reverse transcription quantitative pcr rt qpcr  (Roche)

 
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    Structured Review

    Roche reverse transcription quantitative pcr rt qpcr
    Reverse Transcription Quantitative Pcr Rt Qpcr, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/reverse transcription quantitative pcr rt qpcr/product/Roche
    Average 92 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    reverse transcription quantitative pcr rt qpcr - by Bioz Stars, 2020-07
    92/100 stars

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    SYBR Green Assay:

    Article Title: Signaling Inhibitors Accelerate the Conversion of mouse iPS Cells into Cancer Stem Cells in the Tumor Microenvironment
    Article Snippet: .. Reverse transcription-quantitative PCR (RT-qPCR) was performed with LightCycler 480 SYBR Green I Master Mix (Roche, Switzerland). .. Histological analysis and immunohistochemistryFor histological analysis, tumors were excised from the mice 4–6 weeks after transplantation.

    Article Title: ANXA9 gene expression in colorectal cancer: A novel marker for prognosis
    Article Snippet: .. For quantitative assessment, reverse transcription-quantitative PCR (RT-qPCR) was performed using a LightCycler FastStart DNA Master SYBR Green I kit (Roche Diagnostics, Tokyo, Japan) for cDNA amplification of ANXA9 and GAPDH . .. The amplification protocol consisted of 35 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 10 sec and elongation at 72°C for 10 sec.

    Article Title: Imbalance of M1/M2 macrophages is linked to severity level of knee osteoarthritis
    Article Snippet: .. Reverse transcription-quantitative PCR (RT-qPCR) was performed using FastStart Universal SYBR-Green Master (ROX) Mix (2X) (Roche Diagnostics, Indianapolis, IN, USA), with 2.5 µl cDNA per reaction in a total volume of 20 µl. .. RT-qPCR reactions were run in duplicates on the IQ5™ real-time PCR system (Bio-Rad Laboratories, Hercules, CA, USA).

    Article Title: Unique Microbial Catabolic Pathway for the Human Core N-Glycan Constituent Fucosyl-α-1,6-N-Acetylglucosamine-Asparagine
    Article Snippet: .. Reverse transcription-quantitative PCR (RT-qPCR) was performed for each cDNA sample in triplicate using the LightCycler 480 system (Roche), LC fast-start DNA master SYBR green I (Roche), and the primers pairs q29280for/q29280rev (sugK ), q29290for/q29290rev (asnA2 ), q29300for/q29300rev (pepV ), q29310for/q29310rev (alfR2 ), q29320for/q29320rev (asdA ), q29330for/q29330rev (alfH ), and q29340for/q29340rev (alfC ) ( ). ..

    Amplification:

    Article Title: ANXA9 gene expression in colorectal cancer: A novel marker for prognosis
    Article Snippet: .. For quantitative assessment, reverse transcription-quantitative PCR (RT-qPCR) was performed using a LightCycler FastStart DNA Master SYBR Green I kit (Roche Diagnostics, Tokyo, Japan) for cDNA amplification of ANXA9 and GAPDH . .. The amplification protocol consisted of 35 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 10 sec and elongation at 72°C for 10 sec.

    Quantitative RT-PCR:

    Article Title: Signaling Inhibitors Accelerate the Conversion of mouse iPS Cells into Cancer Stem Cells in the Tumor Microenvironment
    Article Snippet: .. Reverse transcription-quantitative PCR (RT-qPCR) was performed with LightCycler 480 SYBR Green I Master Mix (Roche, Switzerland). .. Histological analysis and immunohistochemistryFor histological analysis, tumors were excised from the mice 4–6 weeks after transplantation.

    Article Title: ANXA9 gene expression in colorectal cancer: A novel marker for prognosis
    Article Snippet: .. For quantitative assessment, reverse transcription-quantitative PCR (RT-qPCR) was performed using a LightCycler FastStart DNA Master SYBR Green I kit (Roche Diagnostics, Tokyo, Japan) for cDNA amplification of ANXA9 and GAPDH . .. The amplification protocol consisted of 35 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 10 sec and elongation at 72°C for 10 sec.

