Using Human Microarray
Contents
1. 4 Using Human Microarray 6 What You Are Going To Do 6 Step 1 Prepare the RNA Sample 7 Step 2 Label the Target 7 Step 3 Pre Hybridize the Microarray 9 Step 4 Complete the Hybridization Protocol 12 Step 5 Wash the Hybridized Microarray 19 Step 6 Scan and Extract Gene Expression Results 20 Step 7 Check Control Probe Data 23 Human Microarray User Manual v3 1 vii Getting Started Please read the introductory information below to help familiarize yourself with Human Microarray before you begin using it Product Contents gt Human Microarray gt Human Microarray Hybridization buffer and tube NOTE Each tube contains buffers sufficient for 5 to 10 microarray hybridization procedures gt Spotted Region Guide gt Human Microarray Product User Guide gt Product Support CD which contains the following gt Sample Images folder gt Human Microarray gal file Gene list and probe sequences for Human Microarray gt List of Human Microarray Control Probes gt Microarray layout of Human Microarray gt Electronic version of the Human Microarray Product User Guide Human Micro2. Do not allow the microarray s to be exposed to air for a significant amount of time otherwise an increased fluorescent background signal could appear D 2 3 4 Submerge the entire labeled target and microarray set up with the cover slide still intact into a large container filled with 42 C 2X SSPE 0 1 SDS solution Carefully remove the cover slide from the glass by gently shaking the glass slide so that the cover slide is freed while the slide is submerged Wash the slide s in the rectangular slide staining dish and slide rack with the excess amount of pre warmed 2X SSPE 0 1 SDS solution for two minutes at 42 C One at a time wash each slide with excess amounts of 0 1X SSPE 0 1 SDS solution for two minutes at room temperature NOTE When working with multiple microarrays keep the arrays you are not currently washing in a 2X SSPE solution while they wait for additional processing 5 6 7 Dip each slide into 0 1X SSPE several times at room temperature and rinse using a squeeze bottle Spin dry with a centrifuge for at least one minute Keep the microarray dry and in the dark until ready to scan Human Microarray User Manual v3 1 19 Using Human Microarray ED Scan and Extract Gene Expression Results There are many scanners available to extract signals from Human Microarray Data extraction using GenePix 4100 from Molecular Devices is described below For instructions for using other co
3. Target Quality Control ITQC Figure 12 Human Microarray User Manual v3 1 40 Using Human Microarray Negative or SPIKE in Controls Ambion ArrayControl RNA SPIKE 1 8 gt Probe ID Numbers AM_c_0000015 to AM_c_0000022 gt Purpose To monitor the specificity of the hybridization To be used with spike in positive controls to determine the sensitivity of the hybridization To be used with spike in controls to trouble shoot the target preparation and labeling reactions gt Sequence information of ArrayControl RNA SPIKE 1 8 from Ambion Inc has been licensed for use with the Human Microarray Using the sequence information eight 60 mer oligonucleotide probes are designed to compliment the respective RNA SPIKES These probes are alien probe sequences that do not cross hybridize with human genomic probes on the Human Microarray and thereby provide a set of negative controls which can be used as spike in positive controls to determine the sensitivity of the hybridization process gt Instructions 1 Convert scanned data into Excel format 2 Sort data by column name ID 3 Copy rows of data with Probe ID numbers above 4 Obtain the average of the 3 6 replicates in the F532 Medium and F635 Medium columns see examples next page Expected Outcome To learn more about the usage of RNA Spikes access Applied Biosystem Ambion s Web site Human Microarray User Manual v3 1 41 Using Human Micro
