Benchmark study: Exiqon mircury LNA microrna arrays vs. Supplier A s DNA based capture probes Includes comparison of: I. II. III. Signal levels Relative quantification False differences For complete experimental set up, please see last slide
Comparison I: Signal levels Comparison of important micrornas All micrornas are robustly detected by the s More than half of the micrornas are undetected or poorly detected by from supplier A 8 Poor detection signal *Signal strength = log signal / amol target mir- mir-8a mir-55 mir- let-7a mir-5 mir- mir- mir- mir-9 Signal strenght [log]* Acceptable detection signal 6 Signals below log are considered under the detection limit whereas signals above 8 log are considered acceptable.
Comparison I: Signal levels Comparison of 66 micrornas With s more than 95% of all studied micrornas are reliably detected 8 Poor detection 5 6 micrornas 5 6 micrornas 6 Acceptable detection 8 Poor detection Signal strenght [log]* 6 Acceptable detection Signal strenght [log]* With DNA capture probes more than 5% of micrornas are either undetected or poorly detected *Signal strength = log signal / amol target Shown is the signal strength from the same 66 synthetic micrornas hybridized to Exiqon's mircury LNA microrna Array and Supplier A's DNA based array.
Comparison II: Relative quantification Comparison of important micrornas s ensures reliable detection of differences in expression for all micrornas TRUE - - - *See last slide for details mir-9 mir- mir- let-7a mir-55 mir-5 mir- mir-8a mir- - mir- Sample A / Sample B* DNA based capture probes from Supplier A show poor correlation between measured and actual differences
Comparison II: Relative quantification Comparison of all micrornas s ensures excellent correlation (R=.9) between expected and measured ratios from supplier A show poor correlation (R=.658) between expected and measured ratios 5-5 - - R² =,658-5 True ratio True ratio R² =,9-5 - - - - - - - - - -5-5 Measured ratio Measured ratio 5
Comparison III: False differences Comparison of 66 micrornas Exqon s array has a low rate of false differences while supplier A s array detects false difference for more than 5% of micrornas studied,5 Significant difference,5 -,5 - -,5 6 8 6 6 8 6,5 Significant difference Measured Ratio,5 Significant difference,5 -,5 - -,5 66 micrornas present in the same amount in sample A + B Significant difference Measured Ratio,5
Summary of benchmark study Comparison of all micrornas Exiqon s s ensures accurate results for all micrornas studied Supplier A s DNA based capture probes gives incorrect results for up to half of all micrornas studied % False differences 8 % 6 % * % of capture probes giving a signal above 8 log per amol synthetic microrna target. % ** % of capture probes that measure the correct ratio between sample A and B. (Correct ratio: log of the true ratio AND going in the same direction). % % Robust detection of mirnas* Reliable relative quantification of micrornas** Detection of false differences*** *** % of capture probes which has differences above,5 log or below -,5 log for microrna targets that were present in the same amounts in sample A and B.
Summary of benchmark study Experimental set-up Samples: Two complex samples of synthetic micrornas: Pool no sample A sample B (amol/ul) (amol/ul) log ratio 76.7.5 55.56.. 8. 66.67. 5 66.67. 6. 65.69.5 7.. 8.. 9.... 65.69. -.5 Data analysis:. 66.67 - Data analysis and determination of calls were done according to manufacturers recomendations. 66.67-8.. - 5. 55.56-6.5 76.7 - Each contain the same set of 7 micrornas (made up from 6 pools of up to 7 micrornas each) MicroRNAs are present at different concentrations in each sample A comparison of the samples gives different expected log ratios in the - to + range Protocol: Each sample labelled and hybirdised either to Exiqon s mircury LNA microrna array or Supplier A s array accordingto manufaturers instructions. Sample A and B have been used in previously published data: RNA (9), 5:8.