SYBR Green Assay:Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection
Article Snippet: E YES Data S1 qPCR OLIGONUCLEOTIDES Primer sequences E YES Included in KRT RTPrimerDB Identification Number D N/A Probe sequences D N/A Location and identity of any modifications E N/A Manufacturer of oligonucleotides D YES Sigma-Aldrich Purification method D YES Desalting qPCR PROTOCOL Complete reaction conditions E YES Initial denaturation 95 ̊ C, 40 cycles of 95 ̊ C of 15 sec, 60 ̊ C for 20 sec Reaction volume and amount of cDNA/DNA E YES 10 μl, 10 ng Primer, (probe), Mg++ and dNTP concentrations E YES 5 μM Polymerase identity and concentration E YES HOT FIREPol® DNA polymerase Buffer/kit identity and manufacturer E YES HOT FIREPol® EvaGreen® qPCR Mix Plus Exact chemical constitution of the buffer D N/A Additives (SYBR Green I, DMSO, etc.) .. E YES SYBR Green Manufacturer of plates/tubes and catalog number D YES Biorad (Catalog: TCS0803, TLS0801) Complete thermocycling parameters E YES Initial denaturation 95 ̊ C, 40 cycles of 95 ̊ C of 15 sec, 60 ̊ C for 20 sec Reaction setup D YES Manual Manufacturer of qPCR instrument E YES Qiagen QIAquant 96 5-plex qPCR VALIDATION Evidence of optimisation (from gradients) D NO Specificity (gel, sequence, melt, or digest) E YES Specific single band For SYBR Green I, Cq of the NTC E YES >38 Standard curves with slope and y-intercept E YES TNF-=-3.1021 -actin=-3.09825 PCR efficiency calculated from slope E YES 110% Confidence interval for PCR efficiency or standard error D NO r2 of standard curve E YES TNF-=0.9928 -actin=0.9984 Linear dynamic range E YES r2 values [TNF=0.9928, Cq variation at lower limit E YES r2 values indicate low variability Confidence intervals throughout range D NO Evidence for limit of detection E NO If multiplex, efficiency and LOD of each assay E N/A DATA ANALYSIS qPCR analysis program (source, version) E YES Graphpad PRISM 8 Cq method determination E YES System generated Outlier identification and disposition E N/A Results of NTCs E YES Cq>38 Justification of number and choice of reference genes E YES -actin Description of normalisation method E YES 2(-delta delta Ct) method Number and concordance of biological replicates D YES Three Number and stage (RT or qPCR) of technical replicates E YES Three Repeatability (intra-assay variation, %CV) E YES 1.73% Reproducibility (inter- assay variation, %CV) D YES 0.6% Power analysis D NO Statistical methods for result significance E YES One-way ANOVA Software (source, version) E YES Q-Rex touch Cq or raw data submission D NO E: essential information; D: desirable information; FFPE: formalin-fixed, paraffin-embedded; RIN: RNA integrity number; RQI: RNA quality indicator; GSP: gene- specific priming; dNTP: deoxynucleoside triphosphate; LOD: limit of detection. ..