4. average 32 Human Microarray User Manual v3 1 Using Human Microarray Intrinsic Hybridization Ladder IHL gt Probe ID Numbers PH_c_0000026 to PH_c_0000033 gt Purpose To monitor the overall quality of the sample processing and hybridization efficiency gt The IHL probes are dispensed in serial dilutions of IHC controls The data extracted from IHL after target hybridization can be used to construct a hybridization intensity response curve depending on different dilutions of the probe mixture which will be able to serve as an indicator to monitor the overall consistency of sample processing and hybridization stringency gt Instructions 1 Convert scanned data into Excel format 2 Sort data by column name ID 3 Copy rows of data with Probe ID numbers above 4 Obtain the average of the 3 6 replicates in the F532 Medium and F635 Medium columns see examples next page NOTE Occasionally some spots will be missing or misaligned so the intensity results can be removed for a more accurate average Expected Outcome When the suggested scanner settings that are listed in Step 6 are used saturated or near saturated signal should be observed for all IHC probes Scanner settings should be adjusted so that the signal increases linearly from the lowest concentration IHL to the highest concentration level of IHL Human Microarray User Manual v3 1 33 Using Human Microarray Table 9 below provides
5. c 0000067 ref NM 122927 2 Entrez Gene ID 833497 PH c 0000068 ref NM 001036207 1 Entrez Gene ID 843854 PH c 0000069 ref NM 104167 4 Entrez Gene ID 841722 PH c 0000070 ref NM 129890 3 Entrez Gene ID 818930 PH c 0000071 ref NM 106286 3 Entrez Gene ID 843969 Human Microarray User Manual v3 1 45 Using Human Microarray Figure 14 below provides a plotted and graphed example of the results of technical repeats of the Intrinsic Target Quality Control ITQC using one labeled sample on two arrays Negative Spike in Controls 1 Take the average 2 Plot a graph of the replicates from the data y _ FS35 Mean B635 Mean F532 Mean B532 Mean PPH_e_0000069 544 so 115 80 m F635 Mean m B635 Mean o F532 Mean g B532 Mean a e CI y i p T a CI 9900000 9 Hd 2900000 9 Hd 8900000 9 Hd 6900000 9 Hd 0200000 9 Hd 1200000 9 Hd Figure 14 Example results of Negative Spike in Controls Additional information about the control probes is included on the Product Support CD and on our Web site at www eurogentec com HHH 46 Human Microarray User Manual v3 1 MA Eurogentec Human Whole Genome DNA Microarray User Guide 2006 07
6. is essential to use at least 2 ug of labeled target for each sample 3 Add 1 ul 10x Fragmentation Buffer and incubate at 70 C for 15 minutes 4 Add 1 ul Stop solution and keep in darkness at 20 C Human Microarray User Manual v3 1 13 Using Human Microarray For cDNA Hybridization Follow the steps below to complete the hybridization process using CDNA 1 Follow the instructions provided by the reagent supplier Indirect labeling with NHS ester dye is recommended Table 3 below contains a list of products used to prepare the labeled cDNA target Table 3 cDNA Preparation Products Manufacturer Product Name and Description Ambion Amino Allyl MessageAmp Il cDNA Kit Invitrogen Superscript Indirect Labeling System Stratagene Fairplay Il Microarray Labeling Kit 2 For cDNA labeling it is recommended that 15 ug of total RNA be used as starting material NOTE For all cDNA preparation it is strongly recommended that you re purify the targets using the Qiagen Mini Elute PCR purification column prior to hybridization according to the manufacturer s protocol Incomplete removal of unincorporated dye from the labeling reaction often contributes to increased background noise The Qiagen Mini Elute PCR has been found to provide consistently more reliable results 14 Human Microarray User Manual v3 1 Using Human Microarray Step 4C 3 Complete the Hybridization Using the Glass Cover Slide Method NOTE If you
7. name Table 10 ITOC Gene Number and Name ITQC Gene Gene Name Number C_ITQC2 Lysosomal associated membrane protein 1 C_ITQC3 Calnexin C_ITQC4 Ubiquitin specific protease 11 C_ITQC5 Damage specific DNA binding protein 1 C_ITQC6 Ubiquitin activating enzyme 28 C_ITQC7 Tripartite motif containing 28 C_ITQC8 Actin Binding LIM protein 1 Table 11 below provides ITQC probe locations and ID numbers Table 11 ITQC Probes C_ITQC2 C_ITQC3 C_ITQC4 C_ITQC5 C_ITQC6 C_ITQC7 C_ITQC8 PH_c_0000038 PH_c_0000042 PH_c_0000046 PH_c_0000050 PH_c_0000054 PH_c_0000058 PH_c_0000062 PH_c_0000039 PH_c_0000043 PH_c_0000047 PH_c_0000051 PH_c_0000055 PH_c_0000059 PH_c_0000063 PH_c_0000040 PH_c_0000044 PH_c_0000048 PH_c_0000052 PH_c_0000056 PH_c_0000060 PH_c_0000064 PH c 0000041 PH c 0000045 PH c 0000049 PH c 0000053 PH c 0000057 PH c 0000061 PH c 0000065 Human Microarray User Manual v3 1 37 Using Human Microarray 38 Figure 10 below provides a plotted and graphed example of the results of the Intrinsic Target Quality Control ITOC Intrinsic Target Quality Control ITQC 1 Take the average of the replicates from the data PH_c_0000040 Ka 78 PH_c_0000041 380 477 gt gt gt PH_c 0000042 7157 9197 PH c 0000043 5533 6163 PH c 0000044 3950 5646 PH c 0000045 2688 2952 PH c 0000046 1309 2365 PH ce 0000048 3 s69 2 Plot a graph SA jo i PH c 0000050 1499 1648 PH c 0000051 1849 1778 4 PH_c_00
8. owners and are the sole property of their respective owners CyDye and Cy are trademarks of Amersham Biosciences AlphaScan is a trademark of Alpha Innotech Inc ArrayWoRx Biochip Reader is a registered trademark of Applied Precision Inc GenePix is a trademark of Molecular Devices GeneTAC is a trademark of Genomic Solutions Inc ScanArray 5000 is a trademark of Perkin Elmer Inc mSerries LifterSlip 25x601 M 5439 is a trademark of Erie Scientific Company Amino Allyl MessageAmp II aRNA is a trademark of Ambion ArrayControl is a trademark of Ambion Inc Amino Allyel cDNA Labeling Kit is a trademark of Ambion Superscript Indirect Labeling System is a trademark of Invitrogen Fairplay II Microarray Labeling Kit is a trademark of Strategene Mini Elute PCR purification is a trademark of Qiagen SpotReport is a registered trademark of Strategene Inc Last updated April 2007 2005 2007 Eurogentec All rights reserved vi Human Microarray User Manual v3 1 Table of Contents Notice TO ING US serres memes i Table 0 Contents sitas vii Getting Started tamara 1 Product Contents 0 0 2220 00 00000000000 022220025 1 Other Necessary Apparatus and Reagents Not Supplied 2 Important Notes on Microarray Handling and Storage 3 Product Description and Overview
9. perform hybridization using methods other than the basic glass cover slide method it is recommended that you validate the protocol experimentally For example the MAUI System from BioMicro Systems or HS Series of Hybridization Stations from TECAN offer a higher throughput and more automated hybridization methods To complete this step you will need to select a type of glass cover slide Table 4 below contains a list of glass cover slides that have been tested and confirmed compatible for use with the Human Microarray Buffer Table 4 Compatible Glass Cover Slide Products Manufacturer Product Name BioRad Laboratories SLS 6001 24x60 mm Erie Scientific Company mSeries LifterSlip 25x601 M 5439 1 Ensure your work and experimentation area as well as the Human Microarray are clean before adding the Hybridization Buffer solution to the target array 2 Thaw and pre warm the Hybridization Buffer with formamide at 42 C for ten minutes 3 Prepare the hybridization mix in a 1 5 ml Eppendorf tube according to the Table 5 below Human Microarray User Manual v3 1 15 Using Human Microarray Table 5 Hybridization Mix Measurements For each slide 55 ul Component Final Volume 1 5X Human Microarray Hybridization Buffer 37 ul Sheared Salmon Sperm DNA 10 ug ul 1 yl Target preparation plus nuclease free ddH 0 Up to 17 ul Alternatives to Salmon Sperm DNA Blocking Mixtures Ambion sheared Salmon Sperm DNA 10 j
