Journal: Nature protocols

Article Title: High-resolution, ultra-sensitive and quantitative DNA double-strand break labeling in eukaryotic cells using i-BLESS

doi: 10.1038/s41596-020-00448-3

Figure Lengend Snippet: Troubleshooting table

Article Snippet: Wash pellet in each tube by resuspending in 1 mL of 1x NEBNext FFPE DNA Repair Buffer (the amount of buffer used in this step is not a critical parameter, if the volume of bead pellet is higher than 0.5 mL, use lower volume of buffer to not exceed the maximum capacity of 1.5 mL tubes) and incubating for 10 min at RT, gently rocking.

Techniques: Centrifugation, Sonication, Amplification

Journal: Nature protocols

Article Title: High-resolution, ultra-sensitive and quantitative DNA double-strand break labeling in eukaryotic cells using i-BLESS

doi: 10.1038/s41596-020-00448-3

Figure Lengend Snippet:

Article Snippet: Wash pellet in each tube by resuspending in 1 mL of 1x NEBNext FFPE DNA Repair Buffer (the amount of buffer used in this step is not a critical parameter, if the volume of bead pellet is higher than 0.5 mL, use lower volume of buffer to not exceed the maximum capacity of 1.5 mL tubes) and incubating for 10 min at RT, gently rocking.

Techniques: Concentration Assay

Journal: Nature protocols

Article Title: High-resolution, ultra-sensitive and quantitative DNA double-strand break labeling in eukaryotic cells using i-BLESS

doi: 10.1038/s41596-020-00448-3

Figure Lengend Snippet:

Article Snippet: Wash pellet in each tube by resuspending in 1 mL of 1x NEBNext FFPE DNA Repair Buffer (the amount of buffer used in this step is not a critical parameter, if the volume of bead pellet is higher than 0.5 mL, use lower volume of buffer to not exceed the maximum capacity of 1.5 mL tubes) and incubating for 10 min at RT, gently rocking.

Techniques: Concentration Assay

Journal: Nature protocols

Article Title: High-resolution, ultra-sensitive and quantitative DNA double-strand break labeling in eukaryotic cells using i-BLESS

doi: 10.1038/s41596-020-00448-3

Figure Lengend Snippet:

Article Snippet: Wash pellet in each tube by resuspending in 1 mL of 1x NEBNext FFPE DNA Repair Buffer (the amount of buffer used in this step is not a critical parameter, if the volume of bead pellet is higher than 0.5 mL, use lower volume of buffer to not exceed the maximum capacity of 1.5 mL tubes) and incubating for 10 min at RT, gently rocking.

Techniques: Concentration Assay

Journal: Radiation research

Article Title: RENEB Inter-Laboratory Comparison 2021: The Gene Expression Assay

doi: 10.1667/RADE-22-00206.1

Figure Lengend Snippet: Overview of Participating Teams, Utilized Platforms, Number and Names of Genes or Gene Combinations Used, the Origin of Calibration Samples, and Further Details

Article Snippet: TaqMan ® assay: DDB2 (Hs00172068_ml), FDXR (HS01031617_ml), ITFG1: Hs01061271_ml SYBR Green assay: CDKN1A F:CCT CAT CCC GTG TTC TCC TTT CDKN1A R: GTA CCA CCC AGC GGA CAA GT GAPDH F: CGA CCA CTT TGT CAA GCT CA GAPDH R: AGG GGT CTA CAT GGC AAC TG HPRT F: TGA CAC TGG CAA AAC AAT GCA HPRT R: GGT CCT TTT CAC CAG CAA GCT FDXR (HS01031617_ml) DDB2 (Hs00172068_ml), FDXR (HS01031617_ml) RNA amount used for cDNA synthesis 0.2 μg; 1.65 μg per array Cycles 1×/50°C/2min, 95°C/10min, 4O×/95°C/15 s, 60°C/1min 1×/95°C/10min, 40×/95°C/15 s, 60°C/1min 1×/95°C/5min, 40×/95°C/10 s, 60°C/30s 1×/95°C/15min, 45×/95°C/15s, 60°C/20s, 72°C/20s, 1×/95°C/5s, 1×/65°C/60s, 1×/97°C, cooling step TaqMan ® assay: 1 ×/50°C/2min, 1×/95°C/20s, 45×/95°C/3s, 60°C/30s SYBR Green assay: 1×/95°C/10min, 40×/95°C/15s, 59°C/30s, 72°C/30s, 1×/72°C/1min 1×/95°C/2min, 40×/95°C/10s, 60°C/1min 1× /50°C/2min, 1× /95°C/10min, 40×/95°C/1min, 60°C/1min cDNA amount used for cRNA synthesis N.D.

Techniques: Generated

Journal: Radiation research

Article Title: RENEB Inter-Laboratory Comparison 2021: The Gene Expression Assay

doi: 10.1667/RADE-22-00206.1

Figure Lengend Snippet: Overview of Methodological Details of Either qRT-PCR (Quantitative Reverse Transcription Polymerase Chain Reaction) or Microarrays Used by the Contributing Teams

Article Snippet: TaqMan ® assay: DDB2 (Hs00172068_ml), FDXR (HS01031617_ml), ITFG1: Hs01061271_ml SYBR Green assay: CDKN1A F:CCT CAT CCC GTG TTC TCC TTT CDKN1A R: GTA CCA CCC AGC GGA CAA GT GAPDH F: CGA CCA CTT TGT CAA GCT CA GAPDH R: AGG GGT CTA CAT GGC AAC TG HPRT F: TGA CAC TGG CAA AAC AAT GCA HPRT R: GGT CCT TTT CAC CAG CAA GCT FDXR (HS01031617_ml) DDB2 (Hs00172068_ml), FDXR (HS01031617_ml) RNA amount used for cDNA synthesis 0.2 μg; 1.65 μg per array Cycles 1×/50°C/2min, 95°C/10min, 4O×/95°C/15 s, 60°C/1min 1×/95°C/10min, 40×/95°C/15 s, 60°C/1min 1×/95°C/5min, 40×/95°C/10 s, 60°C/30s 1×/95°C/15min, 45×/95°C/15s, 60°C/20s, 72°C/20s, 1×/95°C/5s, 1×/65°C/60s, 1×/97°C, cooling step TaqMan ® assay: 1 ×/50°C/2min, 1×/95°C/20s, 45×/95°C/3s, 60°C/30s SYBR Green assay: 1×/95°C/10min, 40×/95°C/15s, 59°C/30s, 72°C/30s, 1×/72°C/1min 1×/95°C/2min, 40×/95°C/10s, 60°C/1min 1× /50°C/2min, 1× /95°C/10min, 40×/95°C/1min, 60°C/1min cDNA amount used for cRNA synthesis N.D.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Microarray, Isolation, Red Blood Cell Lysis, Control, Concentration Assay, Sequencing, cDNA Synthesis, Labeling, SYBR Green Assay, Multiplex Assay, TaqMan Assay, Real-time Polymerase Chain Reaction, Software, Extraction

Journal: Radiation research

Article Title: RENEB Inter-Laboratory Comparison 2021: The Gene Expression Assay

doi: 10.1667/RADE-22-00206.1

Figure Lengend Snippet: The Table Depicts Team Contributions (from Left to Right) Regarding Employed Genes, Reported Dose Estimates per Reference Sample 1–3, Differences among Reported and Reference Dose-Values as well as the Summed Absolute Difference over all Reference Samples (SAD), a Correct (Yes) or Incorrect (No) Order of Dose Estimates (from Lowest to Highest) Corresponding to Three Dose Categories [Unexposed, Low (1.2 Gy) and Highly Exposed (3.5 Gy)], the Use of FDXR Gene Expression Changes for dose estimation, as well as the Report Time

Article Snippet: TaqMan ® assay: DDB2 (Hs00172068_ml), FDXR (HS01031617_ml), ITFG1: Hs01061271_ml SYBR Green assay: CDKN1A F:CCT CAT CCC GTG TTC TCC TTT CDKN1A R: GTA CCA CCC AGC GGA CAA GT GAPDH F: CGA CCA CTT TGT CAA GCT CA GAPDH R: AGG GGT CTA CAT GGC AAC TG HPRT F: TGA CAC TGG CAA AAC AAT GCA HPRT R: GGT CCT TTT CAC CAG CAA GCT FDXR (HS01031617_ml) DDB2 (Hs00172068_ml), FDXR (HS01031617_ml) RNA amount used for cDNA synthesis 0.2 μg; 1.65 μg per array Cycles 1×/50°C/2min, 95°C/10min, 4O×/95°C/15 s, 60°C/1min 1×/95°C/10min, 40×/95°C/15 s, 60°C/1min 1×/95°C/5min, 40×/95°C/10 s, 60°C/30s 1×/95°C/15min, 45×/95°C/15s, 60°C/20s, 72°C/20s, 1×/95°C/5s, 1×/65°C/60s, 1×/97°C, cooling step TaqMan ® assay: 1 ×/50°C/2min, 1×/95°C/20s, 45×/95°C/3s, 60°C/30s SYBR Green assay: 1×/95°C/10min, 40×/95°C/15s, 59°C/30s, 72°C/30s, 1×/72°C/1min 1×/95°C/2min, 40×/95°C/10s, 60°C/1min 1× /50°C/2min, 1× /95°C/10min, 40×/95°C/1min, 60°C/1min cDNA amount used for cRNA synthesis N.D.

Techniques: Gene Expression

Journal: Radiation research

Article Title: RENEB Inter-Laboratory Comparison 2021: The Gene Expression Assay

doi: 10.1667/RADE-22-00206.1

Figure Lengend Snippet: Reported dose estimates are provided per team (teams are numbered as outlined in Table 1) on the left panels and corresponding differences of reported versus reference doses are shown in the right panels. The panels A, C, and E reflect reported dose estimates corresponding to the unexposed, 1.2 Gy and 3.5 Gy irradiated samples, respectively. Panels B, D, and F reflect corresponding differences of reported versus reference doses. Dotted lines refer to the ±0.5 Gy (reference doses below 2.5 Gy) and ±1 Gy (reference doses >3 Gy) uncertainty interval for dose estimation of triage dosimetry. Solid lines reflect values of the reference dose. Circles represent reported dose estimates and squares reflect mean values with error bars showing the standard deviation. Results are provided in descending order of mean reported dose estimates. Stars correspond to dose estimates based on gene expression changes using FDXR.

Article Snippet: TaqMan ® assay: DDB2 (Hs00172068_ml), FDXR (HS01031617_ml), ITFG1: Hs01061271_ml SYBR Green assay: CDKN1A F:CCT CAT CCC GTG TTC TCC TTT CDKN1A R: GTA CCA CCC AGC GGA CAA GT GAPDH F: CGA CCA CTT TGT CAA GCT CA GAPDH R: AGG GGT CTA CAT GGC AAC TG HPRT F: TGA CAC TGG CAA AAC AAT GCA HPRT R: GGT CCT TTT CAC CAG CAA GCT FDXR (HS01031617_ml) DDB2 (Hs00172068_ml), FDXR (HS01031617_ml) RNA amount used for cDNA synthesis 0.2 μg; 1.65 μg per array Cycles 1×/50°C/2min, 95°C/10min, 4O×/95°C/15 s, 60°C/1min 1×/95°C/10min, 40×/95°C/15 s, 60°C/1min 1×/95°C/5min, 40×/95°C/10 s, 60°C/30s 1×/95°C/15min, 45×/95°C/15s, 60°C/20s, 72°C/20s, 1×/95°C/5s, 1×/65°C/60s, 1×/97°C, cooling step TaqMan ® assay: 1 ×/50°C/2min, 1×/95°C/20s, 45×/95°C/3s, 60°C/30s SYBR Green assay: 1×/95°C/10min, 40×/95°C/15s, 59°C/30s, 72°C/30s, 1×/72°C/1min 1×/95°C/2min, 40×/95°C/10s, 60°C/1min 1× /50°C/2min, 1× /95°C/10min, 40×/95°C/1min, 60°C/1min cDNA amount used for cRNA synthesis N.D.

Techniques: Irradiation, Standard Deviation, Gene Expression

Journal: Radiation research

Article Title: RENEB Inter-Laboratory Comparison 2021: The Gene Expression Assay

doi: 10.1667/RADE-22-00206.1

Figure Lengend Snippet: Here we show Team 4 data. Panel A reflects FDXR gene expression changes plotting raw (left graphs) and normalized Cq-values (middle graphs) of reported dose estimates using either BIR (Bundeswehr Institute of Radiobiology) calibration samples (top panels), or two sets of calibration samples generated by Team 4, termed Team A and Team B (bottom panels), respectively. Panels to the right represent reevaluated data generated on newly synthesized cDNA of RNA extracted from calibration samples as indicated. Dashed horizontal lines refer to Cq-values corresponding to blinded, coded samples no. 1 (0 Gy), no. 2 (1.2 Gy), and no. 3 (3.5 Gy) as indicated in the panels. Panel B shows dose estimates generated both during (left) and after the exercise (right) based on the different calibration curves used by Team 4 for FDXR, GADD45A, CDKN1A and MDM2 genes. “Reported dose estimates” corresponds to dose estimates determined during the exercise, and dose estimates based on raw and normalized Cq-values are shown. “Reevaluated dose estimates” include dose estimates generated after the exercise. It comprises raw and normalized Cq-values generated from remaining cDNA as used for the reported dose estimates as well as data generated from newly synthesized cDNA. The table in panel C reflects exposure, age and sex differences between the different sets of calibration samples employed as well as a calculation of fold-differences among the four genes examined, which were generated at the dose which created the maximal fold change from 0–4 Gy irradiated calibration samples employing the 0 Gy Cq-values as the reference.

Article Snippet: TaqMan ® assay: DDB2 (Hs00172068_ml), FDXR (HS01031617_ml), ITFG1: Hs01061271_ml SYBR Green assay: CDKN1A F:CCT CAT CCC GTG TTC TCC TTT CDKN1A R: GTA CCA CCC AGC GGA CAA GT GAPDH F: CGA CCA CTT TGT CAA GCT CA GAPDH R: AGG GGT CTA CAT GGC AAC TG HPRT F: TGA CAC TGG CAA AAC AAT GCA HPRT R: GGT CCT TTT CAC CAG CAA GCT FDXR (HS01031617_ml) DDB2 (Hs00172068_ml), FDXR (HS01031617_ml) RNA amount used for cDNA synthesis 0.2 μg; 1.65 μg per array Cycles 1×/50°C/2min, 95°C/10min, 4O×/95°C/15 s, 60°C/1min 1×/95°C/10min, 40×/95°C/15 s, 60°C/1min 1×/95°C/5min, 40×/95°C/10 s, 60°C/30s 1×/95°C/15min, 45×/95°C/15s, 60°C/20s, 72°C/20s, 1×/95°C/5s, 1×/65°C/60s, 1×/97°C, cooling step TaqMan ® assay: 1 ×/50°C/2min, 1×/95°C/20s, 45×/95°C/3s, 60°C/30s SYBR Green assay: 1×/95°C/10min, 40×/95°C/15s, 59°C/30s, 72°C/30s, 1×/72°C/1min 1×/95°C/2min, 40×/95°C/10s, 60°C/1min 1× /50°C/2min, 1× /95°C/10min, 40×/95°C/1min, 60°C/1min cDNA amount used for cRNA synthesis N.D.

Techniques: Gene Expression, Generated, Synthesized, Irradiation

Journal: Radiation research

Article Title: RENEB Inter-Laboratory Comparison 2021: The Gene Expression Assay

doi: 10.1667/RADE-22-00206.1

Figure Lengend Snippet: The summed absolute difference (Gy) was plotted depending on the application of single genes for dose estimation (FDXR only, other single genes, left and middle part of the graph) or the use of a combined set of genes (right side). Circles represent reported dose estimates and squares are reflecting median values with error bars showing the standard deviation.

Article Snippet: TaqMan ® assay: DDB2 (Hs00172068_ml), FDXR (HS01031617_ml), ITFG1: Hs01061271_ml SYBR Green assay: CDKN1A F:CCT CAT CCC GTG TTC TCC TTT CDKN1A R: GTA CCA CCC AGC GGA CAA GT GAPDH F: CGA CCA CTT TGT CAA GCT CA GAPDH R: AGG GGT CTA CAT GGC AAC TG HPRT F: TGA CAC TGG CAA AAC AAT GCA HPRT R: GGT CCT TTT CAC CAG CAA GCT FDXR (HS01031617_ml) DDB2 (Hs00172068_ml), FDXR (HS01031617_ml) RNA amount used for cDNA synthesis 0.2 μg; 1.65 μg per array Cycles 1×/50°C/2min, 95°C/10min, 4O×/95°C/15 s, 60°C/1min 1×/95°C/10min, 40×/95°C/15 s, 60°C/1min 1×/95°C/5min, 40×/95°C/10 s, 60°C/30s 1×/95°C/15min, 45×/95°C/15s, 60°C/20s, 72°C/20s, 1×/95°C/5s, 1×/65°C/60s, 1×/97°C, cooling step TaqMan ® assay: 1 ×/50°C/2min, 1×/95°C/20s, 45×/95°C/3s, 60°C/30s SYBR Green assay: 1×/95°C/10min, 40×/95°C/15s, 59°C/30s, 72°C/30s, 1×/72°C/1min 1×/95°C/2min, 40×/95°C/10s, 60°C/1min 1× /50°C/2min, 1× /95°C/10min, 40×/95°C/1min, 60°C/1min cDNA amount used for cRNA synthesis N.D.

Techniques: Standard Deviation

Journal: Radiation research

Article Title: RENEB Inter-Laboratory Comparison 2021: The Gene Expression Assay

doi: 10.1667/RADE-22-00206.1

Figure Lengend Snippet: The absolute difference (Gy) was compared per team employing either the different workflows established at Team 1–7 laboratories (white bars) or applying the same workflow for entrusted RNA or cDNA samples by one team (gray filled bars). FDXR gene expression changes after a reference dose of 1.2 Gy (upper panel) and 3.5 Gy (lower panel) were examined. Dose estimates that improved after applying the same workflow are shown on the left (labeled by original team number as outlined in Table 1), and those where dose estimates worsened are shown on the right side of the graph.

Article Snippet: TaqMan ® assay: DDB2 (Hs00172068_ml), FDXR (HS01031617_ml), ITFG1: Hs01061271_ml SYBR Green assay: CDKN1A F:CCT CAT CCC GTG TTC TCC TTT CDKN1A R: GTA CCA CCC AGC GGA CAA GT GAPDH F: CGA CCA CTT TGT CAA GCT CA GAPDH R: AGG GGT CTA CAT GGC AAC TG HPRT F: TGA CAC TGG CAA AAC AAT GCA HPRT R: GGT CCT TTT CAC CAG CAA GCT FDXR (HS01031617_ml) DDB2 (Hs00172068_ml), FDXR (HS01031617_ml) RNA amount used for cDNA synthesis 0.2 μg; 1.65 μg per array Cycles 1×/50°C/2min, 95°C/10min, 4O×/95°C/15 s, 60°C/1min 1×/95°C/10min, 40×/95°C/15 s, 60°C/1min 1×/95°C/5min, 40×/95°C/10 s, 60°C/30s 1×/95°C/15min, 45×/95°C/15s, 60°C/20s, 72°C/20s, 1×/95°C/5s, 1×/65°C/60s, 1×/97°C, cooling step TaqMan ® assay: 1 ×/50°C/2min, 1×/95°C/20s, 45×/95°C/3s, 60°C/30s SYBR Green assay: 1×/95°C/10min, 40×/95°C/15s, 59°C/30s, 72°C/30s, 1×/72°C/1min 1×/95°C/2min, 40×/95°C/10s, 60°C/1min 1× /50°C/2min, 1× /95°C/10min, 40×/95°C/1min, 60°C/1min cDNA amount used for cRNA synthesis N.D.

Techniques: Gene Expression, Labeling