Real-time Polymerase Chain Reaction:Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection
Article Snippet: E YES Data S1 qPCR OLIGONUCLEOTIDES Primer sequences E YES Included in KRT RTPrimerDB Identification Number D N/A Probe sequences D N/A Location and identity of any modifications E N/A Manufacturer of oligonucleotides D YES Sigma-Aldrich Purification method D YES Desalting qPCR PROTOCOL Complete reaction conditions E YES Initial denaturation 95 ̊ C, 40 cycles of 95 ̊ C of 15 sec, 60 ̊ C for 20 sec Reaction volume and amount of cDNA/DNA E YES 10 μl, 10 ng Primer, (probe), Mg++ and dNTP concentrations E YES 5 μM Polymerase identity and concentration E YES HOT FIREPol® DNA polymerase Buffer/kit identity and manufacturer E YES HOT FIREPol® EvaGreen® qPCR Mix Plus Exact chemical constitution of the buffer D N/A Additives (SYBR Green I, DMSO, etc.) .. E YES SYBR Green Manufacturer of plates/tubes and catalog number D YES Biorad (Catalog: TCS0803, TLS0801) Complete thermocycling parameters E YES Initial denaturation 95 ̊ C, 40 cycles of 95 ̊ C of 15 sec, 60 ̊ C for 20 sec Reaction setup D YES Manual Manufacturer of qPCR instrument E YES Qiagen QIAquant 96 5-plex qPCR VALIDATION Evidence of optimisation (from gradients) D NO Specificity (gel, sequence, melt, or digest) E YES Specific single band For SYBR Green I, Cq of the NTC E YES >38 Standard curves with slope and y-intercept E YES TNF-=-3.1021 -actin=-3.09825 PCR efficiency calculated from slope E YES 110% Confidence interval for PCR efficiency or standard error D NO r2 of standard curve E YES TNF-=0.9928 -actin=0.9984 Linear dynamic range E YES r2 values [TNF=0.9928, Cq variation at lower limit E YES r2 values indicate low variability Confidence intervals throughout range D NO Evidence for limit of detection E NO If multiplex, efficiency and LOD of each assay E N/A DATA ANALYSIS qPCR analysis program (source, version) E YES Graphpad PRISM 8 Cq method determination E YES System generated Outlier identification and disposition E N/A Results of NTCs E YES Cq>38 Justification of number and choice of reference genes E YES -actin Description of normalisation method E YES 2(-delta delta Ct) method Number and concordance of biological replicates D YES Three Number and stage (RT or qPCR) of technical replicates E YES Three Repeatability (intra-assay variation, %CV) E YES 1.73% Reproducibility (inter- assay variation, %CV) D YES 0.6% Power analysis D NO Statistical methods for result significance E YES One-way ANOVA Software (source, version) E YES Q-Rex touch Cq or raw data submission D NO E: essential information; D: desirable information; FFPE: formalin-fixed, paraffin-embedded; RIN: RNA integrity number; RQI: RNA quality indicator; GSP: gene- specific priming; dNTP: deoxynucleoside triphosphate; LOD: limit of detection. ..
Biomarker Discovery:Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection
Article Snippet: E YES Data S1 qPCR OLIGONUCLEOTIDES Primer sequences E YES Included in KRT RTPrimerDB Identification Number D N/A Probe sequences D N/A Location and identity of any modifications E N/A Manufacturer of oligonucleotides D YES Sigma-Aldrich Purification method D YES Desalting qPCR PROTOCOL Complete reaction conditions E YES Initial denaturation 95 ̊ C, 40 cycles of 95 ̊ C of 15 sec, 60 ̊ C for 20 sec Reaction volume and amount of cDNA/DNA E YES 10 μl, 10 ng Primer, (probe), Mg++ and dNTP concentrations E YES 5 μM Polymerase identity and concentration E YES HOT FIREPol® DNA polymerase Buffer/kit identity and manufacturer E YES HOT FIREPol® EvaGreen® qPCR Mix Plus Exact chemical constitution of the buffer D N/A Additives (SYBR Green I, DMSO, etc.) .. E YES SYBR Green Manufacturer of plates/tubes and catalog number D YES Biorad (Catalog: TCS0803, TLS0801) Complete thermocycling parameters E YES Initial denaturation 95 ̊ C, 40 cycles of 95 ̊ C of 15 sec, 60 ̊ C for 20 sec Reaction setup D YES Manual Manufacturer of qPCR instrument E YES Qiagen QIAquant 96 5-plex qPCR VALIDATION Evidence of optimisation (from gradients) D NO Specificity (gel, sequence, melt, or digest) E YES Specific single band For SYBR Green I, Cq of the NTC E YES >38 Standard curves with slope and y-intercept E YES TNF-=-3.1021 -actin=-3.09825 PCR efficiency calculated from slope E YES 110% Confidence interval for PCR efficiency or standard error D NO r2 of standard curve E YES TNF-=0.9928 -actin=0.9984 Linear dynamic range E YES r2 values [TNF=0.9928, Cq variation at lower limit E YES r2 values indicate low variability Confidence intervals throughout range D NO Evidence for limit of detection E NO If multiplex, efficiency and LOD of each assay E N/A DATA ANALYSIS qPCR analysis program (source, version) E YES Graphpad PRISM 8 Cq method determination E YES System generated Outlier identification and disposition E N/A Results of NTCs E YES Cq>38 Justification of number and choice of reference genes E YES -actin Description of normalisation method E YES 2(-delta delta Ct) method Number and concordance of biological replicates D YES Three Number and stage (RT or qPCR) of technical replicates E YES Three Repeatability (intra-assay variation, %CV) E YES 1.73% Reproducibility (inter- assay variation, %CV) D YES 0.6% Power analysis D NO Statistical methods for result significance E YES One-way ANOVA Software (source, version) E YES Q-Rex touch Cq or raw data submission D NO E: essential information; D: desirable information; FFPE: formalin-fixed, paraffin-embedded; RIN: RNA integrity number; RQI: RNA quality indicator; GSP: gene- specific priming; dNTP: deoxynucleoside triphosphate; LOD: limit of detection. ..