10. probes provide valuable information to ensure experiments are done correctly resulting in higher guality results for analysis These control probes are explained in detail in the Step 7 Check Control Probe Data section NOTE Additionally detailed control probe information can be found on the Product Support CD that accompanied this product or on our Web site at www eurogentec com Human Microarray User Manual v3 1 5 Using Human Microarray Using Human Microarray This section provides you with detailed information about how to perform the steps necessary to complete the hybridization process to study gene expressions using the Human Microarray What You Are Going To Do Wiese Wu Follow these detailed steps exactly to achieve the best experimentation results gt Step 1 gt Step 2 gt Step 3 gt Step 4 gt Step 5 gt Step 6 gt Step 7 Prepare the RNA Sample Label the Target Pre Hybridize the Microarray Perform the Hybridization Protocol Wash the Hybridized Microarray Scan and Extract Gene Expression Results Check Control Probe Data PROCESS COMPLETE Human Microarray User Manual v3 1 Using Human Microarray Step 1 g Prepare the RNA Sample IMPORTANT High quality intact RNA is essential for all gene expression microarray experiments There are many different RNA isolation protocols and commercially available RNA isolation kits You should choose a solution that meets yo
11. the ID number of the IHL controls and detailed information about their content Table 9 IHL Probe Information Probe ID Probe Name Probe Concentration Units PH_c_0000026 IHL_1 0 39 PH_c_0000027 IHL_2 0 78 PH_c_0000028 IHL_3 1 56 PH_c_0000029 IHL_4 3 13 PH_c_0000030 IHL_5 6 25 PH_c_0000031 IHL_6 12 50 PH_c_0000032 IHL_7 25 00 PH_c_0000033 IHL_8 50 00 PH_c_0000025 IHC 100 00 34 Human Microarray User Manual v3 1 Using Human Microarray Figure 9 below provides a plotted and graphed example of the results of the Intrinsic Hybridization Control and Ladder IHC and IHL Intrinsic Hybridization Control IHC amp Ladder IHL 1 Take the average Table 8 IHL Probe Information of the replicates Probe ID Probe Name Probe Concentration from the data Units PH_c_0000026 IHL 1 PH c 0000027 IHL 2 gt PH_c_0000028 IHL_3 PH_c_0000029 IHL 4 PH c 0000030 IHL 5 PH c 0000031 IHL 6 PH c 0000032 IHL 7 PH c 0000033 IHL 8 PH c 0000025 IHC F635 Medium F532 Medium PH c 0000026 IHL 1 2261 1963 PH c 0000027 IHL 2 4238 3823 PH c 0000028 IHL 3 7812 7465 PH c 0000029 IHL 4 14013 13289 PH c 0000030 IHL 5 24125 22614 PH c 0000031 IHL 6 37162 34060 PH c 0000032 IHL 7 53616 46739 PH c 0000033 IHL 8 59969 54356 PH c 0000025 IHL 57032 v IHC Ladder 2 Plot a graph e F635 Medium m F532 Medium 0501010121 IHL1 IHL 2 IHL 3 IHL 4 IHL 5
12. 00017 PH c 0000017 PH c 0000017 PH c 0000017 0100000 9 Hd 3100000 9 Hd v100000 9 Hd S100000 9 Hd 9100000 9 Hd 100000 9 Hd 1100000 9 Hd 2100000 9 Hd Figure 8 Example Results for Cy5 Intensity Ladder Human Microarray User Manual v3 1 31 Using Human Microarray Positive Controls Intrinsic Hybridization Controls 1 HC gt Probe ID Number PH_c_0000025 gt Purpose To monitor the overall quality of the sample processing and hybridization efficiency gt IHCs contain mixtures of probes from 58 robustly expressed housekeeping genes that serve as built in positive controls The probe mixture is dispensed at a fixed concentration and more than 95 IHC features are evenly distributed across the arrayed area The hybridization signal intensity of IHCs gives the indication of overall quality of sample processing and hybridization efficiency gt Instructions 1 Convert scanned data into Excel format 2 Sort data by column name ID 3 Copy rows of data with Probe ID numbers above 4 Obtain the average of the 3 6 replicates in the F532 Medium and F635 Medium columns see examples next page NOTE Occasionally some spots will be missing or misaligned so the intensity results can be removed for a more accurate
