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1. slides use the cropping tool to crop out each slide and use Save Region of Interest As to create separate image files for them 6 1 2 If Lucidea Microarray ScoreCard is used for data validation and normalization prepare the image files by cropping the left and right half of the image and use Save Region of Interest As to create individual half image files It is recommended to name the files as SlidelD_left ds SlidelD_right ds The half images are now saved in these folders SlidelD_left DIR SlidelD_right DIR Each folder contains these image files UNSEP1 gel channel 1 UNSEP2 gel channel 2 6 2 Use ArrayVision version 5 1 or higher for image quantitation The images processed with ArrayVision are compatible with Lucidea Microarray ScoreCard For more details refer to the mageQuant Utilities Lucidea Microarray ScoreCard and ArrayVision user manuals Q Data Validation and Normalization For the application described in this note Lucidea Microarray ScoreCard was used for data validation and normalization For more details refer to the Lucidea Microarray ScoreCard user manual 63 0043 07 p4 Results Labelled cDNA from human skeletal muscle and liver were hybridized onto a slide spotted with 4224 human genes in duplicate plus the Lucidea M icroarray Scorecard controls in 24 replicate After washing the hybridized slide was scanned on a Typhoon 9410 The spots were about 200 um in diameter To val2. solution from light 2 2 For each slide clean a cover slip by spraying it with 70 ethanol Use a lint free laboratory wipe to dry the cover slips and then blow them completely dry with compressed nitrogen 2 3 Resuspend the dried labelled cDNAs in 6 ul of RNase Free water Add 7 5 ul 4x Amersham Biosciences microarray hybridization solution 15 yl 100 formamide and 1 5 ul PolyA 1 ug pl The final volume is 30 ul per slide 2 4 Denature the labelled cDNAs at 94 C for 2 min and spin for 30 sec Hybridization Option A with a hybridization cabinet and oven 3 1 Pipette 30 ul of the labelled cDNA solution on an area of the slide that does not contain any spots Place the clean cover slip on the slide Make sure bubbles are not trapped under the cover slip and that all of the arrayed area of the slide is covered with hybridization buffer 3 2 Place the slide in a humid hybridization cabinet and place the cabinet in an incubator at 42 C for 12 18 h Hybridization Option B without a hybridization cabinet and oven If a humid hybridization cabinet and oven is not available an alternative hybridization method is described below 3 1 Perform the hybridization as in Option A 3 1 gel blot and microarray analysis system 3 2 Place the slide in a humid hybridization chamber a slide mailer with 50 ul of RNase Free water in the bottom can serve as an adequate humid hybridization chamber Seal the chamber w
3. to the corresponding abundance level The signals were determined to be linear at least to 1 10 000 pg in relative abundance for both Cy3 and Cy5 detection For higher abundance genes such as 3 or 33000 pg the DNA on the microarray spot was hybridized to saturation causing the fluorescence intensity to plateau Please note that Typhoon has a wide linear dynamic range of 5 orders of magnitude from count 1 to 100000 Typically the range of microarray gene expression levels is limited to 2 5 to 3 5 orders of magnitude To determine the relative gene expression from two samples the ratio of the fluorescence intensities of Cy3 and Cy5 needs to be evaluated and normalized The normalization process of the Lucidea M icroarray ScoreCard software is designed to correct for the difference in Cy3 and Cy5 fluo rescence intensities which are caused by factors other than differential gene expression such as variations in Cy3 and Cy5 dye incorporation during labelling and Cy3 and Cy5 imaging sensitivity Table 3 shows the result of the ratio analysis on the ratio and dynamic range controls before and after normalization In general the normalization procedure brings the Cy3 Cy5 ratio much closer to the expected value than the observed unnormalized value Table 3 Lucidea Microarray ScoreCard ratio analysis Cy3 Cy5 RC and DR stand for ratio control and dynamic range control respectively Control Cy3 Cy5
4. 0 1000 10000 NA 4RC 10 1 10000 1000 NA block 1 block 2 Fig 3 A section of a microarray slide imaged on a Typhoon 9410 Human tissue cDNA from skeletal muscle labelled with Cy3 shown in green and cDNA from liver labelled with Cy5 shown in red were mixed and hybridized onto a spotted slide with 4224 human genes printed in duplicate Shown here are two left and right duplicate blocks out of a total of 24 blocks of spots on the whole slide Row 1 of each block contains the set of Lucidea Microarray ScoreCard controls 32 spots Rows 2 through 12 contain a duplicate set of 352 human genes gel blot and microarray analysis system To evaluate the detection limits and dynamic range of this application six dynamic range control samples were used The sixth dynamic range control 6DR has the least relative abundance of 0 0033 33 pg per ug of sample mRNA Even at this low relative abundance level the signal to noise ratios for Cy3 and Cy5 detection for 6DR were determined to be 5 and 13 respectively demonstrat ing Typhoon s high sensitivity for both Cy3 and Cy5 spot detection Typhoon s limit of detection LOD was deter mined to be 0 002 in relative abundance level 20 pg per ug of sample MRNA for imaging Cy3 and 0 0008 8 pg per ug of sample MRNA for Cy5 The LOD was deter mined by converting the fluorescence intensity level of the detection limit at which the background corrected signal to noise ratio is 3
5. 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 3667 5700 Fax 55 11 3667 87 99 Middle East and Africa Tel 30 1 96 00 687 Fax 30 1 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 7 095 232 0250 956 1137 Fax 7 095 230 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 Tel 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 Cy CyScribe ImageQuant Lucidea ScoreCard and Typhoon are trademarks of Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Biosciences Limited iy ee Amersham and Amersham Biosciences are trademarks of Amersham plc Amersham Biosciences AB SE 751 84 Uppsala Sweden ArrayVision is a trademark of Imaging Research Inc SpeedVac is a trademark of Savant Instruments Inc Amersham Biosciences Corp i Taq DNA polymerase and Hot Tub DNA polymerase are sold under licensing arrange 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA ments with Roche Molecular Systems F Hoffmann LaRoche Ltd and the Perkin Elmer marr Corporation P
6. Expected Observed Normalized 1RC 1 3 0 33 0 73 0 31 2RC 3 1 3 7 8 3 2 3RC 1 10 0 1 0 28 0 12 4RC 10 1 10 21 8 6 1DR 1 1 1 2 2 0 97 2DR 1 1 1 2 2 0 96 3DR 1 1 1 2 5 1 0 4DR 1 1 1 2 6 1 1 5DR 1 1 al 2 6 0 95 6DR 1 1 1 3 0 1 1 Conclusion The 10 um pixel size option on Typhoon allows high resolution scanning suitable for microarray applications Typhoon offers high sensitivity for successful detection of genes at very low abundance levels The wide range of excitation sources and emission filters are suitable for multicolor microarray imaging Typhoon also provides flexibility for imaging a variety of fluorescent labels in addition to Cy3 and Cy5 The Typhoon microarray images can be analyzed with spot finding software such as ArrayVision References 1 Brown P O and Botstein D Nat Genet 21 1 Suppl 33 7 1999 2 Fluorescence maging principles and methods Amer sham Biosciences code number 63 0035 28 2000 gel blot and microarray analysis system 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
7. Qiication note gel blot and microarray analysis system Fluorescent microarray imaging and analysis Typhoon Variable Mode Imager Key words microarray gene expression fluorescence imaging Typhoon CyDyes DNA microarray technology is a powerful tool to investi gate global changes in gene expression of cells and tissues 1 In expression profiling experiments using cDN A microarrays thousands of discrete DN A sequences are robotically spotted onto a glass microarray slide and subse quently hybridized to fluorescently labelled cDNAs Differ ential expression profiling is often used to compare the gene expression patterns of two different samples such as diseased versus normal or drug treated versus control This is usually done by labelling the cDN As from two individual samples with two different fluorescent dyes such as Cy 3 and Cy5 followed by hybridizing the labelled cDN As onto the microarray slides After hybridization the slide can be imaged by fluorescence detection The normalized ratio of the fluorescence intensities of the two dyes is then calcu lated and used to determine the relative gene expression from the two samples for each spot The Typhoon Variable M ode Imager is a well proven instrument providing high sensitivity and wide linear dynamic range five orders of magnitude for multicolor fluorescence imaging of gels and blots 2 This applica tion note introduces a new feature on Typhoon the microarra
8. ation v Post hybridization washes x Scan slide on Typhoon imager Data Acquisition Image processing with ArrayVision v Use Lucidea Microarray ScoreCard to normalize and evaluate data y Obtain Lucidea Microarray ScoreCard quality report Data Processing Obtain normalized data output Fig 1 The overall workflow of a microarray application Protocol An example of a microarray application and the preparation protocol are described in this application note Figure 1 shows the overall workflow for preparation data acquisition and data analysis for this application The key products available from Amersham Biosciences used in this process are highlighted Typhoon will also image microarray slides prepared with alterna tive protocols and reagents Please refer to the manufacturer s instructions for experimental details For this application the microarray slides used were robotically spotted with a Generation III Array Spotter from Amersham Biosciences Lucidea Microarray ScoreCard Kit v 1 1 Lucidea Microarray ScoreCard reagents and software were used for data validation and normalization The kit is compatible with human microarray slides spotted with a Generation III Array Spotter A set of 4224 human genes and Lucidea Microarray ScoreCard control reagents were spotted in duplicate on a Corning CMT GAPS microarray slide For non human and human microarra
9. idate the microarray experiment it is important that the variations in the measurements are evaluated so that accurate comparisons can be made within an experi ment and across multiple experiments This is a common requirement with any quantitative microarray application In this application note data validation and normalization is accomplished using the Lucidea M icroarray ScoreCard controls and software The Lucidea M icroarray Score Card controls include negative dynamic range and ratio controls for Cy3 and Cy5 N egative controls are used to evaluate the degree of nonspecific hybridization and provide the detection threshold value Dynamic range controls are mainly used to estimate detection limits and linear range Ratio controls provide a mechanism for verifying the accuracy of calculated gene expression ratios Table 2 illustrates the concentrations and relative abun dance in the labelled sample of the dynamic range control and ratio control elements Table 2 The relative concentration and abundance of Lucidea Microarray ScoreCard dynamic range and ratio controls RC and DR stand for ratio control and dynamic range control respectively Control Cy3 Cy5 Conc in mix pg 5pl mix Relative sample ratio Cy3 Cy5 abundance 1DR Tit 33000 33000 3 30 2DR 1 1 10000 10000 1 3DR 1 1 1000 1000 0 10 4DR 1 1 330 330 0 03 5DR 1 1 100 100 0 01 6DR 1 1 33 33 0 003 1RC 1 3 1000 3000 NA 2RC 3 1 3000 1000 NA 3RC 1 1
10. ith parafilm 3 3 Incubate the slide still in the sealed hybridization chamber for 12 18 hina 42 C water bath Protect the chamber from light 4 Post hybridization washes 4 1 Preheat a solution of 2x SSC 0 1 SDS Buffer 1 anda solution of 1x SSC 0 1 SDS Buffer 2 to 55 C 4 2 Immerse the slide in Buffer 1 at 55 C and remove the cover slip Transfer the slide to a cradle in a staining jar containing Buffer 1 at 55 C Wash the slides in 55 C Buffer 1 and then 55 C Buffer 2 for 5 min each with gentle rocking at room temperature Cover the staining jar with aluminum foil to prevent photobleaching of the dyes 4 3 Fill three staining jars with 0 1x SSC at room temperature and dip the cradle into these three jars for 5 sec each Do not allow the slides to dry between washes 4 4 Use a low speed centrifuge that accommodates microarray slides to spin dry the slides Place a dry paper towel on the plate holder to absorb the excess liquid and then place the cradle with washed slides on the plate holder Balance the rotor and spin for 1 min at lt 500 g Imaging the microarray slide For complete instructions on how to image microarray slides on Typhoon please refer to the Microarray Slide Holder Kit Instruc tions and Typhoon Instrument Guide 5 1 Positioning the slide holder and microarray slide s 5 1 1 Carefully clean the back of the slides to remove any liquid marks finger prints and dust pa
11. n filter settings If imaging Cy3 and Cy5 choose the settings as shown in Table 1 Select Normal for Sensitivity Set an appropriate PMT voltage setting the recommended range is between 450 800 V For quantitative results select Sensitivity mode to perform single channel scans Top view i Back of the Typhoon instrument Coordinates of this grid square J2 Coordinates of this grid square A2 Front of the Typhoon instrument Fig 2 Positioning the microarray slide holder on the glass platen gel blot and microarray analysis system Table 1 Typhoon Scanner Control settings for Cy3 and Cy5 detection The excitation and emission maxima of Cy3 and Cy5 are shown in parentheses along with their appropriate laser and emission filter selections Fluorochrome Ex Em Laser Emission Filter Cy3 550 nm 570 nm Green 532 nm 580 BP 30 Cy5 649 nm 670 nm Red 633 nm 670 BP 30 O Image Processing The following procedures are recommended for image processing 6 1 Use ImageQuant Tools Utility 2 2 IQ Tools to open and process the Typhoon microarray images After the images are opened make sure the image orientation is appropriate for analysis using either the bar code or control spots on the slide as landmarks If necessary change the image orienta tion using the rotating or flipping tools in 1Q Tools 6 1 1 If the image contains two