Sequencing:Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection
Article Snippet: E YES Data S1 qPCR OLIGONUCLEOTIDES Primer sequences E YES Included in KRT RTPrimerDB Identification Number D N/A Probe sequences D N/A Location and identity of any modifications E N/A Manufacturer of oligonucleotides D YES Sigma-Aldrich Purification method D YES Desalting qPCR PROTOCOL Complete reaction conditions E YES Initial denaturation 95 ̊ C, 40 cycles of 95 ̊ C of 15 sec, 60 ̊ C for 20 sec Reaction volume and amount of cDNA/DNA E YES 10 μl, 10 ng Primer, (probe), Mg++ and dNTP concentrations E YES 5 μM Polymerase identity and concentration E YES HOT FIREPol® DNA polymerase Buffer/kit identity and manufacturer E YES HOT FIREPol® EvaGreen® qPCR Mix Plus Exact chemical constitution of the buffer D N/A Additives (SYBR Green I, DMSO, etc.) .. E YES SYBR Green Manufacturer of plates/tubes and catalog number D YES Biorad (Catalog: TCS0803, TLS0801) Complete thermocycling parameters E YES Initial denaturation 95 ̊ C, 40 cycles of 95 ̊ C of 15 sec, 60 ̊ C for 20 sec Reaction setup D YES Manual Manufacturer of qPCR instrument E YES Qiagen QIAquant 96 5-plex qPCR VALIDATION Evidence of optimisation (from gradients) D NO Specificity (gel, sequence, melt, or digest) E YES Specific single band For SYBR Green I, Cq of the NTC E YES >38 Standard curves with slope and y-intercept E YES TNF-=-3.1021 -actin=-3.09825 PCR efficiency calculated from slope E YES 110% Confidence interval for PCR efficiency or standard error D NO r2 of standard curve E YES TNF-=0.9928 -actin=0.9984 Linear dynamic range E YES r2 values [TNF=0.9928, Cq variation at lower limit E YES r2 values indicate low variability Confidence intervals throughout range D NO Evidence for limit of detection E NO If multiplex, efficiency and LOD of each assay E N/A DATA ANALYSIS qPCR analysis program (source, version) E YES Graphpad PRISM 8 Cq method determination E YES System generated Outlier identification and disposition E N/A Results of NTCs E YES Cq>38 Justification of number and choice of reference genes E YES -actin Description of normalisation method E YES 2(-delta delta Ct) method Number and concordance of biological replicates D YES Three Number and stage (RT or qPCR) of technical replicates E YES Three Repeatability (intra-assay variation, %CV) E YES 1.73% Reproducibility (inter- assay variation, %CV) D YES 0.6% Power analysis D NO Statistical methods for result significance E YES One-way ANOVA Software (source, version) E YES Q-Rex touch Cq or raw data submission D NO E: essential information; D: desirable information; FFPE: formalin-fixed, paraffin-embedded; RIN: RNA integrity number; RQI: RNA quality indicator; GSP: gene- specific priming; dNTP: deoxynucleoside triphosphate; LOD: limit of detection. ..