13. 00052 PH A 0000053 HA PH_c_0000056 ED Pe PH_c_0000061 369 407 PH_c_0000062 PH_c_0000063 1580 PH c 0000064 3425 F635 Medium F532 Medium ge ns ge 9600000 9 Hd h 0501010121 8 00000 2 Hd 6 00000 9 Hd 00000 2 Hd v00000 2 Hd 200000 9 Hd 6700000 S Hd B 0500000 9 Hd S00000 9 Hd 36500000 9 Hd S00000 9 Hd E vS00000 9 Hd S900000 9 Hd 0900000 9 Hd 1900000 9 Hd 2900000 9 Hd 900000 9 Hd 900000 2 Hd S900000 9 Hd 000000 S Hd Eb 1 00000 9 Hd jah 200000 9 Hd S 00000 9 Hd 9700000 S Hd 8700000 9 Hd 8500000 9 Hd 6500000 9 Hd 2500000 9 Hd Table 11 ITQC Probes ITQC Gene C_ITQC2 C_ITQC3 C_ITQC4 C_ITQC5 C_ITQC6 C_ITQC7 C_ITQC8 No 300 600 nt PH c 0000038 PH_c_0000042 PH c 0000046 PH c 0000050 PH c 0000054 PH c 0000058 PH_c_0000062 pomi 900 1200 nt PH c 0000039 PH c 0000043 PH c 0000047 PH c 0000051 PH c 0000055 PH c 0000059 PH c 0000063 end 1500 1800 nt PH c 0000040 PH c 0000044 PH c 0000048 PH c 0000052 PH c 0000056 PH c 0000060 PH c 0000064 2100 2400 nt PH c 0000041 PH c 0000045 PH c 0000049 PH c 0000053 PH c 0000057 PH c 0000061 PH c 0000065 Figure 10 Example Results Intrinsic Target Quality Control ITOC Human Microarray User Manual v3 1 Using Human Microarray Figure 11 below provides a plotted and graphed example of t
14. Ambion sheared Salmon Sperm DNA 10 ug ul or e Invitrogen Cot 1 DNA 2 5 10 ug ul or e Invitrogen Poly A 2 5 10 ug ul Important Notes on Microarray Handling and Storage Storage Conditions gt Store Human Microarray product at 4 C gt Store Human Microarray Hybridization Buffer Tube at room temperature NOTE If the product is received with an open bag please contact Eurogentec Customer Service for an immediate replacement Handling Microarrays IMPORTANT x Please read this section carefully and follow the instructions gt Polynucleotide probes are printed on the side of the slide with the barcode gt To avoid irreparable damage of the printing area do not touch the surface with bare hands or with any other objects IMPORTANT ess Open arrays should be used within a week O Human Microarray User Manual v3 1 3 Getting Started Product Description and Overview Human Microarray is made of sense strand polynucleotide probes spotted onto a proprietary chemical layer coated on top of a 1 x 3 25 mm x 75 mm standard format microarray glass slide Updated information of human genome content from public domains is used to design greater than 30 000 highly sensitive 60 mer probes for monitoring the expression level of corresponding human protein coding genes Each probe is spotted onto the array in a highly consistent manner using a proprietary non contact spotting technology adapted fo
15. H c 0000006 PH c 0000006 PH c 0000006 PH c 0000007 PH c 0000007 PH c 0000007 PH c 0000007 PH c 0000007 Cy3 Ladder PH c 0000007 PH c 0000008 PH c 0000008 PH c 0000008 PH c 0000008 PH c 0000008 PH c 0000009 PH c 0000009 PH c 0000009 PH c 0000009 F532 PH c 0000009 Median PH c 0000009 PH c 0000009 2 Plot a graph 2000000 9 Hd 8000000 2 Hd 0501010121 2000000 9 Hd 000000 9 Hd v000000 2 Hd S000000 2 Hd 9000000 2 Hd 6000000 2 Hd Figure 7 Example Results for Cy3 Intensity Ladder 30 Human Microarray User Manual v3 1 Using Human Microarray Cy5 Intensity Ladder C5 IL Il ID 1 Take the average of the replicates PH c 0000010 PH c 0000010 PH c 0000010 Sa PH c 0000010 O F635 Median Pe imo PH c 0000011 PH c 0000011 PH c 0000011 PH c 0000012 mic VI c 0000012 PH c 0000012 PH c 0000012 PH c 0000012 PH c 0000013 PH c 0000013 PH c 0000013 PH c 0000013 2 Plot a graph PH c 0000013 PH c 0000013 PH c 0000014 PH c 0000014 PH c 0000014 PH c 0000014 PH c 0000014 PH c 0000014 PH c 0000015 PH c 0000015 PH c 0000015 PH c 0000015 0501010121 PH c 0000015 Cy5 Ladder PH c 0000015 PH c 0000016 PH c 0000016 PH c 0000016 PH c 0000016 PH c 0000016 PH c 0000016 PH c 0000017 PH c 0000017 PH c 00
