Use of Agilent Feature Extraction Software (v8.1) QC Report to Evaluate Microarray Performance Anthea Dokidis Glenda Delenstarr Abstract The performance of the Agilent microarray system can now be evaluated using statistical results generated in the QC report, a new Feature Extraction (FE) output file (see FE User's Manual). There are three classes of metrics reported in the QC Report: (1) those that are independent of the platform; (2) those that rely on replicated probes; and (3) those that require both the coamplification and co-labeling of E1a spike-in targets, and the presence of E1a probes on the microarray. In this study, microarrays processed with Agilent's Two-Color Microarray- Based Gene Expression Analysis, version 4.0 protocol were experimentally manipulated to produce several images that can be used to examine the trends in the QC Report metrics. These conditions were wash artifacts, ozone exposure, and hybridization of crna signal generated from a degraded total RNA.
Introduction Figure 1 The performance of microarray target labeling, hybridization, and washes can now be evaluated and monitored using control E1a RNA targets supplied in the Agilent RNA Spike-In kit (P/N 5188-5279) in combination with Agilent FE v8.1.these E1a control targets are in vitro synthesized, polyadenylated transcripts premixed at specific concentrations. In this way, when the E1a targets are amplified and labeled in the same reaction as the sample target RNA, they can be used to monitor the entire Agilent Two-Color Microarray-Based Gene Expression Analysis system for uniformity, sensitivity, and accuracy of log ratio signal across microarrays and between microarray experiments. In addition, the E1a targets are an invaluable tool in optimizing and troubleshooting experiments. To accommodate and make this E1a metric information easily available, the new version of the FE software (v8.1) produces an optional output file, the QC Report, which includes statistical results for both the E1a controls, for the non-control probes, and for the calculated backgrounds. These metrics are also available in the FE Stats table output, making it easy for the user to generate tracking charts (e.g. run charts ) of various metrics over time. This report shows examples of a few of these QC metrics, tracking the perturbations to the microarray system caused by manipulation of the experimental conditions. These conditions, described in the Experimental Methods, are: microarray wash artifacts, ozone exposure, and hybridization of crna generated from a degraded total RNA. Experimental Design Total RNA (30 arrays) Ozone (10 arrays) No wash 3 Exposed to ozone crna Targets Degraded RNA (10 arrays) Arrays Hybridizations (40 arrays) Array Washes (40 arrays) (10 arrays) Generate wash artifacts with wash3+10% wash2 Normal Wash (Control) (20 arrays) 10 Control arrays 10 Degraded RNA microarrays Total RNA from human adult and fetal heart were used to generate labeled crna. In addition, degraded total RNA (see Experimental Methods) from both adult and fetal heart were also used to generate labeled crna targets. Forty microarrays were hybridized the same day (Figure 1). Ten of these microarrays were hybridized with crna generated from the degraded total RNA for both adult and fetal heart. After hybridization all microarrays were washed with Gene Expression Wash buffer 1 and 2. Thirty microarrays including the normal wash control and the degraded total RNA microarrays were also washed a third time with the Agilent Stabilization and Drying solution and scanned for fluorescence signal. The remaining ten microarrays were washed with Gene Expression Wash Buffer 1 and 2 but not with the Agilent Stabilization and Drying solution. These microarrays were scanned and then removed from the scanner and were exposed to an ozone dose (see Experimental Methods). These microarrays were scanned, removed from the scanner, exposed to a second ozone dose and then scanned again. Scan Microarrays Microarrays: Whole Human Genome Oligo Microarray -G4112A Wash 1: Gene Expression Wash Buffer 1 (P/N 5188-5325) at room temperature for 1 minute Wash 2: Gene Expression Wash Buffer 2 (P/N 5188-5326) at 37ºC for 1 minute Wash 3: Stabilization and Drying Solution (P/N 5185-5979), at room temperature for 30 seconds 2
Experimental Methods Degraded total RNA: Degraded total RNA was prepared by addding 0.001X RNase I 'A' from the Agilent Low RNA Input Fluorescent Linear Amplification Kit and leaving for 15 minutes at room temperature. After phenol chloroform purification and ethanol precipitation the degraded RNA was purified and spectrophotometrically quantitated while the level of degradation was determined using the RNA 6000 Nanoassay on the Agilent 2100 BioAnalyzer. (Figure 2) Fluorescent crna Targets 200 ng of total RNA from human adult and fetal heart and 12.5 pg of target E1a spike-in RNA were used to generate labeled Cyanine3- and Cyanine5-cRNA targets respectively using the Agilent Low RNA Input Fluorescent Linear Amplification Kit. Hybridization and washes 0.75ng of Cyanine3- and Cyanine5- labeled crna targets and 1X control targets in 1X hybridization buffer were incubated with the human microarrays G4112A, in a rotating oven at 65ºC for 17 hours. The microarrays were washed with Gene Expression Wash Buffer 1 at room temperature for 1 minute followed by a second wash of Gene Expression Wash Buffer 2 at 37ºC for 1 minute. The third wash consisted of Agilent Stabilization and Drying solution for 30 seconds at room temperature. Figure 2: Total RNA from Adult Heart: RNAse Treatment Bioanalyzer Electropherogram Adult Heart Total RNA (No RNAse) Adult Heart Total RNA (treated with 0.001X RNAse I 'A') Microarrays with wash artifacts were generated by washing hybridized microarrays with the Agilent Stabilization and Drying solution which has a 10% added volume of the Gene Expression Wash Buffer 2 for 30 seconds at room temperature. Ozone Exposure Hybridized Microarrays were washed with the first and second wash as described above but not with the stabilization and drying solution. The microarrays were then scanned on the Agilent microarray scanner to obtain a signal baseline. The microarrays were removed from the scanner and were exposed to 50 ppb of ozone for 1 minute while they were still in the scanner B-type slide holder. After this first dose of ozone the microarrays were scanned to obtain a microarray image for the fist ozone dose. The microarrays were removed and then exposed to a second ozone dose of 50 ppb for 1 minute and scanned again to produce microarray images for the second ozone dose. 4,000 2,000 1,000 RNA Ladder No RNase RNase 0.001x 200 25 Adult Heart Total RNA Treatments Bioanalyzer Gel Image 3
Microarray images Figure 3: Log Scale Control, normal microarray Microarray with wash artifacts Microarray with degraded total RNA 4
Figure 4: Log Scale Microarray before ozone dose Same microarray after first ozone dose Same microarray after third ozone dose 5
Figure 5: Linear Scale Microarray before ozone doses Microarray after first ozone dose Microarray after third dose of ozone 6
Trends Observed in QC Report Metric Results To examine and visualize these trends, the QC metric in question is plotted on a graph for each microarray for all of the experimental conditions tested. Signal Dynamic range Net signal statistics can be a good indication of the signal dynamic range for a specific hybridized target. It should be noted that the net signal is independent of the Agilent scanner version. It is calculated as the (MeanSignal - scanner offset) and is also shown as a Features data column in the text output of the Feature Extraction v8.1. In Figures 6 and 7, the green and red net signal intensity at the 99th percentile of all non-control probes are plotted against the microarray experimental condition. From these figures, it is apparent that the microarrays hybridized with the crnas from the degraded total RNA (yellow symbols) show a dramatic decline in their net signal for both channels. A decrease in signal is also seen in the microarrays with the wash artifacts (light blue symbols). Microarrays with exposure to ozone clearly exhibit a significant effect in the red channel but not in the green channel, for both ozone doses (green and black symbols). Background Signal Figures 8 and 9 show the standard deviation for the net signal of the microarray negative controls for both channels. The microarrays with wash artifacts show a higher and wider range of standard deviations in their net signal distribution, as expected, as compared to the control microarrays. These negative control statistics are not shown on the QC Report, but are available in the Stats table output. The standard deviations of the local background inliers are shown on the QC Report. These SD's also increased on the microarrays with wash artifacts compared to control microarrays (data not shown). In comparison, the background standard deviations from the microarrays from the other conditions are similar to the range seen with control microarrays. Microarray Performance Uniformity The average of the signal to noise ratios (S/N) of the hybridized E1a probes can be used as one of the metrics for the microarray uniformity and indicate the amount of up or down regulated differential expression. Each of the ten different E1a probes is present with 30 replicate features on the microarray used for these experiments. An average and standard deviation (SD) of the log ratio for each of the ten E1a probes is calculated. Then, for each E1a probe, the S/N is calculated as the absolute (Avg_LogRatio/ SD_LogRatio). The average S/N across these ten S/N measurements is reported as a QC metric. A high S/N represents differential expression that is reproducible (e.g. a S/N > 3 is commonly used as a significance threshold). In Figure 10, the average of the S/N of the E1a probes is plotted for each microarray in each of the experimental conditions. The E1a Figure 6 Figure 7 Green net Signal At 99 th Percentile for Non-Control Probes Red Net Signal At 99 th Percentile for Non-Control Probes Legend Control Degraded Total RNA Before Ozone Doses Ozone First Dose Ozone Second Dose 7
hybridized to the microarrays with the wash artifacts and to the ozone treated microarrays are considerably closer to zero (less differential expression and/or poorly reproducible log ratios) than the control normal microarrays. This indicates that this metric can be used to show relative changes in the microarray uniformity within microarrays. Note that the microarrays that were scanned before exposure to ozone were not washed with the stabilization and Drying solution and they yield a wider variation of S/N ratios as compared to the control microarrays. Additionally, the E1a S/N's are unaffected by the degradation of the crna, as expected. Thus, this metric can be used to differentiate between problems that affect only the sample RNA from other types of problems that affect the entire sample, E1a amplification, and labeling reactions. Figure 8 Std Dev for Green Net Signal of Negative Controls Microarray Reproducibility The median percent coefficient of variation (%CV) of the background subtracted signals for the replicate features is used as a measure of the intra-array signal reproducibility. The %CV is calculated for each group of replicated probes, as (SD_Signals/Avg_Signals)*100%. The median of this group of %CV's is reported in the QC Report. A lower %CV indicates better reproducibility of signal intensity and hybridization uniformity. In Figures 11-14, the %CV of the background subtracted signal in both channels is shown for the non-control probes and the control E1a probes. Clearly, the %CV of the background subtracted signals for the non-control probes in the microarrays with the wash artifacts is increased in both channels compared to all the other microarrays (Figures 11 and 12). The microarrays with the wash artifacts are not expected to show a reproducible signal across the replicate probes within the microarrays, which is observed with this metric as expected. Figure 9 Std Dev for Red Net Signal of Negative Controls This microarray trend in the slides with wash artifacts is also observed in the %CV of the background subtracted signal for the E1a probes (Figures 13 and 14). Note that the %CV's of the E1a probes in the red channel are also sensitive to ozone (Figure 14). The %CV's seen with the E1a probes are higher, in this experiment, than the %CV's with the non-control probes. The E1a probes are designed to have a wide range of signal intensities across the 9 different probe sequences. The spread observed in the %CV of the background subtracted signal for the E1a replicate probes reflects this signal range; that is, higher %CV's are generally obtained from the E1a probes with the lower signal ranges. This relation is also shown as a plot on the QC Report: E1a %CV's vs. average background subtracted signal (not shown). Legend Control Degraded Total RNA Before Ozone Doses Ozone First Dose Ozone Second Dose 8
Figure 10 E1A S/N Log Ratio Legend Control Degraded Total RNA Before Ozone Doses Ozone First Dose Ozone Second Dose Figure 11 Green Non-Control Median %CV Background Subtracted Signal Figure 12 Red Non-Control Median %CV Background Subtracted Signal 9
Figure 13 Green E1A Median %CV Background Subtracted Signal Figure 14 Red E1A Median %CV Background Subtracted Signal Discussion From the results of this study, it is seen that significant changes in some of the metrics calculated in the FE output QC report appear to be well correlated with certain microarray process defects. For example, in the Ozone-treated hybridized microarrays, the net signal intensity at the 99th percentile of all non-control probes is greatly affected in the red channel (Figure 7) but not affected in the green channel (Figure 6). Similarly, the log ratio for the E1a probes on these microarrays is seen to be closer to zero, compressed and/or non-reproducible, (Figure 10), compared to the control microarrays indicating a change in their signal intensities and an increase in noise. Furthermore, the ozone results also show an increase in the average %CV for the E1a probes in the red channel (and not green channel), indicating a decrease in reproducibility due to the effect of ozone on the Cyanine5-labeled targets (Figure 14). Microarrays exhibiting wash artifacts have the highest number of non feature uniform outliers (graph not shown) and a lower signal dynamic range (Figures 6 and 7). This decrease in performance trend continues in the microarray uniformity metric for E1a controls (Figure 10). We expect the microarrays with wash artifacts to show less signal and log ratio uniformity within microarrays and also across microarrays of this experimental condition (Figure 11 and 12). Additionally, the negative controls on the microarrays with wash artifacts have an increased net signal standard deviation that shows a dramatically wider spread than the control microarrays (Figures 8 and 9). This is particularly evident in the green channel. Interestingly, the microarrays hybridized with crna generated from a degraded sample total RNA (degraded human adult and fetal heart RNA) appear to have greatly decreased signal dynamic ranges in both the red and the green channels, as compared to microarrays from all other conditions (Figure 6). In contrast, the reproducibility of signal and log ratios with the E1a controls is unaffected by the crna degradation condition, as can be with the E1a signal %CV metrics (Figures 13 and 14) and the E1a S/N of log ratio metric (Figures 10). This is expected since the E1a spike-ins were added post sample RNA degradation. Conclusion Legend Control Degraded Total RNA Before Ozone Doses Ozone First Dose Ozone Second Dose In this study, it is demonstrated that the QC report generated using FE 8.1 and the E1A targets is capable of identifying microarray images which exhibit visible hybridized microarray problems such as wash artifacts as well as microarrays with non-visible problems such as exposure to Ozone or hybridization with crna from degraded Total RNA. The ability to detect deviations from normal trends reported in the QC Report enable researchers to identify results which may require closer inspection. The trends of the QC Report metrics observed in this study were as expected for each particular microarray artifact condition proving that it is numerically possible to track the performance of the microarray processes, such as target preparation, microarray hybridization, and washes using the QC Report statistics. 10
www.agilent.com/chem/dna Agilent Technologies, Inc. 2005 Printed in the U.S.A. Agilent Technologies Bioresearch Solutions Unit 3500 Deer Creek Road Palo Alto, CA 94304 E-mail: dna_microarrays@agilent.com Agilent Gene Expression Microarrays Website www.agilent.com/chem/dna Agilent Lab-on-a-Chip Website www.agilent.com/chem/labonachip Information, descriptions and specifications are subject to change without notice. Please register online with Agilent to recieve product updates at: www.agilent.com/chem/dnasupport 5989-3056EN July 20, 2005