ABSTRACT

Proper data analysis is crucial to obtaining valid and relevant results from any experimental system, but is especially critical when assessing variations in mRNA expression of genes, the so-called transcriptome. Unlike DNA which is present in each cell throughout the life of the organism, RNA is transiently expressed and levels vary according to cell type, developmental stage, physiology and pathology. Therefore, quantification of RNA is context dependent and inherently variable. Extra vigilance must be exercised to ensure the data are technically accurate before they can be examined for biological relevance. It should always be remembered that mRNA expression does not necessarily correlate with protein expression and protein expression may not correlate with function. Regardless of the instrument used to perform quantitative real-time PCR, certain basic conditions should be met. The assay should be robust and fulfill all criteria for a good assay. A standard curve using a defined template should result in a slope, coefficient of determination (r2) and y-intercept that demonstrate good efficiency, accuracy and sensitivity. The baseline and threshold should be properly set. The amplification curves should demonstrate exponential amplification and be within the detection limits of the instrument. Proper controls should be included to monitor contamination and sensitivity of the assay. Genomic contamination should be assessed, if applicable. Data should be reported in a manner that allows the variability of results to be seen. Proper statistical analyses should be applied to the data.