16. Human Whole Genome DNA Microarray User Guide MA Eurogentec Notice to the User WE Itis important that users read the entire manual before commencing work Human Microarray User Manual v3 1 Human Microarray User Manual v3 1 Warranty and Liability Eurogentec s products are intended for research use only and not intended for any other uses Human Microarray products are designed and manufactured for research use only Buyers and users agree and understand that they are not granted the right to use Human Microarray products for clinical diagnostic purposes unless they obtain written approval from the appropriate government authority Eurogentec will not be liable for any damages arising from the use of its products in any manner other than their intended use or for the use of its products for clinical diagnostic purposes without written approval from the appropriate government authority The manufacture sale or importation of products from Eurogentec is not permitted without the prior written consent from Eurogentec Buyers and users agree and acknowledge that Eurogentec is the owner and has the copyrights to the probe sequence information of the Human Microarray product and any other Microarray products Eurogentec is founded on the mission to offer researchers high quality and user friendly solutions at an affordable price Your satisfaction in using our products is very important to us Therefore if any of our product
17. IHL 6 IHL 7 IHL 8 IHC Figure 9 Example Results for Cy3 Intensity Ladder Human Microarray User Manual v3 1 35 Using Human Microarray Intrinsic Target Quality Control I TQC gt Probe ID Numbers PH_c_0000038 to PH_c_0000065 gt Purpose To monitor the length of the targets for assessment of the quality of sample integrity and processing gt Four probes per gene were selected from seven consistently expressed housekeeping genes For each gene one probe is designed from regions of 300 600 bp 900 1200 bp 1500 1800 bp and 2100 2400 bp respectively from each 3 end of the transcript gt Instructions 1 Convert scanned data into Excel format 2 Sort data by column name ID 3 Copy rows of data with Probe ID numbers above 4 Obtain the average of the 3 6 replicates in the F532 Medium and F635 Medium columns see examples next page NOTE Occasionally some spots will be missing or misaligned so the intensity results can be removed for a more accurate average Expected Outcome When good quality targets are used signals from all the probes can be observed It is common to observe higher signals from probes designed closer to the 3 end of the transcript 36 Human Microarray User Manual v3 1 Location from 3 end ITQC Gene No 300 600 nt 900 1200 nt 1500 1800 nt 2100 2400 nt Using Human Microarray Table 10 below lists each ITQC gene number and corresponding
18. array Table 12 below lists the ArrayControl RNA SPIKE 1 8 probe ID numbers and their corresponding names Table 12 Ambion ArrayControl RNA SPIKE 1 8 Probe ID Ambion ArrayControl RNA SPIKE ID AM_c_0000015 Array Control RNA SPIKE 1 AM_c_0000016 Array Control RNA SPIKE 2 AM_c_0000017 Array Control RNA SPIKE 3 AM_c_0000018 Array Control RNA SPIKE 4 AM_c_0000019 Array Control RNA SPIKE 5 AM_c_0000020 Array Control RNA SPIKE 6 AM_c_0000021 Array Control RNA SPIKE 7 AM_c_0000022 Array Control RNA SPIKE 8 ArrayControl RNA SPIKES and additional information can be obtained from Ambion Inc at www ambion com 42 Human Microarray User Manual v3 1 Using Human Microarray Figure 13 below provides a plotted and graphed example of the results of the Ambion Negative SPIKE in Controls Ambion Negative SPIKE in Control Take the average JF635Mean B635 Mean F532 Mean B532 Mean of the replicates AM_c_0000015 Y from the data 2 Plota graph y Ambion Negative amp Spike In Controls AM c 0000022 56 121 84 mF635 Mean m B635 Mean o F532 Mean o B532 Mean sl 0501010121 GI00000 9 WV 9100000 9 WV 2100000 9 WV 8100000 9 WV 6100000 9 WV 0400000 9 WY 1200000 9 WY 3200000 9 WV Figure 13 Example results of Ambion Negative Spike in Controls Human Microarray User Manual v3 1 43 Using Human Microarray Negative Control Extrins
