Lucidea Universal ScoreCard Plate Format
Contents
1. Control Cy3 Cy5 Expected Raw Normalized Calib 1 1 00 0 49 0 91 Calib 2 1 00 0 54 1 00 Calib 3 1 00 0 63 1 16 Calib 4 1 00 0 60 1 12 Calib 5 1 00 0 50 0 93 Calib 6 1 00 0 68 1 27 Calib 7 1 00 0 63 1 16 Calib 8 1 00 1 06 1 96 Calib 9 1 00 0 88 1 63 Ratio 3 3 00 1 67 3 11 Ratio 4 0 33 0 16 0 30 Ratio 7 10 00 4 90 9 10 Ratio 8 0 10 0 06 0 12 Utility 1 0 20 0 08 0 15 Asia Pacific Tel 852 2811 8693 Fax 852 2811 5251 Australasia Tel 61 2 9899 0999 Fax 61 2 9899 7511 Austria Tel 01 576 0616 22 Fax 01 576 0616 27 Belgium Tel 0800 73 888 Fax 03 272 1637 Canada Tel 1 800 463 5800 Fax 1 800 567 1008 Central East and Southeast Europe Tel 43 1 982 3826 Fax 43 1 985 8327 Denmark Tel 45 16 2400 Fax 45 16 2424 Finland amp Baltics Tel 358 0 9 512 39 40 Fax 358 0 9 512 17 10 France Tel 01 69 35 67 00 Fax 01 69 41 96 77 Germany Tel 0761 4903 291 Fax 0761 4903 405 Italy Tel 02 27322 1 Fax 02 27302 212 Japan Tel 81 3 5331 9336 Fax 81 3 5331 9370 Latin America Tel 55 11 3933 7300 Fax 55 11 3933 7315 Middle East and Africa Tel 30 210 96 00 687 Fax 30 210 96 00 693 Netherlands Tel 0165 580 410 Fax 0165 580 401 Norway Tel 2318 5800 Fax 2318 6800 Portugal Tel 21 417 70 35 Fax 21 417 31 84 Russia amp other C I S amp N I S Tel 7095 232 0250 956 1137 Fax 7095 230 6377 Southeast Asia Tel 60 3 8024 2080 Fax 60 3 8024 2090 Spain Tel 93 594 49 50 Fax 93 594 49 55 Sweden Te2. O Neg 1 Neg 2 63 0050 26 p2 microarrays Table 1 Spotting parameters for Lucidea Array Spotter The spotting parameters are user definable For more information please refer to the Lucidea Array Spotter User s Guide 1 Parameters cDNA Total slides 27 Total plates 8 Spotting mode Normal Replicates 2 Repeats 1 Complete last row Yes Use default pitch No Row pitch 250 um Column pitch 250 um Slide thickness 0 9 1 1 mm Barcode enable Vv Output file enable Vv Humidity control Vv Humidity setting 65 RH Rows per block 8 Columns per block 16 Spots in last row 16 Spotting complete 7 1 At the end of the spotting run a message window will appear stating The spotting session is complete Please remove your sample plates and slides If you click OK the humidifier will be turned off Leave the humidifier on by not pressing OK 7 2 Allow the slides to remain inside Lucidea Array Spotter for 30 60 min depending on the drying rate of the spotting solution used This step allows all the spots on the slides to dry under the same humidity conditions 7 3 Then press OK and unload the plates and slides and remove the pen set as instructed by the Lucidea Array Spotter User s Guide The slides were fixed and hybridized following the slide manu facturer s protocol The slides were manually hybridized with 17 pmol of Cy3 labelled human heart and 17 pmol
3. of Cy5 labelled human spleen cDNA targets Slides were imaged using an GenePix 4000B Array Scanner The slide images were quantitated using ArrayVision 6 0 software Results From Figure 2 the spotting quality of Lucidea Universal ScoreCard controls and Easy to Spot DNA Human 1 0 genes using Lucidea Array Spotter is shown The spots all have 63 0050 26 p3 good morphology and good reproducibility as seen when visually comparing the spot intensities of duplicate spots The nine calibration controls span a range from 3 to 30 000 pg 2 pl of spike The four ratio controls have a targeted Cy3 CyS ratio of 3 1 and 10 1 and the reverse ratio for both The utility control has a targeted Cy3 CyS ratio of 1 5 Fig 2 A section of the microarray image Cy3 labelled cDNA heart shown in green and Cy5 labelled cDNA spleen shown in red target were mixed and hybridized onto a spotted slide with a total of 2688 human genes printed in duplicate Shown here are two duplicate blocks out of a total of 48 blocks of spots on the whole slide Row 1 of each block contains the calibration yellow boxed negative red boxed ratio green boxed and utility blue boxed controls from Lucidea Universal ScoreCard Rows 2 through 7 contain a duplicate set of 112 human genes from Easy to Spot DNA Human 1 0 The calibration controls can determine the dynamic range of the slide The concentrations of the calibration controls used in this experime