12. rticles Clean the Typhoon platen and microarray slide holder 5 1 2 Gently position the Typhoon microarray slide holder at the front left corner of the clean glass platen Fig 2 5 1 3 Gently place the two slides into the wells of the slide holder with the spotted side face down If scanning only one slide position the slide in the first well closest to A2 to minimize the scan time 5 1 4 Make sure the slides are seated in the grooves of the slide holder and the slide holder is seated firmly against the front left corner of the platen area The correct positions of the slides and the slide holder keep the slides at the appro priate angle and location for proper scanning 5 1 5 Correctly position a slide restraint over each slide an 63 0043 07 p3 5 2 Setting up Typhoon Scanner Control 5 2 1 In the Typhoon Scanner Control window select the following parameters Scan area A2 through D2 inclusive for one slide A2 through J 2 inclusive for two slides Acquisition mode Fluorescence Orientation A Press samplet On box is checked Pixel size 10 um Focal plane 3 mm If the workstation has difficulty performing a scan with this orientation try to perform a scan with orientation and change the image orientation during the image processing step 6 1 The sample is pressed to prevent movement during a scan 5 2 2 In the Fluorescence Setup window select the appro priate laser emissio
13. urchase of these products is accompanied by a limited license to use it Amersham Biosciences Europe GmbH in the Polymerase Chain Reaction PCR process for research in conjunction with a Munzinger Strasse 9 D 79111 Freiburg Germany thermal cycler whose use in the automated performance of the PCR process is covered Amersham Biosciences SV Corp by the up front license fee either by payment to Perkin Elmer or as purchased ie an authorized thermal cycler 928 East Arques Avenue Sunnyvale CA 94085 USA HR d Amersham Biosciences Corp 2002 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 that supplies them A copy of these terms and conditions is available on request Printed in the USA www amershambiosciences com Amersham e Biosciences
14. y imaging capability which is available on the Typhoon 9210 and 9410 models The Typhoon 9210 is equipped with green 532 nm and red 633 nm lasers The Typhoon 9410 has an additional blue laser with two laser lines 457 nm and 488 nm See licensing information 63 0043 07 Rev AA 01 02 pl Products used Typhoon 9210 63 0038 51 Typhoon 9410 63 0038 55 ArrayVision 63 0008 18 Cy3 dCTP PA53021 Cy5 dCTP PA55021 CyScribe First Strand cDNA Labelling Kit RPN6200 HEPES US16926 Humid hybridization cabinet for microarrays RPKO176 Hybridization oven shaker RPN2511 Lucidea Microarray ScoreCard Kit v 1 1 RPK1161 Lucidea Universal ScoreCard 63 0042 85 Microarray hybridization solution RPKO325 RNase Free water US70783 Sodium dodecyl sulphate SDS 17 1313 01 SSC 20x US19629 Other materials required e CMT GAPS microarray slide Corning Compressed nitrogen e Human liver and skeletal muscle mRNA Clontech e Microarray cover slips e QlAquick PCR Purification Kit Qiagen e SpeedVac centrifuge e UV vis Spectrophotometer e Water bath Amersham e Biosciences gel blot and microarray analysis system mRNA sample 1 mRNA sample 2 Spotting materials CyScribe Labeling Kit Generation III Array Spotter Lucidea Microarray ScoreCard Preparation Cy3 cDNA probe Cy5 cDNA probe Microarray Hybridiz
15. y slides prepared by other types of array spotters Lucidea Universal ScoreCard reagents are recommended as universal references for validating and normal izing microarray data The product only contains reagents and is independent of experimental platform and data analysis software The Lucidea Universal ScoreCard reagents display no cross hybridization over a wide range of biological species 63 0043 07 p2 For more details about the Lucidea Microarray ScoreCard reagents and software as well as Lucidea Universal ScoreCard reagents refer to the Lucidea Microarray ScoreCard and Lucidea Universal ScoreCard data files and user manuals O cDNA labelling For this application CyScribe First Strand cDNA Labelling CyScribe Kit was used for cDNA labelling 1 1 Add Lucidea Microarray ScoreCard spike mix to 1 yg of the human skeletal muscle MRNA and 1 yg of the human liver mRNA samples 1 2 Use the CyScribe Kit to label the cDNAs according to the kit instructions Cy3 and Cy5 dCTPs are used to label skeletal muscle and liver cDNAs respectively 1 3 Purify the Cy3 and Cy5 labelled cDNAs using the QIAQuick PCR Purification Kit following the manufacturer s protocol Hybridization preparation 2 1 For each slide combine 30 pmol concentration measured on a spectrophotometer of Cy3 and 30 pmol of Cy5 labelled cDNA into one tube and dry them down in a Speed Vac or other centrifugal vacuum concentrator Protect the
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