Table 1.
Comparison of RNA yield and purity obtained by the different RNA extraction procedures used.
Figure 1.
Effect of cDNA dilution in icaA gene expression quantification.
cDNA from biofilms or planktonic cultures was diluted from 1 10 to 1 1000 fold and icaA transcripts quantified by qPCR. cDNA replicates were synthesized using the same RNA sample but in independent cDNA synthesis reactions. The values represent the mean plus or minus standard deviation of 3 independent experiments. Statistical differences (*p<0.05) were analyzed with ANOVA Tukey's test.
Figure 2.
The impact of different cDNA and qPCR commercial kits in icaA gene expression quantification.
cDNA from biofilms or planktonic cultures was synthesized using different kits. The impact of different qPCR master mixes in icaA quantification was also tested. The values represent the mean plus or minus standard deviation of 3 independent experiments. Statistical differences (p<0.05) between cDNA kits (*) or qPCR master mixes (§) were analyzed with ANOVA Tukey's test.
Figure 3.
Variation in icaA gene expression quantification using different cDNA (top) or qPCR (botton) reaction volumes.
TOP: cDNA, synthesized using 20 µL or 10 µL reaction volumes, was used for icaA transcripts quantification. The transcripts were detected using Maxima® SYBR Green Master Mix. BOTTOM: cDNA (1 100 dilution) synthesized using RevertAid™ First Strand cDNA synthesis kit (20 µL reaction) was used for icaA transcripts quantification by different qPCR master mixes and using different reaction volumes. The values represent the mean plus or minus standard deviation of 3 independent experiments. Statistical differences (*p<0.05) between 10 µL and 20 µL reactions were analyzed with paired t-test.
Figure 4.
SYBR Green I dilution influence in qPCR assay using different Taq polymerase PCR kits.
The tested SYBR Green I concentrations ranged from 0.5 to 4× using the following commercially available PCR kits: Ezyway Direct PCR Mix (top), MyTaq PCR mix (middle) and DyNAzyme™ II PCR Master Mix (bottom). Data shown is representative of two independent experiments.
Table 2.
Comparison of gene expression quantification using a custom qPCR mix and Maxima® SYBR Green Master Mix.
Table 3.
Analysis of the percentage of cost reduction when using the custom RNA extraction and qPCR instead of the commercially available kits.