4. e the data From Table 2 it can be noted that in general the normalization procedure brings the Cy3 CyS ratio much closer to the expected Cy3 Cy5 ratio than when using the raw signal intensities Calib 8 and Calib 9 have a low mRNA spike concentration of 10 and 3 pg 2 pl of spike respectively As a result Calib 8 and Calib 9 have spot signals close to background which causes high signal variation and skewing of the normalization ratios Conclusions Lucidea Universal ScoreCard is an essential tool for microarray analysis Lucidea Universal ScoreCard controls make it possible to validate normalization and generate calibration curves for microarray data Most importantly having Lucidea Universal ScoreCard controls on microarray slides allows the comparison of data from multiple experiments Together with Lucidea Array Spotter microarray data is generated with excellent accuracy and quality References 1 Lucidea Array Spotter User s Manual Amersham Biosciences pp 375 752 2002 Table 2 Lucidea Universal ScoreCard ratio analysis Cy3 Cy5 Calib stands for calibration Expected ratios are determined from the known mRNA spike concentrations Raw ratios are calculated from the observed Cy3 and Cy5 signal intensities Normalized ratios are calculated from the constant factor normalized Cy3 and Cy5 signal intensities The ratios listed in the table are the average of the Cy3 Cy5 ratios for each control Ratio Analysis Cy3 Cy5
5. f Utility 1 spike to the human heart mRNA labelling reaction Add 1 ul of Utility 1 spike to the human spleen mRNA labelling reaction 1 4 Proceed with direct labelling and purification of the targets using CyScribe First Strand cDNA Labelling Kit 1 5 Quantitate the concentration of the target Lucidea Universal ScoreCard spotting sample preparation Lucidea Universal ScoreCard spotting samples are lyophilized in individual tubes to allow users the flexibility to resuspend the samples in their choice of spotting buffer and customize the format of the spotting plate pattern Amersham Biosciences microarrays 2 1 Resuspend the samples in 600 ul of 50 DMSO to a final concentration of 20 ng ul 50 DMSO is the recommended spotting solution for Lucidea Microarray Reflective Slides For other spotting buffers and slide types the required final concentration of Lucidea Universal ScoreCard spotting samples may be different Spotting plate pattern 3 1 Transfer 25 ul of each sample to a well in the barcoded 384 well microplate following the format shown in Figure 1 3 2 Centrifuge the plate for 1 min at 2750 x g to spin down the samples Spotting parameters 4 1 Create or edit a spotting protocol to the spotting parameters in Table 1 Spotter setup 5 1 Install the Lucidea Spotting Pens pen set Be careful not to damage the pens 5 2 Load the plates for spotting Place the plate containing L
6. l 018 612 1900 Fax 018 612 1910 Switzerland Tel 01 802 81 50 Fax 01 802 81 51 UK Tel 0800 616928 Fax 0800 616927 USA Tel 1 800 526 3593 Fax 1 877 295 8102 Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Biosciences AB SE 751 84 Uppsala Sweden Amersham Biosciences Corp 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA Amersham Biosciences GmbH Munzinger Strasse 9 D 79111 Freiburg Germany Amersham Biosciences SV Corp 928 East Arques Avenue Sunnyvale CA 94085 USA 63 0050 26 p4 Cy CyDye CyScribe Lucidea and ScoreCard are trademarks of the Amersham Biosciences Ltd Amersham and Amersham Biosciences are trademarks of Amersham plc ArrayVision is a trademark of Imaging Research Inc Easy to Spot is a trademark of Incyte Genomics GenePix is a trademark of Axon Instruments Inc Amersham Biosciences Corp 2003 All rights reserved All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group which supplies them A copy of these terms and conditions is available on request www amershambiosciences com Amersham Biosciences
7. lication note microarrays Lucidea Universal ScoreCard Plate Format Lucidea Array Spotter Key words microarray gene expression Lucidea cDNA Gene expression profiling with microarrays is being widely used to compare the expression levels of thousands of genes using a single microarray slide To properly evaluate microarray results it is necessary to minimize the variations in the measurements so that accurate comparisons can be made within an experiment and across multiple experiments One method to minimize variations is the use of control samples to quantitatively relate the experimental data to the control data The control samples also provide a means of validating normalization Normalization is necessary to factor out differences in dye incorporation hybridization efficiency scanner dye sensitivity and other experimental factors Lucidea Universal ScoreCard is a set of 23 microarray controls consisting of calibration ratio negative and utility controls All the controls except the utility controls are artificial genes that generate pre determined signal intensities and ratios that do not change across samples or experiments While the utility controls are also artificial genes their signal intensities and ratios are user definable Utility controls can be used to troubleshoot and estimate sample recovery during the RNA purification process as well All the controls are used for validating and normalizing microa
