infection, Reverse Transcription Polymerase Chain Reaction, Expressing

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: Addressing fluorogenic real-time qPCR inhibition using the novel custom Excel file system 'FocusField2-6GallupqPCRSet-upTool-001' to attain consistently high fidelity qPCR reactions
    Article Snippet: The tubes are incubated for 2 minutes at room temperature with intermittent vortexing every 10 to 15 seconds, and then centrifuged at 10,000 × g for 3 minutes to pellet the Inactivation Reagent. .. Next, if RNA is to be used directly in one-step qPCR applications, 80 ?l is carefully recovered from each DNase-treatment reaction; the upper transparent layer containing the RNA is transferred to a new tube (care is taken to avoid ~15-25% of the solution on the bottom of each tube – which is the pelleted Ambion DNase Inactivation Reagent polymer complex that can inhibit PCR reactions) and diluted 1:10 with nuclease-free water (Ambion) resulting in 800 ?l of each RNA isolate to use for [FF2-6-001-calibrated] real-time qPCR analyses. .. However, for one-step qPCR analyses, it is important to note two things at this point: 1.) even at 1:10 dilution post DNase treatment, the RNA samples are still too concentrated to generate uninhibited qPCR target signals, and 2.) we never freeze the RNA samples from this point on before their use in qPCR; they are stored at 4°C in nuclease-free 1.5 ml vials.

    Polymerase Chain Reaction:

    Article Title: Addressing fluorogenic real-time qPCR inhibition using the novel custom Excel file system 'FocusField2-6GallupqPCRSet-upTool-001' to attain consistently high fidelity qPCR reactions
    Article Snippet: The tubes are incubated for 2 minutes at room temperature with intermittent vortexing every 10 to 15 seconds, and then centrifuged at 10,000 × g for 3 minutes to pellet the Inactivation Reagent. .. Next, if RNA is to be used directly in one-step qPCR applications, 80 ?l is carefully recovered from each DNase-treatment reaction; the upper transparent layer containing the RNA is transferred to a new tube (care is taken to avoid ~15-25% of the solution on the bottom of each tube – which is the pelleted Ambion DNase Inactivation Reagent polymer complex that can inhibit PCR reactions) and diluted 1:10 with nuclease-free water (Ambion) resulting in 800 ?l of each RNA isolate to use for [FF2-6-001-calibrated] real-time qPCR analyses. .. However, for one-step qPCR analyses, it is important to note two things at this point: 1.) even at 1:10 dilution post DNase treatment, the RNA samples are still too concentrated to generate uninhibited qPCR target signals, and 2.) we never freeze the RNA samples from this point on before their use in qPCR; they are stored at 4°C in nuclease-free 1.5 ml vials.

    Amplification:

    Article Title: MutL? Heterodimers Modify the Molecular Phenotype of Friedreich Ataxia
    Article Snippet: .. Total RNA was treated to prevent DNA contamination using DNase I (amplification grade, Invitrogen). .. Further to suitable RNA quality determination by A 260 /A 280 ratio analysis by NanoDrop spectrophotometry (Invitrogen) and agarose gel electrophoresis, total RNA samples were adjusted to 500 ng/µl with DEPC water and cDNA was subsequently prepared by applying AMV reverse transcriptase (Invitrogen) and oligo-dT primers.

    Staining:

    Article Title: Small RNAs derived from tRNAs and rRNAs are highly enriched in exosomes from both old and new world Leishmania providing evidence for conserved exosomal RNA Packaging
    Article Snippet: RNA was resuspended in 4 μL ddH2 O and RNA length profiles were obtained with the Agilent Bioanalyzer using the RNA 6000 Pico kit according to the manufacturer’s instructions (Agilent). .. Alternatively, DNase-treated RNA was run on a 15% polyacrylamide gel, stained with SYBR green (Life Technologies) and imaged with UV-imaging. .. To confirm identity of nucleic acid purified from exosomes as RNA, 1–2 μg of phenol/chloroform extracted RNA was treated with DNase (as above), followed by treatment with either 0.4 mg/mL RNase A (Thermo) for 15 mins at 37°C or hydrolysis with 50 mM KOH (Sigma-Aldrich) for 15 min at 95°C.