    Article Title: Characterization and comparative analysis of microRNAs in the rice pest Sogatella furcifera
    Article Snippet: .. Reverse transcription quantitative PCR (RT-qPCR) was conducted on a Light Cycler 96 (Roche) with FastStart Essential DNA Green Master mix. .. All reactions were performed in triplicate, and the expression level of RNAs was calculated using the 2−ΔΔCt method [ ].

    Article Title: Imbalance of M1/M2 macrophages is linked to severity level of knee osteoarthritis
    Article Snippet: .. Reverse transcription-quantitative PCR (RT-qPCR) was performed using FastStart Universal SYBR-Green Master (ROX) Mix (2X) (Roche Diagnostics, Indianapolis, IN, USA), with 2.5 µl cDNA per reaction in a total volume of 20 µl. .. RT-qPCR reactions were run in duplicates on the IQ5™ real-time PCR system (Bio-Rad Laboratories, Hercules, CA, USA).

    Article Title: Transcriptome Analysis of Landrace Pig Subcutaneous Preadipocytes during Adipogenic Differentiation
    Article Snippet: .. Validation of DEGs Using Reverse Transcription Quantitative PCR To validate the sequencing results, DEGs (acetyl-CoA acyltransferase 2 (ACAA2 ), angiopoietin-like 4 (ANGPTL4 ), aldehyde dehydrogenase 2 family member (ALDH2 ), perilipin 2 (PLIN2 ), solute carrier family 27 member 1 (SLC27A1 ), lipoprotein lipase (LPL ), carnitine palmitoyltransferase 1A (CPT1A ), and lipase A lysosomal acid type (LIPA )) were selected for further analysis using reverse transcription quantitative PCR (RT-qPCR) with the Light Cycler® 480 Real-Time PCR system (Roche, Hercules, CA, USA), according to the manufacturer’s instructions. .. The primers used for the RT-qPCR detection of selected genes are listed in .

    Article Title: Unique Microbial Catabolic Pathway for the Human Core N-Glycan Constituent Fucosyl-α-1,6-N-Acetylglucosamine-Asparagine
    Article Snippet: .. Reverse transcription-quantitative PCR (RT-qPCR) was performed for each cDNA sample in triplicate using the LightCycler 480 system (Roche), LC fast-start DNA master SYBR green I (Roche), and the primers pairs q29280for/q29280rev (sugK ), q29290for/q29290rev (asnA2 ), q29300for/q29300rev (pepV ), q29310for/q29310rev (alfR2 ), q29320for/q29320rev (asdA ), q29330for/q29330rev (alfH ), and q29340for/q29340rev (alfC ) ( ). ..

    Real-time Polymerase Chain Reaction:

    Article Title: Characterization and comparative analysis of microRNAs in the rice pest Sogatella furcifera
    Article Snippet: .. Reverse transcription quantitative PCR (RT-qPCR) was conducted on a Light Cycler 96 (Roche) with FastStart Essential DNA Green Master mix. .. All reactions were performed in triplicate, and the expression level of RNAs was calculated using the 2−ΔΔCt method [ ].

    Article Title: Transcriptome Analysis of Landrace Pig Subcutaneous Preadipocytes during Adipogenic Differentiation
    Article Snippet: .. Validation of DEGs Using Reverse Transcription Quantitative PCR To validate the sequencing results, DEGs (acetyl-CoA acyltransferase 2 (ACAA2 ), angiopoietin-like 4 (ANGPTL4 ), aldehyde dehydrogenase 2 family member (ALDH2 ), perilipin 2 (PLIN2 ), solute carrier family 27 member 1 (SLC27A1 ), lipoprotein lipase (LPL ), carnitine palmitoyltransferase 1A (CPT1A ), and lipase A lysosomal acid type (LIPA )) were selected for further analysis using reverse transcription quantitative PCR (RT-qPCR) with the Light Cycler® 480 Real-Time PCR system (Roche, Hercules, CA, USA), according to the manufacturer’s instructions. .. The primers used for the RT-qPCR detection of selected genes are listed in .

    Polymerase Chain Reaction:

    Article Title: Signaling Inhibitors Accelerate the Conversion of mouse iPS Cells into Cancer Stem Cells in the Tumor Microenvironment
    Article Snippet: .. Reverse transcription-quantitative PCR (RT-qPCR) was performed with LightCycler 480 SYBR Green I Master Mix (Roche, Switzerland). .. Histological analysis and immunohistochemistryFor histological analysis, tumors were excised from the mice 4–6 weeks after transplantation.