Polymerase Chain Reaction:Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection
Article Snippet: E YES Data S1 qPCR OLIGONUCLEOTIDES Primer sequences E YES Included in KRT RTPrimerDB Identification Number D N/A Probe sequences D N/A Location and identity of any modifications E N/A Manufacturer of oligonucleotides D YES Sigma-Aldrich Purification method D YES Desalting qPCR PROTOCOL Complete reaction conditions E YES Initial denaturation 95 ̊ C, 40 cycles of 95 ̊ C of 15 sec, 60 ̊ C for 20 sec Reaction volume and amount of cDNA/DNA E YES 10 μl, 10 ng Primer, (probe), Mg++ and dNTP concentrations E YES 5 μM Polymerase identity and concentration E YES HOT FIREPol® DNA polymerase Buffer/kit identity and manufacturer E YES HOT FIREPol® EvaGreen® qPCR Mix Plus Exact chemical constitution of the buffer D N/A Additives (SYBR Green I, DMSO, etc.) .. E YES SYBR Green Manufacturer of plates/tubes and catalog number D YES Biorad (Catalog: TCS0803, TLS0801) Complete thermocycling parameters E YES Initial denaturation 95 ̊ C, 40 cycles of 95 ̊ C of 15 sec, 60 ̊ C for 20 sec Reaction setup D YES Manual Manufacturer of qPCR instrument E YES Qiagen QIAquant 96 5-plex qPCR VALIDATION Evidence of optimisation (from gradients) D NO Specificity (gel, sequence, melt, or digest) E YES Specific single band For SYBR Green I, Cq of the NTC E YES >38 Standard curves with slope and y-intercept E YES TNF-=-3.1021 -actin=-3.09825 PCR efficiency calculated from slope E YES 110% Confidence interval for PCR efficiency or standard error D NO r2 of standard curve E YES TNF-=0.9928 -actin=0.9984 Linear dynamic range E YES r2 values [TNF=0.9928, Cq variation at lower limit E YES r2 values indicate low variability Confidence intervals throughout range D NO Evidence for limit of detection E NO If multiplex, efficiency and LOD of each assay E N/A DATA ANALYSIS qPCR analysis program (source, version) E YES Graphpad PRISM 8 Cq method determination E YES System generated Outlier identification and disposition E N/A Results of NTCs E YES Cq>38 Justification of number and choice of reference genes E YES -actin Description of normalisation method E YES 2(-delta delta Ct) method Number and concordance of biological replicates D YES Three Number and stage (RT or qPCR) of technical replicates E YES Three Repeatability (intra-assay variation, %CV) E YES 1.73% Reproducibility (inter- assay variation, %CV) D YES 0.6% Power analysis D NO Statistical methods for result significance E YES One-way ANOVA Software (source, version) E YES Q-Rex touch Cq or raw data submission D NO E: essential information; D: desirable information; FFPE: formalin-fixed, paraffin-embedded; RIN: RNA integrity number; RQI: RNA quality indicator; GSP: gene- specific priming; dNTP: deoxynucleoside triphosphate; LOD: limit of detection. ..
Multiplex Assay:Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection
Article Snippet: E YES Data S1 qPCR OLIGONUCLEOTIDES Primer sequences E YES Included in KRT RTPrimerDB Identification Number D N/A Probe sequences D N/A Location and identity of any modifications E N/A Manufacturer of oligonucleotides D YES Sigma-Aldrich Purification method D YES Desalting qPCR PROTOCOL Complete reaction conditions E YES Initial denaturation 95 ̊ C, 40 cycles of 95 ̊ C of 15 sec, 60 ̊ C for 20 sec Reaction volume and amount of cDNA/DNA E YES 10 μl, 10 ng Primer, (probe), Mg++ and dNTP concentrations E YES 5 μM Polymerase identity and concentration E YES HOT FIREPol® DNA polymerase Buffer/kit identity and manufacturer E YES HOT FIREPol® EvaGreen® qPCR Mix Plus Exact chemical constitution of the buffer D N/A Additives (SYBR Green I, DMSO, etc.) .. E YES SYBR Green Manufacturer of plates/tubes and catalog number D YES Biorad (Catalog: TCS0803, TLS0801) Complete thermocycling parameters E YES Initial denaturation 95 ̊ C, 40 cycles of 95 ̊ C of 15 sec, 60 ̊ C for 20 sec Reaction setup D YES Manual Manufacturer of qPCR instrument E YES Qiagen QIAquant 96 5-plex qPCR VALIDATION Evidence of optimisation (from gradients) D NO Specificity (gel, sequence, melt, or digest) E YES Specific single band For SYBR Green I, Cq of the NTC E YES >38 Standard curves with slope and y-intercept E YES