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.

Figure 1

Comparison of oligonucleotide probe and PCR probe sensitivity. Microarray sensitivity was measured by detecting known fold changes at varying RNA levels using oligonucleotide probe and PCR probe microarrays. thr, dap and trp mRNAs were added to total rat liver RNA at varying concentrations and assayed using two-color microarray analysis. Oligonucleotide data represents two oligonucleotide probes per transcript in duplicate using three different transcripts (thr, dap and trp), PCR probe data represents one PCR probe per transcript in duplicate using these three transcripts. The x-axis is labeled with the ratio of added (‘spiked’) transcripts (e.g. 3/1 represents the measured, normalized ratio of 3 copies/cell to 1 copy/cell).

Figure 2

Oligonucleotide probe specificity as a function of contiguous complementary base pairs. Oligonucleotide probes (50mers) were designed to detect dap cDNA with varying degrees of complementary sequence to the engineered deletion (dapΔ cDNA). Probe set 1 (two oligonucleotide probes) includes a 25 bp complementary sequence with the conserved region present in dapΔ, probe set 2 contains 30 bp of complementary sequence, probe set 3 contains 35 bp of complementary sequence and so on. Note that probe set 3 includes the largest complementary sequence (35 bp) while probe set 5 contains the smallest (15 bp).

Figure 3

The ratio of target signal (dap) to non-target signal (dapΔ) using the probe sets outlined in Figure 2. (A). The graph shows that the presence of 15 continuous complementary base pairs results in a target/non-target (dap/dapΔ) signal ratio slightly greater than 100, i.e. the target signal is ∼100 times greater than the non-target signal. The target signal for the 20 bp complementary probe set is ∼25 times greater than the non-target signal. Thus the contribution of non-target signal is ∼1, 4, 15, 30 and 50% of the target signal for the complementary sequences of 15, 20, 25, 30 and 35 bp, respectively. (B) Raw data from one microarray where dap cDNA is labeled with Cy5 and dapΔ is labeled with Cy3. The length of continuous complementary base pairs is noted above the features.

Figure 4

Microarray specificity (i.e. cross-hybridization) using varying degrees of non-target cDNA similarity (see Table 2). Microarrays were constructed which included oligonucleotide probes to detect yicK, yeiO and yabM cDNA. Three different microarrays are shown hybridized with yicK, yeiO or yabM cDNA. The arrow indicates non-target detection of yeiO cDNA with probe Yick_337 that is 18% of the target signal (yicK cDNA hybridized to probe Yick_337).