Two of the 17 samples, Mex-1225 and Mex-730, exhibited very weak microarray probe intensities on Virochip analysis and were grouped with the negative control samples.
A comparison between microarray probe intensities and qPCR fold changes indicated that different miRNAs had different levels of correlation, varying from R2 = 0.81 to R2 = 0.95.
Correlation coefficients of microarray probe intensities between biological duplicates were greater than 0.93.
However, the correlation between RNA sequencing reads and microarray probe intensities tends to be quite reasonable [ 27] so major changes seem unlikely.
One expects that the loss of RNA material in general and particularly, RNA-fragments probed far away from the 3′-end, systematically decreases the apparent expression degree extracted from microarray probe intensities.
For example, while many microarray probe intensities overestimate gene expression levels by several orders of magnitude, SSMIs for nearly all probes much more closely match TPM values determined by RNA-Seq.
Affymetrix microarray probe intensities were normalised using RMA normalisation via the Bioconductor/R package oligo (version 1.18.1) (Carvalho and Irizarry, 2010) and differences tested using the Bioconductor/R package limma (version 3.10.3) (Smyth, 2004).
Third, and most importantly, our study proposes for the first time the use of high throughput sequencing to scale microarray probe intensities to more closely reflect absolute gene expression levels.
We systematically analysed the effect of varying RNA quality on microarray probe intensities using a physico-chemical hybridization model and propose (i) new measures to assess RNA quality and (ii), a simple method to correct probe intensities for the degradation bias.
As reference against the PS algorithm, we developed a novel statistical method in MATLAB, Probe Intensity Means (PIM), that uses the per-chromosome means and standard deviations of normalized microarray probe intensities to call chromosome copy number (Rabinowitz et al., 2007).