    SYBR Green Assay:

    Article Title: Small RNAs derived from tRNAs and rRNAs are highly enriched in exosomes from both old and new world Leishmania providing evidence for conserved exosomal RNA Packaging
    Article Snippet: RNA was resuspended in 4 μL ddH2 O and RNA length profiles were obtained with the Agilent Bioanalyzer using the RNA 6000 Pico kit according to the manufacturer’s instructions (Agilent). .. Alternatively, DNase-treated RNA was run on a 15% polyacrylamide gel, stained with SYBR green (Life Technologies) and imaged with UV-imaging. .. To confirm identity of nucleic acid purified from exosomes as RNA, 1–2 μg of phenol/chloroform extracted RNA was treated with DNase (as above), followed by treatment with either 0.4 mg/mL RNase A (Thermo) for 15 mins at 37°C or hydrolysis with 50 mM KOH (Sigma-Aldrich) for 15 min at 95°C.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: A 9 bp cis-element in the promoters of class I small heat shock protein genes on chromosome 3 in rice mediates L-azetidine-2-carboxylic acid and heat shock responses
    Article Snippet: Total RNA was extracted with use of TRIZOL reagent (Invitrogen, Rockville, MD, USA) according to the manufacturer's protocol. .. A 16 ng aliquot of DNase I-treated total RNA was used for RT-PCR analyses with use of the Superscript one-step RT-PCR kit (Invitrogen) according to the manufacturer's protocol. .. For all treatments, RT-PCR was repeated three times, with three batches of total RNA samples isolated independently.

    Quantitative RT-PCR:

    Article Title: Grapevine Grafting: Scion Transcript Profiling and Defense-Related Metabolites Induced by Rootstocks
    Article Snippet: Metabolic pathways were visualized using MapMan software Version 3.6.0RC1 (Thimm et al., ; http://mapman.gabipd.org/web/guest/mapman), integrated for grapevine with information from Nimblegen and Affymetrix platforms, as previously reported (Rotter et al., ; Dal Santo et al., ). .. RT-qPCR analysis of gene expression Total RNA was treated with DNase I (Invitrogen, Thermo Fisher Scientific) in accordance with the manufacturer's instructions. .. For each biological replicate, first-strand cDNA was synthesized starting from 2 μg of total RNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Thermo Fisher Scientific) according to the manufacturer's instructions.

    Expressing:

    Article Title: Grapevine Grafting: Scion Transcript Profiling and Defense-Related Metabolites Induced by Rootstocks
    Article Snippet: Metabolic pathways were visualized using MapMan software Version 3.6.0RC1 (Thimm et al., ; http://mapman.gabipd.org/web/guest/mapman), integrated for grapevine with information from Nimblegen and Affymetrix platforms, as previously reported (Rotter et al., ; Dal Santo et al., ). .. RT-qPCR analysis of gene expression Total RNA was treated with DNase I (Invitrogen, Thermo Fisher Scientific) in accordance with the manufacturer's instructions. .. For each biological replicate, first-strand cDNA was synthesized starting from 2 μg of total RNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Thermo Fisher Scientific) according to the manufacturer's instructions.

    Purification:

    Article Title: An Optimized Lentiviral Vector Efficiently Corrects the Human Sickle Cell Disease Phenotype
    Article Snippet: Viral Transcript Quantification Total RNA was extracted from HEK293T cells transfected as described above (“Viral vector preparation”), using the RNeasy kit (QIAGEN, Venlo, Netherlands), which included an in-column DNase I treatment step. .. An additional DNase treatment was performed after RNA purification using the TURBO DNA-free kit (Ambion, Waltham, MA, USA). .. Then, RNA was reverse-transcribed by extension of random hexamers using the SuperScript III First-Strand Synthesis kit (Invitrogen, Carlsbad, CA, USA).

    Activity Assay:

    Article Title: Capsular Type, Sequence Type and Microbial Resistance Factors Impact on DNase Activity of Streptococcus agalactiae Strains from Human and Bovine Origin
    Article Snippet: .. DNase Activity – Qualitative Assays Qualitative evaluation of DNase production was performed for all S. agalactiae strains; this was done by inoculation of DNA–methyl green agar plates (Oxoid, Basingstoke, England) or DNase test agar with toluidine blue plates (Sigma-Aldrich, USA). .. S. agalactiae strains, namely, 090R, COH1, or NEM316, were used as positive controls, and 2603V/R as a negative control.