TNF-=-3.1021 -actin=-3.09825 PCR efficiency calculated from slope E YES 110% Confidence interval for PCR efficiency or standard error D NO r2 of standard curve E YES TNF-=0.9928 -actin=0.9984 Linear dynamic range E YES r2 values [TNF=0.9928, Cq variation at lower limit E YES r2 values indicate low variability Confidence intervals throughout range D NO Evidence for limit of detection E NO If multiplex, efficiency and LOD of each assay E N/A DATA ANALYSIS qPCR analysis program (source, version) E YES Graphpad PRISM 8 Cq method determination E YES System generated Outlier identification and disposition E N/A Results of NTCs E YES Cq>38 Justification of number and choice of reference genes E YES -actin Description of normalisation method E YES 2(-delta delta Ct) method Number and concordance of biological replicates D YES Three Number and stage (RT or qPCR) of technical replicates E YES Three Repeatability (intra-assay variation, %CV) E YES 1.73% Reproducibility (inter- assay variation, %CV) D YES 0.6% Power analysis D NO Statistical methods for result significance E YES One-way ANOVA Software (source, version) E YES Q-Rex touch Cq or raw data submission D NO E: essential information; D: desirable information; FFPE: formalin-fixed, paraffin-embedded; RIN: RNA integrity number; RQI: RNA quality indicator; GSP: gene- specific priming; dNTP: deoxynucleoside triphosphate; LOD: limit of detection. ..
Generated:Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection
Article Snippet: E YES Data S1 qPCR OLIGONUCLEOTIDES Primer sequences E YES Included in KRT RTPrimerDB Identification Number D N/A Probe sequences D N/A Location and identity of any modifications E N/A Manufacturer of oligonucleotides D YES Sigma-Aldrich Purification method D YES Desalting qPCR PROTOCOL Complete reaction conditions E YES Initial denaturation 95 ̊ C, 40 cycles of 95 ̊ C of 15 sec, 60 ̊ C for 20 sec Reaction volume and amount of cDNA/DNA E YES 10 μl, 10 ng Primer, (probe), Mg++ and dNTP concentrations E YES 5 μM Polymerase identity and concentration E YES HOT FIREPol® DNA polymerase Buffer/kit identity and manufacturer E YES HOT FIREPol® EvaGreen® qPCR Mix Plus Exact chemical constitution of the buffer D N/A Additives (SYBR Green I, DMSO, etc.) .. E YES SYBR Green Manufacturer of plates/tubes and catalog number D YES Biorad (Catalog: TCS0803, TLS0801) Complete thermocycling parameters E YES Initial denaturation 95 ̊ C, 40 cycles of 95 ̊ C of 15 sec, 60 ̊ C for 20 sec Reaction setup D YES Manual Manufacturer of qPCR instrument E YES Qiagen QIAquant 96 5-plex qPCR VALIDATION Evidence of optimisation (from gradients) D NO Specificity (gel, sequence, melt, or digest) E YES Specific single band For SYBR Green I, Cq of the NTC E YES >38 Standard curves with slope and y-intercept E YES TNF-=-3.1021 -actin=-3.09825 PCR efficiency calculated from slope E YES 110% Confidence interval for PCR efficiency or standard error D NO r2 of standard curve E YES TNF-=0.9928 -actin=0.9984 Linear dynamic range E YES r2 values [TNF=0.9928, Cq variation at lower limit E YES r2 values indicate low variability Confidence intervals throughout range D NO Evidence for limit of detection E NO If multiplex, efficiency and LOD of each assay E N/A DATA ANALYSIS qPCR analysis program (source, version) E YES Graphpad PRISM 8 Cq method determination E YES System generated Outlier identification and disposition E N/A Results of NTCs E YES Cq>38 Justification of number and choice of reference genes E YES -actin Description of normalisation method E YES 2(-delta delta Ct) method Number and concordance of biological replicates D YES Three Number and stage (RT or qPCR) of technical replicates E YES Three Repeatability (intra-assay variation, %CV) E YES 1.73% Reproducibility (inter- assay variation, %CV) D YES 0.6% Power analysis D NO Statistical methods for result significance E YES One-way ANOVA Software (source, version) E YES Q-Rex touch Cq or raw data submission D NO E: essential information; D: desirable information; FFPE: formalin-fixed, paraffin-embedded; RIN: RNA integrity number; RQI: RNA quality indicator; GSP: gene- specific priming; dNTP: deoxynucleoside triphosphate; LOD: limit of detection. ..
Intra Assay:Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection
Article Snippet: E YES Data S1 qPCR OLIGONUCLEOTIDES Primer sequences E YES Included in KRT RTPrimerDB Identification Number D N/A Probe sequences D N/A Location and identity of any modifications E N/A Manufacturer of oligonucleotides D YES Sigma-Aldrich Purification method D YES Desalting qPCR PROTOCOL Complete reaction conditions E YES Initial denaturation 95 ̊ C, 40 cycles of 95 ̊ C of 15 sec, 60 ̊ C for 20 sec Reaction volume and amount of cDNA/DNA E YES 10 μl, 10 ng Primer, (probe), Mg++ and dNTP concentrations E YES 5 μM Polymerase identity and concentration E YES HOT FIREPol® DNA polymerase Buffer/kit identity and manufacturer E YES HOT FIREPol® EvaGreen® qPCR Mix Plus Exact chemical constitution of the buffer D N/A Additives (SYBR Green I, DMSO, etc.) .. E YES SYBR Green Manufacturer of plates/tubes and catalog number D YES Biorad (Catalog: TCS0803, TLS0801) Complete thermocycling parameters E YES Initial denaturation 95 ̊ C, 40 cycles of 95 ̊ C of 15 sec, 60 ̊ C for 20 sec Reaction setup D YES Manual Manufacturer of qPCR instrument E YES Qiagen QIAquant 96 5-plex qPCR VALIDATION Evidence of optimisation (from gradients) D NO Specificity (gel, sequence, melt, or digest) E YES Specific single band For SYBR Green I, Cq of the NTC E YES >38 Standard curves with slope and y-intercept E YES TNF-=-3.1021 -actin=-3.09825 PCR efficiency calculated from slope E YES 110% Confidence interval for PCR efficiency or standard error D NO r2 of standard curve E YES TNF-=0.9928 -actin=0.9984 Linear dynamic range E YES r2 values [TNF=0.9928, Cq variation at lower limit E YES r2 values indicate low variability Confidence intervals throughout range D NO Evidence for limit of detection E NO If multiplex, efficiency and LOD of each assay E N/A DATA ANALYSIS qPCR analysis program (source, version) E YES Graphpad PRISM 8 Cq method determination E YES System generated Outlier identification and disposition E N/A Results of NTCs E YES Cq>38 Justification of number and choice of reference genes E YES -actin Description of normalisation method E YES 2(-delta delta Ct) method Number and concordance of biological replicates D YES Three Number and stage (RT or qPCR) of technical replicates E YES Three Repeatability (intra-assay variation, %CV) E YES 1.73% Reproducibility (inter- assay variation, %CV) D YES 0.6% Power analysis D NO Statistical methods for result significance E YES One-way ANOVA Software (source, version) E YES Q-Rex touch Cq or raw data submission D NO E: essential information; D: desirable information; FFPE: formalin-fixed, paraffin-embedded; RIN: RNA integrity number; RQI: RNA quality indicator; GSP: gene- specific priming; dNTP: deoxynucleoside triphosphate; LOD: limit of detection. ..