    Article Title: ANXA9 gene expression in colorectal cancer: A novel marker for prognosis
    Article Snippet: .. For quantitative assessment, reverse transcription-quantitative PCR (RT-qPCR) was performed using a LightCycler FastStart DNA Master SYBR Green I kit (Roche Diagnostics, Tokyo, Japan) for cDNA amplification of ANXA9 and GAPDH . .. The amplification protocol consisted of 35 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 10 sec and elongation at 72°C for 10 sec.

    Article Title: Imbalance of M1/M2 macrophages is linked to severity level of knee osteoarthritis
    Article Snippet: .. Reverse transcription-quantitative PCR (RT-qPCR) was performed using FastStart Universal SYBR-Green Master (ROX) Mix (2X) (Roche Diagnostics, Indianapolis, IN, USA), with 2.5 µl cDNA per reaction in a total volume of 20 µl. .. RT-qPCR reactions were run in duplicates on the IQ5™ real-time PCR system (Bio-Rad Laboratories, Hercules, CA, USA).

    Article Title: Unique Microbial Catabolic Pathway for the Human Core N-Glycan Constituent Fucosyl-α-1,6-N-Acetylglucosamine-Asparagine
    Article Snippet: .. Reverse transcription-quantitative PCR (RT-qPCR) was performed for each cDNA sample in triplicate using the LightCycler 480 system (Roche), LC fast-start DNA master SYBR green I (Roche), and the primers pairs q29280for/q29280rev (sugK ), q29290for/q29290rev (asnA2 ), q29300for/q29300rev (pepV ), q29310for/q29310rev (alfR2 ), q29320for/q29320rev (asdA ), q29330for/q29330rev (alfH ), and q29340for/q29340rev (alfC ) ( ). ..

    Sequencing:

    Article Title: Transcriptome Analysis of Landrace Pig Subcutaneous Preadipocytes during Adipogenic Differentiation
    Article Snippet: .. Validation of DEGs Using Reverse Transcription Quantitative PCR To validate the sequencing results, DEGs (acetyl-CoA acyltransferase 2 (ACAA2 ), angiopoietin-like 4 (ANGPTL4 ), aldehyde dehydrogenase 2 family member (ALDH2 ), perilipin 2 (PLIN2 ), solute carrier family 27 member 1 (SLC27A1 ), lipoprotein lipase (LPL ), carnitine palmitoyltransferase 1A (CPT1A ), and lipase A lysosomal acid type (LIPA )) were selected for further analysis using reverse transcription quantitative PCR (RT-qPCR) with the Light Cycler® 480 Real-Time PCR system (Roche, Hercules, CA, USA), according to the manufacturer’s instructions. .. The primers used for the RT-qPCR detection of selected genes are listed in .

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    Roche real time quantitative reverse transcription pcr rt qpcr reactions
    Quantitative <t>RT-PCR</t> analysis of candidate genes. Mock infected plants were used to normalize fold change of early and late samples in leaf and tuber tissues as indicated. White bars represent the <t>RT-qPCR</t> analysis; grey bars show the microarray data. Error bars represent standard error (SE). L23 and PP2A genes were used as internal normalization controls. Asterisk indicates that the means of fold change of the mock and infected samples are significantly different (Student’s t-test).
    Real Time Quantitative Reverse Transcription Pcr Rt Qpcr Reactions, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/real time quantitative reverse transcription pcr rt qpcr reactions/product/Roche
    Average 90 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    real time quantitative reverse transcription pcr rt qpcr reactions - by Bioz Stars, 2020-07
    90/100 stars
      Buy from Supplier

    92
    Roche quantitative reverse transcription pcr rt qpcr total rna
    Notch signaling augments TLR4-stimulated IL-6 and TNFα expression. Monocytes were pretreated with DAPT (2.5 μM) for 1 h before LPS stimulation and E. coli infection. 2 h after infection, medium was changed and DAPT was added again. (A) The next day <t>RNA</t> was isolated, cDNA produced and gene expression was analyzed by qRT <t>PCR</t> using sequence-specific primer for HES and Hey and SYBR Green. Results were normalized against actin. (B) Supernatants were used for ELISA analysis to quantify released IL-6, IL-12p40, and TNFα. (C) For western blot analysis cells were harvested 2 h after infection. Nuclear lysates were produced and equal amounts of lysates were blotted and probed with antibodies against p65 or B23 (control). Shown is one representative experiment out of three and the associated quantification of three experiments. (A–C) mean ± std n = 3, * p ≤ = 0.05 by Mann–Whitney U -test.
    Quantitative Reverse Transcription Pcr Rt Qpcr Total Rna, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quantitative reverse transcription pcr rt qpcr total rna/product/Roche
    Average 92 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    quantitative reverse transcription pcr rt qpcr total rna - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

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    Quantitative RT-PCR analysis of candidate genes. Mock infected plants were used to normalize fold change of early and late samples in leaf and tuber tissues as indicated. White bars represent the RT-qPCR analysis; grey bars show the microarray data. Error bars represent standard error (SE). L23 and PP2A genes were used as internal normalization controls. Asterisk indicates that the means of fold change of the mock and infected samples are significantly different (Student’s t-test).

    Journal: PLoS ONE

    Article Title: Insight on Genes Affecting Tuber Development in Potato upon Potato spindle tuber viroid (PSTVd) Infection

    doi: 10.1371/journal.pone.0150711

    Figure Lengend Snippet: Quantitative RT-PCR analysis of candidate genes. Mock infected plants were used to normalize fold change of early and late samples in leaf and tuber tissues as indicated. White bars represent the RT-qPCR analysis; grey bars show the microarray data. Error bars represent standard error (SE). L23 and PP2A genes were used as internal normalization controls. Asterisk indicates that the means of fold change of the mock and infected samples are significantly different (Student’s t-test).

    Article Snippet: Three technical replicates of each three biological replicates (for infected tuber tissue only two biological replicates) were used in subsequent Real-Time Quantitative Reverse Transcription PCR (RT-qPCR) reactions using FastStart Universal Probe Master with Rox (Roche Diagnostics, Switzerland).

    Techniques: Quantitative RT-PCR, Infection, Microarray

    Comparison of the multiplex endpoint RT-PCR and RT-qPCR assays for protocol 2 (P2) . The bottom axis identifies the samples. The band sizes obtained for each of the sample replicate are shown as bars. The values on the top of the graph provide both the C q value measured for each sample replicate in the TBP RT-qPCR assay and the mean C q value. Samples labelled with a * symbol indicate a standard deviation between replicates that is greater than 0.5.

    Journal: BMC Biotechnology

    Article Title: A multiplex endpoint RT-PCR assay for quality assessment of RNA extracted from formalin-fixed paraffin-embedded tissues

    doi: 10.1186/1472-6750-10-89

    Figure Lengend Snippet: Comparison of the multiplex endpoint RT-PCR and RT-qPCR assays for protocol 2 (P2) . The bottom axis identifies the samples. The band sizes obtained for each of the sample replicate are shown as bars. The values on the top of the graph provide both the C q value measured for each sample replicate in the TBP RT-qPCR assay and the mean C q value. Samples labelled with a * symbol indicate a standard deviation between replicates that is greater than 0.5.

    Article Snippet: Reverse transcription - quantitative real-time PCR (RT-qPCR) PCR was performed on the LightCycler 480 Instrument (Roche).

    Techniques: Multiplex Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation

    Primer placement for the multiplex endpoint RT-PCR and the RT-qPCR assay . Both assays use the TBP mRNA (NM_003194) as the target sequence. The eight exons are shown as rectangles labelled from E1 to E8. The primer locations are shown as horizontal arrow heads. The positions of the primers for the multiplex endpoint RT-PCR assay (92-F, 92-R, 161-F, 161-R, 252-F, 252-R, 300-F and 300-R) and for the RT-qPCR assay (RT-qPCR-F and RT-qPCR-R) are shown. Each primer pair was designed to span at least one intron.