19. array User Manual v3 1 1 Getting Started Other Necessary Apparatus and Reagents Not Supplied Apparatus Reagents gt Water bath heating block gt Powder free gloves gt Clean blunt forceps gt Micropipettors gt Sterilized and nuclease free pipet tips gt Sterilized and nuclease free microcentrifuge tubes gt Microcentrifuge gt Vortex mixer gt Clips and forceps gt Hybridization oven gt Hybridization accessories chamber cover slides etc gt Rectangular slide staining dish and slide rack for washing microarrays gt PCR polymerase chain reaction machine gt Microarray scanner for standard 1 x 3 format see Table 7 on page 21 under Human Microarray Scanner Specifications for a list of compatible scanners gt Hybridization systems optional gt Automated hybridization station optional gt De ionized nuclease free water gt Cyanine 3 or 5 labeled amplified aRNA sample or Cyanine 3 or 5 labeled cDNA sample gt Wash solutions four types all necessary e 2X SSPE 0 1 SDS solution e 2X SSPE e 0 1X SSPE 0 1 SDS solution e 0 1X SSPE Human Microarray User Manual v3 1 Getting Started Reagents Continued gt BSA bovine serum albumin or albumin bovine serum NOTE BSA must be molecular tested gt Pre hybridization Buffer e 5X SSPE 0 1 SDS solution e 1 BSA gt Deionized formamide gt Alternatives to Salmon Sperm DNA Blocking Mixtures e
20. ce across the scanned region gt GAM control probe is a Cy3 labeled 60 mer oligonucleotide probe designed from an alien sequence that does not cross hybridize with human targets More than 90 GAM features are evenly distributed across the arrayed area and these features can be excited by a 532 nm laser without hybridization GAM probes can serve as orientation or grid alignment landmarks gt Instructions 1 Convert scanned data into Excel format 2 Sort data by column name ID 3 Copy 90 rows of data with Probe ID number PH_c_0000001 4 Obtain the medium average and standard deviation of the signals in the F532 Medium and F532 Mean columns Expected Outcome When the suggested scanner settings that are listed in Step 6 are used saturated or near saturated signal should be observed for all 90 plus GAM probes Figure 6 below provides and example of scanned Grid Alignment Mark data for the Human Microarray 26 Human Microarray User Manual v3 1 Using Human Microarray Grid Alignment Marks GAM column row OGOD ODO OD 0 0 ODA POD DTO DO OOOO ES GO OO OC TOO OOO NC COCO OC ON OR Ore OO O ONCE OO O UND gt OOO DO O OOOO O OOO DO DOVO O DO OOO O OD O OO OO oO OIGO CASO ON Ov SROTOES ONO PECERA OOTO os CARRO Or OO Or OO deme eo ORC OF Die Orn Gi Bi Se O MNAE SR OOOO 0 O OO O OOOO CA DD ASES ASAS Ne OSA GAM Position Index GAM Position Index Example
21. dry the slides for two minutes It is recommended that you use the slides in the hybridization protocol within one hour of completing the pre hybridization process Figure 1 below provides an illustration of Step 3 the pre hybridization procedure 10 Human Microarray User Manual v3 1 Using Human Microarray Replace the slides in the same location in which they were removed NS 1 Remove the glass slide s then pour in the pre hybridization buffer 2 Replace the slide s in the plastic tube in the same place from which it was removed Try to get them in the correct location the first time Figure 1 Diagram of Round Cap Tube Pre Hybridization Procedure Human Microarray User Manual v3 1 11 Using Human Microarray Step4 Complete the Hybridization Protocol Once you have completed the pre hybridization step using one of the methods outlined in the Step 3 Pre Hybridize the Microarray section you are ready to complete the hybridization protocol There are many different hybridization protocols apparatus and instruments available that may be compatible for use with the Human Microarray Detailed instructions for using the glass cover slide method are described below For best performance and consistent hybridization results it is recommended that you use the Human Microarray Hybridization Buffer included with this product to complete the hybridization process Step 4A 3 Prepare Hybridization Buf