8. nt span over four orders of magnitude The average signal intensities from the calibration control spots of the experiment show that this slide has a dynamic range of approximately 3 3 5 orders of magnitude as seen in Figure 3 For higher expressing genes the DNA on the spot was hybridized to saturation causing the fluorescence intensity to plateau For lower expressing genes the signal of the spots was not high enough above background to be quantitated accurately Calibration Curve 10 000 000 cys 1000000 Cy3 3 100 000 o Ty s S 10 000 lt 1000 1 10 100 1000 10 000 100 000 Amount of RNA pg Fig 3 Calibration curve from Lucidea Universal ScoreCard calibration controls for the microarray slide The average signal was calculated for each calibration control The signal intensities for Cy3 and Cy5 have been background subtracted Rfu is an abbreviation for relative fluorescent unit and is a measure of fluorescence intensity microarrays The accuracy of the normalization protocol can be determined from Lucidea Universal ScoreCard controls This can be done by comparing the normalized Cy3 CyS ratios against the expected Cy3 CyS ratio of the controls For each CyDye label constant factor normalization was performed on the quantitated data using the average signal from the spots as the constant factor The constant factor is divided into the signal of each spot to normaliz
9. rray data They can also be used to generate a calibration curve for determining limits of detection linear range and the amount of mRNA in each spot Using Lucidea Universal ScoreCard with Lucidea Array Spotter ensures high quality and accurate microarray results This application note describes how Lucidea Universal ScoreCard can be used in association with Lucidea Array Spotter to determine the quality of microarray data Products used Lucidea Array Spotter 63 0040 09 Lucidea Spotting Pens 24 pen set 63 0040 10 Lucidea Universal ScoreCard 1000 hybridizations 63 0042 86 Lucidea Microarray Reflective Slides inquire CyScribe First Strand cDNA Labelling Kit 25 8007 67 384 well barcoded microplate 25 0199 34 63 0050 26 Rev A 2003 02 p1 Other materials required e Easy to Spot DNA Human 1 0 Incyte Genomics e 50 DMSO Protocol The protocol below represents an example for spotting cDNA on Lucidea Microarray Reflective Slides The protocol can be modified by the user to accommodate the needs of the experiment It is recommended to use only 384 well barcoded microplates from Amersham Biosciences for spotting Lucidea Universal ScoreCard target labelling 1 1 Add 2 ul of reference spike mix to the Cy 3 target labelling of human heart mRNA 1 2 Add 2 ul of test spike mix to the Cy5 target labelling of human spleen mRNA 1 3 Create an additional 1 5 ratio control using Utility 1 control Add 1 ul of a 1 5 dilution o
10. ucidea Universal ScoreCard in plate position 1 5 3 Load the slides onto the slide tray 5 4 Make sure that the plates and slides are seated properly in the correct orientation Start spotting 6 1 To start the spotting session click Start in the Lucidea Array Spotter Control window 6 2 If more than 12 plates are being spotted the Change Plates In timer will include when to change the plates drain the waste container and fill the wash solutions and humidifier if necessary Fig 1 Spotting Plate Pattern setup for the Lucidea Universal ScoreCard control plate on the Lucidea Array Spotter The arrangement of the calibration Calib ratio negative Neg and utility controls are shown OOOOCOOOCOOCOOCSSOOO8008800080 OOOO OOOOOO8ODODO0D0DO000000 COCOOCOCOOCOCOCO SOOO O808008 SOOOOOOOOOOOOOOOOCOOCOCOO OOOO OOO OOOO OOOOO8OO8OOO di DDD D DDD DDI DDD L ILILILILIL II I OOOO OOOOOOOOOOOOOOOOOO DQOOODOOODDOODSDOOOSOOOOSOO 01010101000000 D S LIL ILILILILILLL IJ LLALL LLLLILILLLLILLLLLLLL LA Ii 01001000D DIDID D LL LILALLLAL ALA A LLALLA LLLLILLLLLLLLLLLL AA di ODOOCOCCOCOCO0EOOO08OO0888 SOOOOOOO OOOO 1000000000 00O DOOOSCCOOOCCOCESOOOOSOOOOO8 OOOO a a OOOO OOOOCOOOCOCOO oor out ON _ _ Calib 01 calib 02 Calib 03 Calib 04 calib 05 calib 06 Calin 07 Calib 08 Calin 09 O Ratio 3 Ratio 4 Ratio 7 O Ratio 8 Utility 1
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