    Journal: BMC Biotechnology

    Article Title: A multiplex endpoint RT-PCR assay for quality assessment of RNA extracted from formalin-fixed paraffin-embedded tissues

    doi: 10.1186/1472-6750-10-89

    Figure Lengend Snippet: Primer placement for the multiplex endpoint RT-PCR and the RT-qPCR assay . Both assays use the TBP mRNA (NM_003194) as the target sequence. The eight exons are shown as rectangles labelled from E1 to E8. The primer locations are shown as horizontal arrow heads. The positions of the primers for the multiplex endpoint RT-PCR assay (92-F, 92-R, 161-F, 161-R, 252-F, 252-R, 300-F and 300-R) and for the RT-qPCR assay (RT-qPCR-F and RT-qPCR-R) are shown. Each primer pair was designed to span at least one intron.

    Article Snippet: Reverse transcription - quantitative real-time PCR (RT-qPCR) PCR was performed on the LightCycler 480 Instrument (Roche).

    Techniques: Multiplex Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Sequencing

    Comparison of the multiplex endpoint RT-PCR and RT-qPCR assays for protocol 1 (P1) . The bottom axis identifies the samples. The band sizes obtained for each of the sample replicate are shown as bars. The values on the top of the graph provide both the C q value measured for each sample replicate in the TBP RT-qPCR assay and the mean C q value. Symbols are as follows: * indicates a standard deviation between replicates that is greater than 0.5; ** one out of two replicates amplified.

    Journal: BMC Biotechnology

    Article Title: A multiplex endpoint RT-PCR assay for quality assessment of RNA extracted from formalin-fixed paraffin-embedded tissues

    doi: 10.1186/1472-6750-10-89

    Figure Lengend Snippet: Comparison of the multiplex endpoint RT-PCR and RT-qPCR assays for protocol 1 (P1) . The bottom axis identifies the samples. The band sizes obtained for each of the sample replicate are shown as bars. The values on the top of the graph provide both the C q value measured for each sample replicate in the TBP RT-qPCR assay and the mean C q value. Symbols are as follows: * indicates a standard deviation between replicates that is greater than 0.5; ** one out of two replicates amplified.

    Article Snippet: Reverse transcription - quantitative real-time PCR (RT-qPCR) PCR was performed on the LightCycler 480 Instrument (Roche).

    Techniques: Multiplex Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation, Amplification

    Spearman correlation between fragment size in the multiplex endpoint RT-PCR assay and the appropriate C q value obtained in the RT-qPCR assay . The x-axis shows the fragment size and the y-axis shows the C q value. Panel A corresponds to protocol 1 (P1) and Panel B corresponds to protocol 2 (P2).

    Journal: BMC Biotechnology

    Article Title: A multiplex endpoint RT-PCR assay for quality assessment of RNA extracted from formalin-fixed paraffin-embedded tissues

    doi: 10.1186/1472-6750-10-89

    Figure Lengend Snippet: Spearman correlation between fragment size in the multiplex endpoint RT-PCR assay and the appropriate C q value obtained in the RT-qPCR assay . The x-axis shows the fragment size and the y-axis shows the C q value. Panel A corresponds to protocol 1 (P1) and Panel B corresponds to protocol 2 (P2).

    Article Snippet: Reverse transcription - quantitative real-time PCR (RT-qPCR) PCR was performed on the LightCycler 480 Instrument (Roche).

    Techniques: Multiplex Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

    Notch signaling augments TLR4-stimulated IL-6 and TNFα expression. Monocytes were pretreated with DAPT (2.5 μM) for 1 h before LPS stimulation and E. coli infection. 2 h after infection, medium was changed and DAPT was added again. (A) The next day RNA was isolated, cDNA produced and gene expression was analyzed by qRT PCR using sequence-specific primer for HES and Hey and SYBR Green. Results were normalized against actin. (B) Supernatants were used for ELISA analysis to quantify released IL-6, IL-12p40, and TNFα. (C) For western blot analysis cells were harvested 2 h after infection. Nuclear lysates were produced and equal amounts of lysates were blotted and probed with antibodies against p65 or B23 (control). Shown is one representative experiment out of three and the associated quantification of three experiments. (A–C) mean ± std n = 3, * p ≤ = 0.05 by Mann–Whitney U -test.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: The Interplay of Notch Signaling and STAT3 in TLR-Activated Human Primary Monocytes