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    Thermo Fisher dnase treated total rna
    RT-PCR validation of 21 predicted novel genes. a Amplification of a fragment of PfAlba3 (PF3D7_1006200) using genomic DNA (middle lane) or cDNA prepared from <t>DNase-treated</t> total <t>RNA</t> (right lane) as a template. Primers were designed on both sides of intron 1, yielding a 429 bp PCR product from genomic DNA and a 164 bp PCR product from cDNA. The presence of a single 164 bp PCR product amplified from cDNA confirms the absence of gDNA contamination. b Out of our 231 novel candidate genes, we chose 21 regions for validation using reverse transcription polymerase chain reaction (RT-PCR). The top panel shows amplification products using DNase-treated cDNA as a template, while the bottom panel shows the control reactions using genomic DNA as a template. Of the 21 gene tested, we were able to amplify 20 of the predicted regions. As a control, we were unable to amplify a fragment of intergenic region that was not predicted to contain any genes (marked as “intergenic”)
    Dnase Treated Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase treated total rna/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dnase treated total rna - by Bioz Stars, 2021-04
    97/100 stars
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    99
    Thermo Fisher rna
    Detection of mRNAs encoding invertase in both Leishmania sp. promastigote and axenic amastigote developmental forms. a Northern blot of total <t>RNA</t> isolated from L. donovani ( Ld ) promastigotes (Pro) and axenic amastigotes (AxAm) probed with the LdINV -DIG858 probe. Molecular mass markers in kb are shown at the left ( arrows ). b Ethidium bromide stained gel showing equivalent amounts of Pro and AxAm RNA were used to generate the Northern blot shown in a above. Molecular mass markers in kb are shown at the left ( arrows ). c Ethidium bromide-stained agarose gel showing the ~500 bp product ( arrow ) obtained from RT-PCR reactions using RNA isolated from L. <t>mexicana</t> ( Lmex ) promastigotes (Pro) and axenic amastigotes (AxAm). This image was inverted using Adobe Photoshop CS2 to accentuate the RT-PCR product obtained in these reactions
    Rna, 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/rna/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rna - by Bioz Stars, 2021-04
    99/100 stars
      Buy from Supplier

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    RT-PCR validation of 21 predicted novel genes. a Amplification of a fragment of PfAlba3 (PF3D7_1006200) using genomic DNA (middle lane) or cDNA prepared from DNase-treated total RNA (right lane) as a template. Primers were designed on both sides of intron 1, yielding a 429 bp PCR product from genomic DNA and a 164 bp PCR product from cDNA. The presence of a single 164 bp PCR product amplified from cDNA confirms the absence of gDNA contamination. b Out of our 231 novel candidate genes, we chose 21 regions for validation using reverse transcription polymerase chain reaction (RT-PCR). The top panel shows amplification products using DNase-treated cDNA as a template, while the bottom panel shows the control reactions using genomic DNA as a template. Of the 21 gene tested, we were able to amplify 20 of the predicted regions. As a control, we were unable to amplify a fragment of intergenic region that was not predicted to contain any genes (marked as “intergenic”)

    Journal: BMC Genomics

    Article Title: Analysis of nucleosome positioning landscapes enables gene discovery in the human malaria parasite Plasmodium falciparum

    doi: 10.1186/s12864-015-2214-9

    Figure Lengend Snippet: RT-PCR validation of 21 predicted novel genes. a Amplification of a fragment of PfAlba3 (PF3D7_1006200) using genomic DNA (middle lane) or cDNA prepared from DNase-treated total RNA (right lane) as a template. Primers were designed on both sides of intron 1, yielding a 429 bp PCR product from genomic DNA and a 164 bp PCR product from cDNA. The presence of a single 164 bp PCR product amplified from cDNA confirms the absence of gDNA contamination. b Out of our 231 novel candidate genes, we chose 21 regions for validation using reverse transcription polymerase chain reaction (RT-PCR). The top panel shows amplification products using DNase-treated cDNA as a template, while the bottom panel shows the control reactions using genomic DNA as a template. Of the 21 gene tested, we were able to amplify 20 of the predicted regions. As a control, we were unable to amplify a fragment of intergenic region that was not predicted to contain any genes (marked as “intergenic”)

    Article Snippet: DNase-treated total RNA was then mixed with 0.1 μg of random hexamers, 0.6 μg of oligo-dT(20), and 2 μl 10 mM dNTP mix (Life Technologies) in total volume of 10 μl, incubated for 10 min at 70 °C and then chilled on ice for 5 min.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction

    qPCR of novel differentially expressed Rp _sRs Confluent monolayers of human microvascular endothelial cells (HMECs) cultured at 37°C or 34°C or Amblyomma americanum cells (AAE2) cultured at 34°C were infected with R. prowazekii for 0.5h, 3h, and 24h. Total RNA was extracted using Trizol, DNaseI treated, and reverse transcribed for RT-PCR (n≥3). AAE2 and HMEC expression were baselined to 0.5h and normalized to 16S rRNA. The data is presented as mean±SEM for four trans-acting Rp _sRs, namely, Rp _sR76, Rp_ sR83, Rp_ sR86, and Rp_ sR159. Asterisks indicate *p

