Assessment of the sensitivity and specificity of oligonucleotide (50mer) microarrays

Abstract

To examine the utility and performance of 50mer oligonucleotide (oligonucleotide probe) microarrays, gene-specific oligonucleotide probes were spotted along with PCR probes onto glass microarrays and the performance of each probe type was evaluated. The specificity of oligonucleotide probes was studied using target RNAs that shared various degrees of sequence similarity. Sensitivity was defined as the ability to detect a 3-fold change in mRNA. No significant difference in sensitivity between oligonucleotide probes and PCR probes was observed and both had a minimum reproducible detection limit of approximately 10 mRNA copies/cell. Specificity studies showed that for a given oligonucleotide probe any 'non-target' transcripts (cDNAs) >75% similar over the 50 base target may show cross-hybridization. Thus non-target sequences which have >75-80% sequence similarity with target sequences (within the oligonucleotide probe 50 base target region) will contribute to the overall signal intensity. In addition, if the 50 base target region is marginally similar, it must not include a stretch of complementary sequence >15 contiguous bases. Therefore, knowledge about the target sequence, as well as its similarity to other mRNAs in the target tissue or RNA sample, is required to design successful oligonucleotide probes for quality microarray results. Together these results validate the utility of oligonucleotide probe (50mer) glass microarrays.

Comprehensive genetic exploration of skeletal dysplasia using targeted exome sequencing

“…26 During preparation of the library, polymerase chain reaction is used to amplify target regions; however, high GC content reduces the efficiency of such amplification. 27 Moreover, the hybridization of capture probes to the target sequences can be hindered by high or low GC content 28 (Supplementary Table S4 online). In our study, we confirmed that both sequencing depth and coverage were dependent on GC content (Supplementary Figure S2 online).…”

Swedish mutation within amyloid precursor protein modulates global gene expression towards the pathogenesis of Alzheimer's disease

“…RNA-Seq overcomes several shortcomings of microarray-based detection of transcripts, including probe cross-hybridization (9), restricted signal dynamic range, and low sensitivity and specificity, which often lead to difficulties in the detection of low abundance transcripts and discrimination between similar sequences. Sequence level transcript information has much greater power to distinguish between paralogous genes, better detection of low abundance transcripts, and allows replicable digital quantification based upon counting sequence reads (10)(11)(12)(13)(14).…”

Comparison of oligonucleotide-microarray and serial analysis of gene expression (SAGE) in transcript profiling analysis of megakaryocytes derived from CD34+ cells

“…DNA microarrays are technology to profile the expression of thousands of transcripts simultaneously (Brown and Botstein, 1999;Lockhart and Winzeler, 2000;Noordewier and Warren, 2001), and two different types of microarray technologies are available; Genechips (Affymetrix platform) and spotted microarrays. The Genechips produced by photolithography and hybridize the single cRNA probe have certain advantages over spotted microarray, such as that crosshybridization could be avoided, quality control by sequence validation of PCR clones is not required, and noncompetitive nature of hybridization make small amount of RNA sample detectable (Kane et al, 2000;Lockhart and Winzeler, 2000). As this platform is not accessible for every laboratory, spotted microarrays of longer oligonucleotides (50-70 mer) are becoming attractive and alternative platform (Hughes et al, 2001).…”