Journal: Expert Review of Molecular Diagnostics
Article Title: Properties of targeted preamplification in DNA and cDNA quantification
Figure Lengend Snippet: The effect of additives on preamplification specificity and efficiency. The yield and specificity of preamplification were evaluated at 35 conditions using 18 different additives. Analysis of preamplification: (A, B) preamplification response curves and corresponding melting curves for reactions in the presence of 1-µg/µl bovine serum albumin with 2.5% glycerol or water. (C) Average Cq ± SD for positive (n = 3) and negative samples (n = 3) applying different conditions for preamplification. ΔCq refers to the difference in Cq-values between positive and negative samples for each condition. Conditions are sorted according to ΔCq-value. (F) High-throughput qPCR data of individual assays for nine selected conditions. Average Cq ± SD (n = 3) is shown. The right y-axis indicates the percentage of negative controls positive for nonspecific PCR product formation, calculated from the 91 assays performing accurately in the preamplification (n total = 273, 3 negative qPCR controls per assay). 7-deaza-dGTP: 7-deaza-2′-deoxyguanosine 5′-triphosphate lithium salt; BSA: Bovine serum albumin; DMSO: Dimethyl sulfoxide; DTT: Dithiothreitol; LPA: GenElute-LPA; NTC: Non-template control; Poly(I:C): Polyinosinic–polycytidylic acid potassium salt; TMA Cl: Tetramethylammonium chloride.
Article Snippet: Preamplification additives The effects of 18 different PCR additives on the preamplification reaction were evaluated (final tested concentrations are shown): 7-deaza-2′-deoxyguanosine 5′-triphosphate lithium salt (50 and 100 µM; Sigma-Aldrich), ammonium sulfate (10 and 15 mM; Sigma-Aldrich), betaine (0.25 and 0.50 M; Sigma-Aldrich), bovine serum albumin supplied at 20 mg/ml in 10 mM Tris-HCl, 100 mM KCl, 1 mM EDTA and 50% glycerol (1 and 2 µg/µl; Thermo Scientific), D-(+)-trehalose dehydrate (0.15 and 0.30 M; Sigma-Aldrich), dimethyl sulfoxide (1%; Sigma-Aldrich), dithiothreitol (1.5 and 3.0 mM; Life Technologies), formamide (0.5 M; Sigma-Aldrich), gelatin (0.01 and 0.10%; Sigma-Aldrich), glycerol (2.5 and 5.0%; Sigma-Aldrich), IGEPAL CA-630 (0.25 and 0.50%, Sigma-Aldrich), l -carnitine inner salt (0.25 and 0.50 M; Sigma-Aldrich), GenElute-LPA (50 and 100 ng/µl; Sigma-Aldrich), polyinosinic:polycytidylic acid potassium salt (5 and 50 ng/µl; Sigma-Aldrich), tetramethylammonium chloride (30 and 60 mM; Sigma-Aldrich), Triton X-100 (0.2 and 0.4%; Sigma-Aldrich), TWEEN 20 (0.10 and 0.50%, Sigma-Aldrich) and yeast tRNA (50 and 100 ng/µl, Life Technologies).
Techniques: High Throughput Screening Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction