Journal: Clinical Cancer Research

Article Title: Highly Sensitive EGFRvIII Detection in Circulating Extracellular Vesicle RNA of Glioma Patients

doi: 10.1158/1078-0432.CCR-22-0444

Figure Lengend Snippet: EGFR transcript variant vIII droplet digital PCR (ddPCR) optimization. A–C, ddPCR 1D plots with mutant channel amplitude (blue, top) and GAPDH channel amplitude (green, bottom), run in parallel with (right) and without (left) ethanol precipitation of EGFRvIII cDNA from three different reverse transcription protocols: (a) standard, (b) standard + 1 μL 7-deaza-GTP, (c) standard + 2 μL. D–F, Two-tailed t test results depicting the difference in copies/20 μL from ddPCR using EGFRvIII cDNA treated with and without ethanol precipitation from different reverse transcription protocols; (d) standard, (e) standard + 1 μL 7-deaza-GTP, (f) standard + 2 μL. G, Two-tailed t test results comparing the statistically significant difference in copies/20 μL in EGFRvIII cDNA from standard and (standard + 1 μL 7-deaza-dGTP) RT protocols, both untreated with ethanol precipitation. H, Two-tailed t test results comparing the statistically significant difference in copies/20 μL in EGFRvIII cDNA from (standard + 2 μL 7-deaza-GTP) and (standard + 1 μL 7-deaza-dGTP) RT protocols, both untreated with ethanol precipitation. I, QQ plot obtained from one-way ANOVA results depicting statistically significant difference in average copies/20 μL in EGFRvIII cDNA across different conditions overall. J, Two-tailed t test results comparing the statistically significant difference in copies/20 μL in EGFRvIII cDNA from standard and (standard + 1 μL 7-deaza-dGTP) RT protocols, treated with ethanol precipitation. K, Two-tailed t test results comparing the statistically significant difference in copies/20 μL in EGFRvIII cDNA from (standard + 2 μL 7-deaza-GTP) and (standard + 1 μL 7-deaza-dGTP) RT protocols, treated with ethanol precipitation. L, Two-tailed t test results depicting statistically significant difference in copies/20 μL in EGFRvIII cDNA (standard + 1 μL 7-deaza-dGTP, RT protocol) purified using different cleanup protocols. M, ddPCR 1D plots demonstrating change in separation of mutant events from the baseline at different annealing/extension temperatures (low vs. high). N, Quantitative difference in copies/20 μL of mutant events across different annealing/extension temperatures. O, Violin plots demonstrating statistically significant difference in copies/20 μL with the addition of 0.5 mmol/L betaine, 0.5 mmol/L EDTA, 0.5 mmol/L (betaine + EDTA) versus no addition of ddPCR additive (control). P, ddPCR 2D plots (top row) and 1D plots depicting cluster density, tightness, and separation of mutant events (blue) and GAPDH events (green) at different concentrations of betaine versus no betaine addition to ddPCR. Q, Varying numbers of EGFRvIII synthetic RNA were spiked into the reverse transcription reaction. The resulting cDNA was then amplified using the optimized ddPCR (top) and qPCR (bottom) cycling conditions. For ddPCR, copies per 20 μL are plotted against EGFRvIII copies spike-in (top). Limit of detection (LOD, dashed line) is plotted, defined as 3 standard deviations over average copies per 20 μL obtained when only a small concentration of EGFRvIII was run along with blanks. Limit of blank (LOB, dashed line) is plotted, defined as the apparent highest copy number expected to be found when replicates of a blank sample containing no EGFRvIII (or small concentration of EGFRvIII ) are tested. For qPCR cycle threshold ( C t ) is plotted against EGFRvIII copies spike-in (bottom).

Article Snippet: In the first part, 1.0 μL 50 μmol/L Oligo d(T) 20 , 1.0 μL 50 μmol/L random hexamers, 1.5 μL 10 mmol/L dNTP mix, and 1.0 μL 7-deaza-dGTP (New England BioLabs) were combined with template RNA in a 0.2 mL PCR reaction tube.

Techniques: Variant Assay, Digital PCR, Mutagenesis, Ethanol Precipitation, Reverse Transcription, Two Tailed Test, Purification, Control, Amplification, Concentration Assay

Journal: Nucleic Acids Research

Article Title: A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects

doi: 10.1093/nar/gkq341

Figure Lengend Snippet: Overview of chemically modified ASs and SSs

Article Snippet: Total RNA was harvested after 24 h using Trizol (Invitrogen) and reverse transcription was performed using the SuperScript ™ III Reverse Transcriptase (Invitrogen) and random hexamer primers. qPCR quantification of mRNA levels was performed using the Taqman Gene Expression Master mix (Applied Biosystems) on a Stratagene Mx3005 qPCR thermocycler (Stratagene, La Jolla CA, USA) using the following Taqman Gene Expression Assays (Applied Biosystems): GRK4 (HS00178384_m1); HIF1A (hs00153153_m1); ICAM1 (hs99999152_m1); TNFR1 (hs00533560_m1); GAPDH (Hs99999905_m1).

Techniques: Modification, Sequencing

Journal: Nucleic Acids Research

Article Title: A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects

doi: 10.1093/nar/gkq341

Figure Lengend Snippet: UNA-modification of siRNA AS and SS reduces off-targeting of well-characterized endogenous off-targets while preserving on-target activity. ( A ) Evaluation of on-target (GRK4 mRNA) and off-target activity ( hif-1 α mRNA) for a siRNA directed against GRK4. ( B ) Evaluation of on-target (ICAM1 mRNA) and off-target activity (TNFR1 mRNA) for a siRNA directed against ICAM1. H1299 were transfected with the siRNAs at the indicated concentrations and mRNA levels were evaluated after 24 h by qPCR. The presented values are normalized to values from cells transfected with the unrelated siEGFP1.

Article Snippet: Total RNA was harvested after 24 h using Trizol (Invitrogen) and reverse transcription was performed using the SuperScript ™ III Reverse Transcriptase (Invitrogen) and random hexamer primers. qPCR quantification of mRNA levels was performed using the Taqman Gene Expression Master mix (Applied Biosystems) on a Stratagene Mx3005 qPCR thermocycler (Stratagene, La Jolla CA, USA) using the following Taqman Gene Expression Assays (Applied Biosystems): GRK4 (HS00178384_m1); HIF1A (hs00153153_m1); ICAM1 (hs99999152_m1); TNFR1 (hs00533560_m1); GAPDH (Hs99999905_m1).

Techniques: Modification, Preserving, Activity Assay, Transfection