Inter Assay:Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection
Article Snippet: E YES Data S1 qPCR OLIGONUCLEOTIDES Primer sequences E YES Included in KRT RTPrimerDB Identification Number D N/A Probe sequences D N/A Location and identity of any modifications E N/A Manufacturer of oligonucleotides D YES Sigma-Aldrich Purification method D YES Desalting qPCR PROTOCOL Complete reaction conditions E YES Initial denaturation 95 ̊ C, 40 cycles of 95 ̊ C of 15 sec, 60 ̊ C for 20 sec Reaction volume and amount of cDNA/DNA E YES 10 μl, 10 ng Primer, (probe), Mg++ and dNTP concentrations E YES 5 μM Polymerase identity and concentration E YES HOT FIREPol® DNA polymerase Buffer/kit identity and manufacturer E YES HOT FIREPol® EvaGreen® qPCR Mix Plus Exact chemical constitution of the buffer D N/A Additives (SYBR Green I, DMSO, etc.) .. E YES SYBR Green Manufacturer of plates/tubes and catalog number D YES Biorad (Catalog: TCS0803, TLS0801) Complete thermocycling parameters E YES Initial denaturation 95 ̊ C, 40 cycles of 95 ̊ C of 15 sec, 60 ̊ C for 20 sec Reaction setup D YES Manual Manufacturer of qPCR instrument E YES Qiagen QIAquant 96 5-plex qPCR VALIDATION Evidence of optimisation (from gradients) D NO Specificity (gel, sequence, melt, or digest) E YES Specific single band For SYBR Green I, Cq of the NTC E YES >38 Standard curves with slope and y-intercept E YES TNF-=-3.1021 -actin=-3.09825 PCR efficiency calculated from slope E YES 110% Confidence interval for PCR efficiency or standard error D NO r2 of standard curve E YES TNF-=0.9928 -actin=0.9984 Linear dynamic range E YES r2 values [TNF=0.9928, Cq variation at lower limit E YES r2 values indicate low variability Confidence intervals throughout range D NO Evidence for limit of detection E NO If multiplex, efficiency and LOD of each assay E N/A DATA ANALYSIS qPCR analysis program (source, version) E YES Graphpad PRISM 8 Cq method determination E YES System generated Outlier identification and disposition E N/A Results of NTCs E YES Cq>38 Justification of number and choice of reference genes E YES -actin Description of normalisation method E YES 2(-delta delta Ct) method Number and concordance of biological replicates D YES Three Number and stage (RT or qPCR) of technical replicates E YES Three Repeatability (intra-assay variation, %CV) E YES 1.73% Reproducibility (inter- assay variation, %CV) D YES 0.6% Power analysis D NO Statistical methods for result significance E YES One-way ANOVA Software (source, version) E YES Q-Rex touch Cq or raw data submission D NO E: essential information; D: desirable information; FFPE: formalin-fixed, paraffin-embedded; RIN: RNA integrity number; RQI: RNA quality indicator; GSP: gene- specific priming; dNTP: deoxynucleoside triphosphate; LOD: limit of detection. ..
Software:Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection
Article Snippet: E YES Data S1 qPCR OLIGONUCLEOTIDES Primer sequences E YES Included in KRT RTPrimerDB Identification Number D N/A Probe sequences D N/A Location and identity of any modifications E N/A Manufacturer of oligonucleotides D YES Sigma-Aldrich Purification method D YES Desalting qPCR PROTOCOL Complete reaction conditions E YES Initial denaturation 95 ̊ C, 40 cycles of 95 ̊ C of 15 sec, 60 ̊ C for 20 sec Reaction volume and amount of cDNA/DNA E YES 10 μl, 10 ng Primer, (probe), Mg++ and dNTP concentrations E YES 5 μM Polymerase identity and concentration E YES HOT FIREPol® DNA polymerase Buffer/kit identity and manufacturer E YES HOT FIREPol® EvaGreen® qPCR Mix Plus Exact chemical constitution of the buffer D N/A Additives (SYBR Green I, DMSO, etc.) .. E YES SYBR Green Manufacturer of plates/tubes and catalog number D YES Biorad (Catalog: TCS0803, TLS0801) Complete thermocycling parameters E YES Initial denaturation 95 ̊ C, 40 cycles of 95 ̊ C of 15 sec, 60 ̊ C for 20 sec Reaction setup D YES Manual Manufacturer of qPCR instrument E YES Qiagen QIAquant 96 5-plex qPCR VALIDATION Evidence of optimisation (from gradients) D NO Specificity (gel, sequence, melt, or digest) E YES Specific single band For SYBR Green I, Cq of the NTC E YES >38 Standard curves with slope and y-intercept E YES TNF-=-3.1021 -actin=-3.09825 PCR efficiency calculated from slope E YES 110% Confidence interval for PCR efficiency or standard error D NO r2 of standard curve E YES TNF-=0.9928 -actin=0.9984 Linear dynamic range E YES r2 values [TNF=0.9928, Cq variation at lower limit E YES r2 values indicate low variability Confidence intervals throughout range D NO Evidence for limit of detection E NO If multiplex, efficiency and LOD of each assay E N/A DATA ANALYSIS qPCR analysis program (source, version) E YES Graphpad PRISM 8 Cq method determination E YES System generated Outlier identification and disposition E N/A Results of NTCs E YES Cq>38 Justification of number and choice of reference genes E YES -actin Description of normalisation method E YES 2(-delta delta Ct) method Number and concordance of biological replicates D YES Three Number and stage (RT or qPCR) of technical replicates E YES Three Repeatability (intra-assay variation, %CV) E YES 1.73% Reproducibility (inter- assay variation, %CV) D YES 0.6% Power analysis D NO Statistical methods for result significance E YES One-way ANOVA Software (source, version) E YES Q-Rex touch Cq or raw data submission D NO E: essential information; D: desirable information; FFPE: formalin-fixed, paraffin-embedded; RIN: RNA integrity number; RQI: RNA quality indicator; GSP: gene- specific priming; dNTP: deoxynucleoside triphosphate; LOD: limit of detection. ..
Formalin-fixed Paraffin-Embedded:Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection
Article Snippet: E YES Data S1 qPCR OLIGONUCLEOTIDES Primer sequences E YES Included in KRT RTPrimerDB Identification Number D N/A Probe sequences D N/A Location and identity of any modifications E N/A Manufacturer of oligonucleotides D YES Sigma-Aldrich Purification method D YES Desalting qPCR PROTOCOL Complete reaction conditions E YES Initial denaturation 95 ̊ C, 40 cycles of 95 ̊ C of 15 sec, 60 ̊ C for 20 sec Reaction volume and amount of cDNA/DNA E YES 10 μl, 10 ng Primer, (probe), Mg++ and dNTP concentrations E YES 5 μM Polymerase identity and concentration E YES HOT FIREPol® DNA polymerase Buffer/kit identity and manufacturer E YES HOT FIREPol® EvaGreen® qPCR Mix Plus Exact chemical constitution of the buffer D N/A Additives (SYBR Green I, DMSO, etc.) .. E YES SYBR Green Manufacturer of plates/tubes and catalog number D YES Biorad (Catalog: TCS0803, TLS0801) Complete thermocycling parameters E YES Initial denaturation 95 ̊ C, 40 cycles of 95 ̊ C of 15 sec, 60 ̊ C for 20 sec Reaction setup D YES Manual Manufacturer of qPCR instrument E YES Qiagen QIAquant 96 5-plex qPCR VALIDATION Evidence of optimisation (from gradients) D NO Specificity (gel, sequence, melt, or digest) E YES Specific single band For SYBR Green I, Cq of the NTC E YES >38 Standard curves with slope and y-intercept E YES TNF-=-3.1021 -actin=-3.09825 PCR efficiency calculated from slope E YES 110% Confidence interval for PCR efficiency or standard error D NO r2 of standard curve E YES TNF-=0.9928 -actin=0.9984 Linear dynamic range E YES r2 values [TNF=0.9928, Cq variation at lower limit E YES r2 values indicate low variability Confidence intervals throughout range D NO Evidence for limit of detection E NO If multiplex, efficiency and LOD of each assay E N/A DATA ANALYSIS qPCR analysis program (source, version) E YES Graphpad PRISM 8 Cq method determination E YES System generated Outlier identification and disposition E N/A Results of NTCs E YES Cq>38 Justification of number and choice of reference genes E YES -actin Description of normalisation method E YES 2(-delta delta Ct) method Number and concordance of biological replicates D YES Three Number and stage (RT or qPCR) of technical replicates E YES Three Repeatability (intra-assay variation, %CV) E YES 1.73% Reproducibility (inter- assay variation, %CV) D YES 0.6% Power analysis D NO Statistical methods for result significance E YES One-way ANOVA Software (source, version) E YES Q-Rex touch Cq or raw data submission D NO E: essential information; D: desirable information; FFPE: formalin-fixed, paraffin-embedded; RIN: RNA integrity number; RQI: RNA quality indicator; GSP: gene- specific priming; dNTP: deoxynucleoside triphosphate; LOD: limit of detection. ..
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