Fig. 1From: Detecting eukaryotic microbiota with single-cell sensitivity in human tissueOptimization of blocking primer annealing temperature and concentration. The EMP protocol utilizes a mammal blocking primer annealing temperature of 65Ā Ā°C and blocking primer concentration of 1.6Ā Ī¼M. a Different blocking primer annealing temperatures were tested for the 18S rRNA gene amplification. In all reactions, 1.6Ā Ī¼M blocking primer was used. The effect of increasing annealing temperature was determined by quantifying the yield of product by Q-PCR. A higher Ct indicates more effective blocking of amplification of the human 18S rRNA gene. An annealing temperature of 61Ā Ā°C resulted in a significantly higher Ct than at 67Ā Ā°C or 68Ā Ā°C (repeated measures ANOVA followed by Tukeyās multiple comparisons test). Data presented as meanā+āSEM, nā=ā3; different letters above bars indicate Pā<ā0.05. b Comparison of blocking primer concentration and annealing temperature (65Ā Ā°C) with the original sample of S. cerevisiae of 0.0001%. Data presented as meanā+āSEM, nā=ā4. Different letters above the bars indicate Pā<ā0.05 (repeated measures ANOVA followed by Tukeyās multiple comparisons test)Back to article page