Computational protocol: Quantification noise in single cell experiments

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Protocol publication

[…] Primers were designed using published nucleic-acid sequences of the human genome GenBank (NCBI, Bethesda, USA) (). Primer design and optimization was done using primer design program Primer 3 v.0.4.0 ( and checked with Netprimer (Premier Biosoft, Silicon Valey, USA) with regard to primer dimer formation, self-priming formation and similar primer annealing temperature. Newly designed primers were ordered and synthesized by TIB Molbiol (Berlin, Germany). Primer testing was performed on human white blood cells sample pool and a negative control (RNAse free water) for each primer set. Specificity of the primer was controlled by melting curve analysis and agarose gel electrophoresis. Primer sequence and location was checked with PrimerBLAST (NCBI, Bethesda, USA). Primer design was done MIQE compliant (). [...] To compare individual samples and slides within the DNA and RNA standard array, a standard curve was drawn for each 48-well AmpliGrid slide and the actual number of copies per sample was figured out using the linear regression formula: where Y = Cq-value; a = slope of curve; x = copy number in log-scale; b = y-intercept of curve.Mean value and standard deviation for each dilution step was determined and intra-run variance (n = 4) and inter-run variance (n = 4) was calculated as the mean variation (standard deviation SD divided by mean of copies) of each slide. For calculation of mean copy numbers in the gene expression profiling the geometric mean was taken (,).For statistical evaluation of significance in LPS induced gene regulation in expression profiling experiments the ‘Mann–Whitney Rank Sum Test’ was applied. Test was chosen due to none normal (Gaussian) distribution of the expression data and the inhomogeneous data groups in all analyzed genes and treatments. For normality testing of cellular copy numbers and log-normal distribution the robust ‘Kolmogorow Smirnow Test’ (KS-test) was used. The KS-test has the advantage of making no assumption about the distribution of data. All calculations were performed in Microsoft Excel (Microsoft, Redmond, USA), illustrated using SigmaPlot 11.0 (Systat Software Inc., Richmond, USA) and statistical evaluations were performed in Sigma Stat version 3 (Systat Software Inc.). […]

Pipeline specifications

Software tools Primer3, NetPrimer, SigmaPlot
Applications Miscellaneous, qPCR