Methodology article
Optimization and analysis of a quantitative real-time PCR-based technique to determine microRNA expression in formalin-fixed paraffin-embedded samples
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* Corresponding author: Suzanne Kamel-Reid suzanne.kamel-reid@uhn.on.ca
- † Equal contributors
1 Division of Applied Molecular Oncology, Ontario Cancer Institute, University Health Network, Toronto, ON, Canada
2 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada
3 Division of Signaling Biology, Ontario Cancer Institute
4 Campbell Family Institute for Cancer Research, University Health Network, Toronto, ON, Canada
5 Department of Biostatistics, Princess Margaret Hospital, University Health Network, Toronto, ON, Canada
6 Department of Pathology, Toronto General Hospital, University Health Network, Toronto, ON, Canada
7 Department of Computer Science, University of Toronto, Toronto, ON, Canada
8 Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada
9 Division of Medical Oncology, Princess Margaret Hospital, University Health Network, Toronto, ON, Canada
BMC Biotechnology 2010, 10:47 doi:10.1186/1472-6750-10-47
Published: 23 June 2010Additional files
Additional file 1:
Figure S1. Absolute value of the difference between duplicate measurements as a function of the duplicate mean for all input RNA concentrations. Measurements where at least one of the duplicate Ct values equals 40 are removed. The red line shows a cubic spline fit using default settings for the smooth spline function in the R statistics package (R Development Core Team, 2008). The apparent decrease in variability for mean Ct>37 is an artifact caused by the absence of Ct values greater or equal to 40.
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Additional file 2:
Figure S2. A) Graph of average percentage of well failures according to input RNA concentrations. Note that cDNA dilution factors were kept constant (15×). B) Percentage of well failures as they relate to input RNA concentration and cDNA dilution factor. cDNA dilution factors are shown on the x-axis and percentage of well failures are shown on the y-axis.
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Additional file 3:
Table S1. Table of Pearson correlation coefficients between equivalent input RNA concentration/cDNA dilution factor combinations (equivalent samples).
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Additional file 4:
Table S2. Table depicting number of miRs in each abundance stratum for equivalent dilutions. No significant difference exists in the abundance distributions of the columns by chi-square analysis (χ2 = 3.3, df = 6, p = 0.77).
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