    Journal: Ticks and tick-borne diseases

    Article Title: Transcriptional profiling of Rickettsia prowazekii coding and non-coding transcripts during in vitro host-pathogen and vector-pathogen interactions

    doi: 10.1016/j.ttbdis.2017.06.008

    Figure Lengend Snippet: qPCR of novel differentially expressed Rp _sRs Confluent monolayers of human microvascular endothelial cells (HMECs) cultured at 37°C or 34°C or Amblyomma americanum cells (AAE2) cultured at 34°C were infected with R. prowazekii for 0.5h, 3h, and 24h. Total RNA was extracted using Trizol, DNaseI treated, and reverse transcribed for RT-PCR (n≥3). AAE2 and HMEC expression were baselined to 0.5h and normalized to 16S rRNA. The data is presented as mean±SEM for four trans-acting Rp _sRs, namely, Rp _sR76, Rp_ sR83, Rp_ sR86, and Rp_ sR159. Asterisks indicate *p

    Article Snippet: One microgram (1μg) of DNaseI treated total RNA was reverse transcribed using High Capacity Reverse Transcription Kit (Applied Biosystems) with random primers following the manufacturer’s instructions. q-RT PCR was performed using SYBR® Green PCR Master Mix (Life Technologies).

    Techniques: Real-time Polymerase Chain Reaction, Cell Culture, Infection, Reverse Transcription Polymerase Chain Reaction, Expressing

    Identification of alternative MIPS transcript variants in tilapia gill epithelium. (A) Amplification of MIPS cDNA using the initial set of primers yields two PCR products, but the shorter product (MIPS-160) is much more abundant than the longer product (MIPS-250). Lanes 1–5: gill RNA from different biological replicates of 70 ppt acclimated fish and treated with TURBO DNase (2U) prior to cDNA synthesis; Lane 6: control 1 indicating effectiveness of DNase treatment (gDNA was added to RNA at a 1:3 ratio, which did not affect the PCR result); Lane 7: control indicating complete removal of gDNA by DNase treatment (RNA was substituted with gDNA prior to DNase treatment and cDNA synthesis); Lane 8: negative control (no template); Lane 9: tilapia genomic DNA amplification (same as Lane 7 but without TURBO DNase treatment); Lane 10: positive RT-PCR control using HeLa cell RNA for cDNA synthesis followed by amplification of human beta-actin; Lane M: 100 bp DNA ladder. (B) Schematic representation of MIPS post-transcriptional processing due to alternative in-frame splicing of a 86 bp nucleotide fragment (indicated by a gray arrow) yielding two distinct MIPS transcripts whose PCR products are 164 and 251 bp long. (C) Sequence of the amplicons obtained using the initial MIPS primer pair and a primer that is specific for amplifying only the long transcript variant of MIPS. Annealing sites of each PCR primer are indicated as blue and green arrows, and the 86 bp fragment that distinguishes the transcript variants is shown italicized and with gray shading. The corresponding amino acid sequence is also shown italicized and with gray shading in the box below. (D) Agarose gel containing PCR products from gill cDNA samples of tilapia exposed to 34 ppt salinity stress. Lane M: 100 bp DNA ladder; Lanes 1–5: different MIPS primer pairs that are specific to only amplifying the long MIPS transcript variant (including the pair shown in C, which was selected for qPCR) yield only a single PCR product (MIPS250); Lane NC: negative PCR control (no primers).

    Journal: The Journal of Experimental Biology

    Article Title: Salinity-induced regulation of the myo-inositol biosynthesis pathway in tilapia gill epithelium

    doi: 10.1242/jeb.093823

    Figure Lengend Snippet: Identification of alternative MIPS transcript variants in tilapia gill epithelium. (A) Amplification of MIPS cDNA using the initial set of primers yields two PCR products, but the shorter product (MIPS-160) is much more abundant than the longer product (MIPS-250). Lanes 1–5: gill RNA from different biological replicates of 70 ppt acclimated fish and treated with TURBO DNase (2U) prior to cDNA synthesis; Lane 6: control 1 indicating effectiveness of DNase treatment (gDNA was added to RNA at a 1:3 ratio, which did not affect the PCR result); Lane 7: control indicating complete removal of gDNA by DNase treatment (RNA was substituted with gDNA prior to DNase treatment and cDNA synthesis); Lane 8: negative control (no template); Lane 9: tilapia genomic DNA amplification (same as Lane 7 but without TURBO DNase treatment); Lane 10: positive RT-PCR control using HeLa cell RNA for cDNA synthesis followed by amplification of human beta-actin; Lane M: 100 bp DNA ladder. (B) Schematic representation of MIPS post-transcriptional processing due to alternative in-frame splicing of a 86 bp nucleotide fragment (indicated by a gray arrow) yielding two distinct MIPS transcripts whose PCR products are 164 and 251 bp long. (C) Sequence of the amplicons obtained using the initial MIPS primer pair and a primer that is specific for amplifying only the long transcript variant of MIPS. Annealing sites of each PCR primer are indicated as blue and green arrows, and the 86 bp fragment that distinguishes the transcript variants is shown italicized and with gray shading. The corresponding amino acid sequence is also shown italicized and with gray shading in the box below. (D) Agarose gel containing PCR products from gill cDNA samples of tilapia exposed to 34 ppt salinity stress. Lane M: 100 bp DNA ladder; Lanes 1–5: different MIPS primer pairs that are specific to only amplifying the long MIPS transcript variant (including the pair shown in C, which was selected for qPCR) yield only a single PCR product (MIPS250); Lane NC: negative PCR control (no primers).

    Article Snippet: Two micrograms of DNase-treated total RNA were reverse transcribed in the presence of oligo(dT) and random hexamers in a 1:1 ratio using SuperScript III First-Strand Synthesis kit (Life Technologies).

    Techniques: Amplification, Polymerase Chain Reaction, Fluorescence In Situ Hybridization, Negative Control, Reverse Transcription Polymerase Chain Reaction, Sequencing, Variant Assay, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction

    Detection of mRNAs encoding invertase in both Leishmania sp. promastigote and axenic amastigote developmental forms. a Northern blot of total RNA isolated from L. donovani ( Ld ) promastigotes (Pro) and axenic amastigotes (AxAm) probed with the LdINV -DIG858 probe. Molecular mass markers in kb are shown at the left ( arrows ). b Ethidium bromide stained gel showing equivalent amounts of Pro and AxAm RNA were used to generate the Northern blot shown in a above. Molecular mass markers in kb are shown at the left ( arrows ). c Ethidium bromide-stained agarose gel showing the ~500 bp product ( arrow ) obtained from RT-PCR reactions using RNA isolated from L. mexicana ( Lmex ) promastigotes (Pro) and axenic amastigotes (AxAm). This image was inverted using Adobe Photoshop CS2 to accentuate the RT-PCR product obtained in these reactions

    Journal: Molecular and cellular biochemistry

    Article Title: A unique, highly conserved secretory invertase is differentially expressed by promastigote developmental forms of all species of the human pathogen, Leishmania

    doi: 10.1007/s11010-015-2366-6

    Figure Lengend Snippet: Detection of mRNAs encoding invertase in both Leishmania sp. promastigote and axenic amastigote developmental forms. a Northern blot of total RNA isolated from L. donovani ( Ld ) promastigotes (Pro) and axenic amastigotes (AxAm) probed with the LdINV -DIG858 probe. Molecular mass markers in kb are shown at the left ( arrows ). b Ethidium bromide stained gel showing equivalent amounts of Pro and AxAm RNA were used to generate the Northern blot shown in a above. Molecular mass markers in kb are shown at the left ( arrows ). c Ethidium bromide-stained agarose gel showing the ~500 bp product ( arrow ) obtained from RT-PCR reactions using RNA isolated from L. mexicana ( Lmex ) promastigotes (Pro) and axenic amastigotes (AxAm). This image was inverted using Adobe Photoshop CS2 to accentuate the RT-PCR product obtained in these reactions

    Article Snippet: For these experiments, 1.5 μg of total RNA (pre-treated with DNase) from L. mexicana M379 Pro- and AxAm were reverse transcribed into cDNA using the Superscript® First Strand Synthesis System for RT-PCR (Invitrogen) and oligo d(t) primers as per manufacturer’s instructions.

    Techniques: Northern Blot, Isolation, Staining, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction