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

Journal: The Journal of Lipid Research

Article Title: Prediction of PPAR- ligand-mediated physiological changes using gene expression profiles

doi: 10.1194/jlr.d300239-jlr200

Figure Lengend Snippet: Fig. 4. Relationship between regulated target genes and the relative drug peroxisome proliferator-acti- vated receptor- (PPAR-) efficacy. A: Comparison of quantitative expression levels as measured in microar- rays and Northern blot analysis and the relative PPAR- efficacies of the tested compounds using linear regres- sion analysis. Fold regulations as calculated in Table 2 were used for the microarray data, whereas signal in- tensities for the genes in Northern blots were col- lected using a PhosphorImager and normalized to the signals for ribosomal phosphoprotein 36B4. For apoli- poprotein C-III (apoC-III), the log fold change was used because this gene is downregulated and its ex- pression ratios lie between 1 and 0. B: Northern blots showing the expression of peroxisomal enoyl-CoA:hy- drotase-3-hydroxyacyl-CoA bifunctional enzyme (bi- functional enzyme), apoC-III, and ribosomal phos- phoprotein 36B4.

Article Snippet: The purified PCR products were mixed 1:1 in DMSO (Sigma, St. Louis, MO) and rearranged in 384-well plates. cDNA targets were spotted in duplicate onto silanized glass slides (Amersham Biosciences, Buckinghamshire, UK) at 55% relative humidity using the Molecular Dynamics Gen. III microarray spotter.

Techniques: Comparison, Expressing, Northern Blot, Microarray, Functional Assay

Journal: RNA

Article Title: Generation of a neuro-specific microarray reveals novel differentially expressed noncoding RNAs in mouse models for neurodegenerative diseases

doi: 10.1261/rna.047225.114

Figure Lengend Snippet: Biotype and genome location of ncRNAs from the neuro-ncRNA microarray. ncRNAs are sorted by ncRNA biotype and genomic locations. Frequencies are illustrated in log 2 scale. Frequency of zero is indicated in gray. Strandness of ncRNAs is included in the biotype names (sense: “_SE,” antisense: “_AS”). Biotypes follow GENCODE/ENSEMBL annotation. Missing annotations are designated as “unknown.”

Article Snippet: The neuro-ncRNA microarray was generated using the MicroGrid II Microarray Spotter (Zinsser Analytic).

Techniques: Microarray

Journal: RNA

Article Title: Generation of a neuro-specific microarray reveals novel differentially expressed noncoding RNAs in mouse models for neurodegenerative diseases

doi: 10.1261/rna.047225.114

Figure Lengend Snippet: Comparison of expression analysis between Northern blot (black bars) and microarray analysis (gray bars) of 14 randomly chosen ncRNAs. ( A ) Expression changes of ncRNAs between brain and muscle tissues. ( B ) Expression changes of ncRNAs between brain and liver tissues. Candidate IDs are indicated on top . Details are listed in Supplemental Table 2. Fold changes from Northern blot analysis were quantified by using ImageQuant 8.1 (GE Healthcare).

Article Snippet: The neuro-ncRNA microarray was generated using the MicroGrid II Microarray Spotter (Zinsser Analytic).

Techniques: Comparison, Expressing, Northern Blot, Microarray

Journal: RNA

Article Title: Generation of a neuro-specific microarray reveals novel differentially expressed noncoding RNAs in mouse models for neurodegenerative diseases

doi: 10.1261/rna.047225.114

Figure Lengend Snippet: Differential expression analysis of ncRNAs from selected brain regions of mouse models for voltage-gated calcium channel activity compared with wild type. ( A ) Cacnb4 mutant mouse: cerebellum. ( B ) Ca V 1.3 −/− : hippocampus. ( C ) Ca V 1.3 −/− : striatum. Box plots represent fold changes of ncRNAs (colored data points), which showed significant changes in expression relative to wild-type controls in microarray experiments. Fold changes are illustrated in log 2 scale. Whiskers extend to ±1.5-fold of the interquartile range (IQR). Biotypes are designated by color code: miRNAs (red), snoRNA (orange), tRF (blue), other biotypes (green), and unknown biotype (gray). ( D ) Venn diagram illustrating the overlap of differentially expressed candidates between the models analyzed. ( E ) Fold change and annotation of ncRNA candidates highlighted in D .

Article Snippet: The neuro-ncRNA microarray was generated using the MicroGrid II Microarray Spotter (Zinsser Analytic).

Techniques: Quantitative Proteomics, Activity Assay, Mutagenesis, Expressing, Microarray

Journal: RNA

Article Title: Generation of a neuro-specific microarray reveals novel differentially expressed noncoding RNAs in mouse models for neurodegenerative diseases

doi: 10.1261/rna.047225.114

Figure Lengend Snippet: Differential expression of ncRNAs from cortices of a triple-transgenic (3xTG) mouse model for Alzheimer's disease compared with wild-type controls at the age of 3, 10, and 20 mo, respectively. ( A ) Box plots represent fold changes of ncRNAs (colored data points), which showed significant changes in expression relative to their wild-type controls in microarray experiments. The experiments are illustrated from left to right : 3-, 10-, and 20-mo-old mice. Fold changes are illustrated in log 2 scale. Whiskers extend to ±1.5-fold of the interquartile range (IQR). Biotypes are designated by color code: miRNAs (red), snoRNA (orange), tRF (blue), other biotypes (green), and unknown biotype (gray). ( B ) Venn diagram illustrating the overlap of differentially expressed candidates between the different age groups. ( C ) Fold change and annotation of ncRNA candidates highlighted in B .

Article Snippet: The neuro-ncRNA microarray was generated using the MicroGrid II Microarray Spotter (Zinsser Analytic).

Techniques: Quantitative Proteomics, Transgenic Assay, Expressing, Microarray

Journal: RNA

Article Title: Generation of a neuro-specific microarray reveals novel differentially expressed noncoding RNAs in mouse models for neurodegenerative diseases

doi: 10.1261/rna.047225.114

Figure Lengend Snippet: Differentially expressed snoRNAs in young 3xTG AD mice. ( A ) Candidate e307: sequence similarity of mouse (mm9 assembly) to the human (hg19 assembly) snoRNA homologs (located on chromosome 5), and ( left ) predicted secondary structure illustrated by VARNA ( http://varna.lri.fr/ ). ( Right ) Northern blot analysis of e307 from total RNA of cortices of 10-mo-old 3xTG mice compared with wild-type controls. ( B ) Candidate e470: genome positions of the two (red and green bars) potential loci of transcription (on chromosome 5), which differ by a single SNP (indicated in bold). Sequence similarity of HBII-336 and MBII-336; ( left ) predicted secondary structure illustrated by VARNA. ( Right ) Northern blot analysis from total RNA of cortices of 10-mo-old 3xTG mice compared with wild-type controls. C/D boxes are illustrated in red and sequences targeted by the oligonucleotide from the microarray e307 and e470, respectively, are highlighted in blue.

Article Snippet: The neuro-ncRNA microarray was generated using the MicroGrid II Microarray Spotter (Zinsser Analytic).

Techniques: Sequencing, Northern Blot, Microarray

Journal: Cancer Research

Article Title: Transcriptional Switch of Dormant Tumors to Fast-Growing Angiogenic Phenotype

doi: 10.1158/0008-5472.can-08-2590

Figure Lengend Snippet: Figure 1. Characterization of the experimental model. A, left, schematic presentation of the experimental model. Pairs of fast-growing or dormant tumor cell lines were generated from various tumor types. Whereas fast-growing tumor cells (red) generate tumors that expand rapidly, injection of dormant tumor cells (gray) resulted in occult lesions that were difficult to be detected by gross examination. Bar, 1 mm. The pictures are of tumors injected s.c. on the right flank of SCID mice. The top picture is of dormant glioblastoma, 111 d after the injection of the tumor cells. The bottom picture is of an angiogenic, fast-growing osteosarcoma, 48 d after the injection of the tumor cells. Right, schematic presentation of the in vivo tumor growth patterns of the human tumor models used. Red lines, growth of tumors generated from the fast-growing tumor cell lines; gray lines, tumors generated from the dormant tumor cell lines. These lines represent the earliest time point at which a tumor is detected after a s.c. tumor cells injection into the right flank of SCID mice. B, tumor sizes at experimental end points. There is a significant difference in the size of dormant tumors (gray columns) compared with fast-growing tumor cell lines (red columns). Dormant tumors at these time points were undetectable by gross examination (size values are zero). Each column represents one tumor. C, experimental strategy for the identification of the consensus gene expression signature of dormant tumors. Total RNA from each cell line was extracted and hybridized to genome-wide microarrays. Gene expression in every dormant tumor cell line (gray; D) was compared with that from the angiogenic fast-growing cell line (red; A) of the same tumor type. Each assay was done in duplicate and therefore is represented as two columns in the heat map. The heat map includes only genes that have the same pattern of expression in all tumor types analyzed. D, the top 10 functional processes differentially regulated between dormant versus fast-growing tumors. Data analysis of differentially regulated genes, in dormant versus fast-growing tumor cells, indicates the most significant enrichment for genes related to the regulation of angiogenesis process. Bars represent log P values for the probability of enrichment for a gene ontology process arising by chance. Bars are colored based on the direction of gene regulation: red, up-regulated in dormant tumors; blue, up-regulated in fast-growing tumors.

Article Snippet: To detect differential expression of genes on a genome-wide scale, we generated and used the Human Unigene III c-DNA microarrays (13, 14).

Techniques: Generated, Injection, In Vivo, Gene Expression, Genome Wide, Expressing, Functional Assay

Journal: Frontiers in Microbiology

Article Title: A Panel of CircRNAs in the Serum Serves as Biomarkers for Mycobacterium tuberculosis Infection

doi: 10.3389/fmicb.2020.01215

Figure Lengend Snippet: circRNAs selected in Biomarker-screening phase by Arraystar Human CircRNA microarray V2 of pooled samples (three active TB patients, three healthy controls) with fold change = 2, and p < 0.01.

Article Snippet: CircRNA expression profiles of serum for three active pulmonary TB patients and three healthy controls were generated using the 074301 Arraystar Human CircRNA microarray V2.

Techniques: Biomarker Discovery, Microarray

Journal: Frontiers in Microbiology

Article Title: A Panel of CircRNAs in the Serum Serves as Biomarkers for Mycobacterium tuberculosis Infection

doi: 10.3389/fmicb.2020.01215

Figure Lengend Snippet: Serum circRNA expression signature for TB diagnosis in the validation phase. (A–C) The expression of circRNA_051239, circRNA_029965, and circRNA_404022 in serum from active TB patients ( N = 72), CAP patients ( N = 30), and healthy controls ( N = 30) by qRT-PCR. (D–F) ROC curve analysis for circRNA_051239, circRNA_029965, and circRNA_404022 in serum from active TB patients ( N = 128, including training set and validation set), CAP patients ( N = 50, including training set and validation set), and healthy controls ( N = 50, including training set and validation set). (G) ROC curve analysis for the combined circRNAs panel in serum from active TB patients ( N = 128, including training set and validation set), CAP patients ( N = 50, including training set and validation set), and healthy controls ( N = 50, including training set and validation set). Each value is the mean ± SD; *** P < 0.001.

Article Snippet: CircRNA expression profiles of serum for three active pulmonary TB patients and three healthy controls were generated using the 074301 Arraystar Human CircRNA microarray V2.

Techniques: Expressing, Biomarker Discovery, Quantitative RT-PCR

Journal: Frontiers in Microbiology

Article Title: A Panel of CircRNAs in the Serum Serves as Biomarkers for Mycobacterium tuberculosis Infection

doi: 10.3389/fmicb.2020.01215

Figure Lengend Snippet: The expression level of circRNA_051239 and miR-320a in the serum of drug-resistant TB patients and drug-susceptible patients. (A,B) The expression level of circRNA_051239 and miR-320a in the serum of drug-resistant TB patients and drug-susceptible patients. (C) Spearman’s rank correlation scatter plot of circRNA_051239 and miR-320a in the serum of drug-resistant TB patients. Each value is the mean ± SD; *** P < 0.001. (D) Schematic descriptions of the hypothetical duplexes formed by miR-320a with circRNA_051239.

Article Snippet: CircRNA expression profiles of serum for three active pulmonary TB patients and three healthy controls were generated using the 074301 Arraystar Human CircRNA microarray V2.

Techniques: Expressing

Journal: eLife

Article Title: FAK/PYK2 promotes the Wnt/β-catenin pathway and intestinal tumorigenesis by phosphorylating GSK3β

doi: 10.7554/eLife.10072

Figure Lengend Snippet: ( A ) HEK293T cells were pre-treated with or without PF-562271 (4 µM) for 4 hr followed by incubation with control-CM or Wnt3a-CM containing PF-562271 (4 µM) for additional 4 hr as indicated. The whole cell lysates were subjected for IB with indicated antibodies. ( B ) FAK and PYK2 expressions were evaluated by IB analysis. ( C ) Parental, FAK KD and shRNA control HEK293T cells were treated with control-CM or Wnt3a-CM for 4 hr before harvesting for IB analysis using indicated antibodies. ( D – F ) FAK KD, PYK2 KD or FAK/PYK2 KD cell lines and shRNA control cell lines were generated using lentiviral-based shRNA targeting FAK or PYK2 or control shRNA. The whole cell lysates were used to evaluate the levels of p-GSK3β Y216 and β-catenin by IB with indicated antibodies. ( G ) GSK3β in SW480 and HT29 cells was immunoprecipitated with anti-GSK3β followed by IB with anti-PYK2 and anti-FAK, respectively. Normal IgG was used as IP control. ( H ) In vitro synthesized HA-GSK3β was immunoprecipitated with anti-HA. Beads-bound HA-GSK3β was then incubated with purified FAK or PYK2 recombined protein (or kinase buffer as a negative control) in the presence of ATP at 37°C for 2 hr. The reaction mixtures were subjected to IB analysis using indicated antibodies. DOI: http://dx.doi.org/10.7554/eLife.10072.005

Article Snippet: The bead-bound immunoprecipitates were washed with lysis buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP40 and 10% Glycerol for three times followed by a final kinase buffer wash, then incubated with 20 ng full length recombinant FAK (SignalChem, Canada) or PYK2 (Enzo, Farmingdale, NY), 2 mM ATP, kinase buffer (Cell Signaling, Danvers, MA) in a total volume of 15 µl at 37°C for 2 hrs.

Techniques: Incubation, shRNA, Generated, Immunoprecipitation, In Vitro, Synthesized, Purification, Negative Control

Journal: eLife

Article Title: FAK/PYK2 promotes the Wnt/β-catenin pathway and intestinal tumorigenesis by phosphorylating GSK3β

doi: 10.7554/eLife.10072

Figure Lengend Snippet: ( A ) Time courses of PF-562271 treatment-induced inhibition of phosphorylation of PYK2, GSK3β and β-catenin in SW480 cells. The whole cell lysates were used for the IB analysis using indicated antibodies. ( B ) The whole cell lysates from indicated cells were used for IB analysis. ( C ) The WT MEFs, FAK R454/R454 /PYK2 KD MEFs transfected with empty vector or vector expressing indicated GSK3β mutants were treated with Wnt-3a CM or control-CM for 6 hr before harvesting for IB analysis. ( D ) The WT MEFs and FAK R454/R454 /PYK2 KD MEFs transfected with empty vector or vector expressing HA-GSK3β Y216E were treated with Wnt3a-CM and proteasome inhibitor MG132 for 4 hr and then harvested for IP and IB analyses. KI/KD: FAK R454/R454 /PYK2 KD MEFs expressing empty vector. KI/KD/Y216E: FAK R454/R454 /PYK2 KD MEFs expressing HA-GSK3β Y216E . ( E ) Parental SW480 cells and SW480 cell lines expressing vector control, HA-GSK3β or its mutants were treated with 2 µM PF-562271 for 4 days. Cell viability was determined by CCK-8 assay, using SW480 parental cell treated with DMSO as control. Data are presented as mean ±SD. * denotes a statistically significant difference (p < 0.01) between PF-562271-treated parental SW480 cells and cells overexpressing HA-GSK3βY 216E . ( F ) Upper left panel: indicated HT-29 cell lines were injected into both flanks of nude mice (5 mice in each cell group). Animals were treated with either vehicle (5% Gelucire) or PF-562271 (33 mg/kg in vehicle) as indicated by oral gavage twice daily for 3 weeks. Tumor sizes were measured at indicated time points. Tumor volumes were calculated and plotted. * denotes a statistically significant difference (p < 0.01) comparing with vehicle-treated group. Upper right panel, tumor weighs from different groups at the end of the experiment. Values are means ±SD (n = 10). Bottom panel, representative tumors from different groups. DOI: http://dx.doi.org/10.7554/eLife.10072.006

Article Snippet: The bead-bound immunoprecipitates were washed with lysis buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP40 and 10% Glycerol for three times followed by a final kinase buffer wash, then incubated with 20 ng full length recombinant FAK (SignalChem, Canada) or PYK2 (Enzo, Farmingdale, NY), 2 mM ATP, kinase buffer (Cell Signaling, Danvers, MA) in a total volume of 15 µl at 37°C for 2 hrs.

Techniques: Inhibition, Transfection, Plasmid Preparation, Expressing, CCK-8 Assay, Injection

Journal: eLife

Article Title: FAK/PYK2 promotes the Wnt/β-catenin pathway and intestinal tumorigenesis by phosphorylating GSK3β

doi: 10.7554/eLife.10072

Figure Lengend Snippet: ( A ) Hematoxylin and eosin (H&E) and immunohistochemical staining of the colorectal adenoma, carcinoma and adjacent normal tissues from a single patient. ( B ) 34 tumor samples on tissue microarray were immunohistochemical stained and scored. Score distribution and average score of each antibody staining were summarized. ( C ) Scoring differences between tumor and matched normal tissues from twenty-six patients. * Denotes p-GSK3β Y216 , FAK or PYK2 scores in different directions. _ highlights the numbers of patients whose TMA scores of p-GSK3β Y216 , FAK or PYK2, and β-catenin move in the same direction. ( D ) Hematoxylin and eosin (H&E) and immunohistochemical staining of colorectal carcinoma and adjacent normal tissues from a familial adenomatous polyposis patient. ( E ) Schematic diagram of the FKA/PYK2/p-GSK3β Y216 /β-catenin axis and its role in intestinal tumorigenesis. DOI: http://dx.doi.org/10.7554/eLife.10072.008

Article Snippet: The bead-bound immunoprecipitates were washed with lysis buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP40 and 10% Glycerol for three times followed by a final kinase buffer wash, then incubated with 20 ng full length recombinant FAK (SignalChem, Canada) or PYK2 (Enzo, Farmingdale, NY), 2 mM ATP, kinase buffer (Cell Signaling, Danvers, MA) in a total volume of 15 µl at 37°C for 2 hrs.

Techniques: Immunohistochemical staining, Staining, Microarray

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Discovery of RNA Binding Small Molecules Using Small Molecule Microarrays

doi: 10.1007/978-1-4939-6584-7_11

Figure Lengend Snippet: Overview of SMM strategy to identify RNA-binding small molecules. Small molecule libraries prepared in 384-well plates are utilized to print onto isocyanate-functionalized slides by the microarrayer. Prepared SMMs are next incubated with the labeled RNA of interest and scanned to detect hits

Article Snippet: Printing of Small Molecule Microarrays MicroGrid II Microarrayer (BioRobotics) or equivalent.

Techniques: RNA Binding Assay, Incubation, Labeling