    doi: 10.3389/fcimb.2018.00241

    Figure Lengend Snippet: Notch signaling augments TLR4-stimulated IL-6 and TNFα expression. Monocytes were pretreated with DAPT (2.5 μM) for 1 h before LPS stimulation and E. coli infection. 2 h after infection, medium was changed and DAPT was added again. (A) The next day RNA was isolated, cDNA produced and gene expression was analyzed by qRT PCR using sequence-specific primer for HES and Hey and SYBR Green. Results were normalized against actin. (B) Supernatants were used for ELISA analysis to quantify released IL-6, IL-12p40, and TNFα. (C) For western blot analysis cells were harvested 2 h after infection. Nuclear lysates were produced and equal amounts of lysates were blotted and probed with antibodies against p65 or B23 (control). Shown is one representative experiment out of three and the associated quantification of three experiments. (A–C) mean ± std n = 3, * p ≤ = 0.05 by Mann–Whitney U -test.

    Article Snippet: Quantitative reverse transcription PCR (RT-qPCR) Total RNA was extracted from 2 × 106 cultured primary human monocytes using the high pure RNA isolation kit (Roche, Mannheim, Germany) according to the manufacturer's protocol.

    Techniques: Expressing, Infection, Isolation, Produced, Quantitative RT-PCR, Sequencing, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Western Blot, MANN-WHITNEY

    TLR signaling induces DLL1 in primary human monocytes. CD14 + monocytes, isolated from blood of healthy donors were stimulated with 100 ng/ml LPS or infected with Escherichia (E.) coli, Enterococcus (E.) faecalis, Klesiella (K.) pneumoniae, Pseudomonas (P.) aeruginosa in a concentration of 10 6 bacteria per 10 6 monocytes/ml. After 2 h bacteria were killed by gentamicin. The next day cells and supernatant were analyzed. (A) RNA was isolated and cDNA produced. Induction of gene expression was analyzed by qRT PCR using sequence-specific primer for DLL1 and JAG1 (gene encoding Jagged-1) and SYBR Green Master mix. Actin was detected as endogenous control for normalization. (B) For western blot analysis equal amounts of protein lysates were blotted and probed with antibodies against DLL1 or GAPDH (loading control). Shown is one representative blot and the associated quantification. Quantification was performed using the Image Analysis System Bioprofil (Fröbel, Germany). The intensity of signals were calculated against loading control and presented as percent of untreated samples. (C) Supernatants were used for ELISA analysis to quantify shedded DLL1 extracellular domain. (A,C) depict the mean and standard deviation of at least three donors. Statistics: * p ≤ 0.05 by Mann–Whitney U -test.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: The Interplay of Notch Signaling and STAT3 in TLR-Activated Human Primary Monocytes

    doi: 10.3389/fcimb.2018.00241

    Figure Lengend Snippet: TLR signaling induces DLL1 in primary human monocytes. CD14 + monocytes, isolated from blood of healthy donors were stimulated with 100 ng/ml LPS or infected with Escherichia (E.) coli, Enterococcus (E.) faecalis, Klesiella (K.) pneumoniae, Pseudomonas (P.) aeruginosa in a concentration of 10 6 bacteria per 10 6 monocytes/ml. After 2 h bacteria were killed by gentamicin. The next day cells and supernatant were analyzed. (A) RNA was isolated and cDNA produced. Induction of gene expression was analyzed by qRT PCR using sequence-specific primer for DLL1 and JAG1 (gene encoding Jagged-1) and SYBR Green Master mix. Actin was detected as endogenous control for normalization. (B) For western blot analysis equal amounts of protein lysates were blotted and probed with antibodies against DLL1 or GAPDH (loading control). Shown is one representative blot and the associated quantification. Quantification was performed using the Image Analysis System Bioprofil (Fröbel, Germany). The intensity of signals were calculated against loading control and presented as percent of untreated samples. (C) Supernatants were used for ELISA analysis to quantify shedded DLL1 extracellular domain. (A,C) depict the mean and standard deviation of at least three donors. Statistics: * p ≤ 0.05 by Mann–Whitney U -test.

    Article Snippet: Quantitative reverse transcription PCR (RT-qPCR) Total RNA was extracted from 2 × 106 cultured primary human monocytes using the high pure RNA isolation kit (Roche, Mannheim, Germany) according to the manufacturer's protocol.

    Techniques: Isolation, Infection, Concentration Assay, Produced, Expressing, Quantitative RT-PCR, Sequencing, SYBR Green Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Standard Deviation, MANN-WHITNEY