An evaluation of the performance of cDNA microarrays for detecting changes in global mRNA expression

Abstract

The cDNA microarray is one technological approach that has the potential to accurately measure changes in global mRNA expression levels. We report an assessment of an optimized cDNA microarray platform to generate accurate, precise and reliable data consistent with the objective of using microarrays as an acquisition platform to populate gene expression databases. The study design consisted of two independent evaluations with 70 arrays from two different manufactured lots and used three human tissue sources as samples: placenta, brain and heart. Overall signal response was linear over three orders of magnitude and the sensitivity for any element was estimated to be 2 pg mRNA. The calculated coefficient of variation for differential expression for all non-differentiated elements was 12-14% across the entire signal range and did not vary with array batch or tissue source. The minimum detectable fold change for differential expression was 1.4. Accuracy, in terms of bias (observed minus expected differential expression ratio), was less than 1 part in 10 000 for all non-differentiated elements. The results presented in this report demonstrate the reproducible performance of the cDNA microarray technology platform and the methods provide a useful framework for evaluating other technologies that monitor changes in global mRNA expression.

Nicotine coregulates multiple pathways involved in protein modification/degradation in rat brain

“…This array contains 638 sequence-verified genes selected from rat cDNA clones (Research Genetics, AL) and the 15K NIA mouse cDNA gene set [52] on the basis of our previous studies [28,37]. It was shown that cross-species hybridization (i.e., mouse clones against rat target samples) using a cDNA platform is accurate and specific [59]. Cells containing the cDNA clones of interest were grown overnight, and 10 Al of the culture medium plus 90 Al of water were incubated at 95 8C for 10 min to release plasmid DNA.…”

Characterization of a method for profiling gene expression in cells recovered from intact human prostate tissue using RNA linear amplification

“…The mean (range) yield of aRNA for PC3 and PC3-M cells was 3.0 mg (1.8-4.0 mg) and 3.1 mg (1.9-4.8 mg), respectively, Table 1. To evaluate the effect of operator experience, aRNA yields from reactions performed early in the 3-month period (reactions 1-6) were compared to those performed late (7)(8)(9)(10)(11)(12), for both PC3 and PC3-M cells. For PC3 cells, mean yields for early and late reactions were 2.9 and 3.0, respectively, whereas for PC3-M cells, they were 3.1 and 3.1, respectively.…”

“…[3][4][5][6] Standard protocols require B5 mg of total RNA per sample as starting material for high-density oligonucleotide microarrays; B20 mg is required for cDNA-based microarrays. 7,8 Whereas microgram quantities of RNA are readily extracted from whole biopsy specimens, individual specimens consist of a heterogeneous mixture of cell types. This, in turn, imposes practical limitations upon interpretation of resultant array data, particularly when one seeks to identify changes in a single-cell type, as is typically the case.…”

Downregulation of gene expression in the ageing lens: a possible contributory factor in senile cataract

“…This software generates significant differences between gene expressions where, a two-fold difference is equal to a P-value of 0.05. [14][15][16][17] All differential expression values were log transformed and balanced, essentially as previously described. 17 Briefly, the differential values were calculated only if the signal to background ratio exceeded 2.0, the signal intensity was above 200 U in at least one of the two channels, and the element diameter exceeded 40% of the mean element diameter for the array.…”