22. fer Solution Using the Human Microarray Buffer Included MEA For correct use of this buffer you must add a specific O amount of formamide and labeled target Please follow the instructions below carefully 1 Spin down the stock Human Microarray Buffer 410 ul in each tube 2 Add 90 ul of deionized formamide 3 Warm the mixture to 42 C to completely dissolve the solution Yield 500 ul of 1 5X Hybridization Buffer solution 4 Aliquot 250 ul of the solution into individual tubes and freeze in darkness at 20 C Each aliquot is sufficient for five hybridizations using the glass cover slide 12 Human Microarray User Manual v3 1 Using Human Microarray Step 4B Prepare Hybridization Hybridization Using Labeled Targets from aRNA or cDNA Labeling Approaches eee Not all RNA labels with the same efficiency for Cy3 and Cy5 For aRNA Hybridization Follow the steps below to complete the hybridization process using aRNA 5 Follow the instructions provided by the reagent supplier Indirect labeling with NHS ester dye is recommended Table 2 below contains a list of products that can be used to prepare the labeled aRNA target Table 2 aRNA Preparation Products Manufacturer Product Name and Description Ambion Amino Allyl MessageAmp II aRNA Kit Ambion aRNA Fragmentation Reagent Mere 2 Mix 2 ug of Cy3 aRNA or 2 ug of Cy5 aRNA samples with 0 nuclease free H20 to yield a final volume of 9 ug NOTE It
23. he results of technical repeats of the Intrinsic Target Quality Control ITQC using one labeled sample on two arrays E F635 Medium F532 Medium 0501010121 PH_c_0000065 PH_c_0000064 PH_c_0000063 PH_c_0000062 PH_c_0000061 PH_c_0000060 PH_c_0000059 PH_c_0000058 mF635 Medium m F532 Medium PH_c_0000065 PH_c_0000064 PH_c_0000063 PH_c_0000062 PH_c_0000057 PH_c_0000056 PH_c_0000055 PH_c_0000054 PH_c_0000061 PH_c_0000060 PH_c_0000053 PH c 0000059 PH_c_0000052 PH_c_0000058 PH_c_0000051 0 PH_c_0000050 PH_c_0000057 PH_c_0000056 PH_c_0000055 PH_c_0000054 PH_c_0000049 PH_c_0000048 PH_c_0000047 PH_c_0000046 PH_c_0000053 PH_c_0000052 PH_c_0000051 PH_c_0000050 MAA al ITQC 0501010120 PH_c_0000045 PH_c_0000044 PH_c_0000043 PH_c_0000042 PH_c_0000049 PH_c_0000048 PH_c_0000047 PH_c_0000046 PH_c_0000041 PH_c_0000040 PH_c_0000039 PH_c_0000045 PH_c_0000038 PH_c_0000044 PH_c_0000043 PH_c_0000042 O O ES S O O T 3 O O D pe E PH_c_0000041 PH_c_0000040 PH_c_0000039 PH_c_0000038 T o Technical Repeats 1 labeled sample on 2 arrays 39 Example results of technical repeats of Intrinsic Human Microarray User Manual v3 1 Target Quality Control ITQC Figure 11 Figure 12 below provides a plotted and graphed example of the result
24. ic Target Quality Control ETQC gt Probe ID Numbers PH_c_0000066 to PH_c_0000071 gt Purpose To monitor the specificity of the hybridization To be used with spike in positive controls to determine the sensitivity of the hybridization To be used with spike in controls to trouble shoot the target preparation and labeling reactions gt ETQC probes are alien probe sequences that do not cross hybridize with human genomic probes on Human Microarray The targets to these probes can be prepared separately as RNA containing a poly A tail and spiked into the experimental RNA sample to monitor target preparation quality and determine the sensitivity of the hybridization reaction Instructions gt Instructions 1 Convert scanned data into Excel format 2 Sort data by column name ID 3 Copy rows of data with Probe ID numbers above 4 Obtain the average of the 3 6 replicates in the F532 Medium and F635 Medium columns see examples next page Expected Outcome These probes emit no or low signal Targets matching to these probes can be obtained see Table 13 below and used in a manner similar to how they are used in the Ambion RNA Spike controls above 44 Human Microarray User Manual v3 1 Using Human Microarray Probe ID Clones ID Reference Table 13 Prob ID Clones ID Reference for ETQC Probes Probe ID Reference PH c 0000066 ref NM 202341 1 Entrez Gene ID 842522 PH
25. ilities 1 x 3 one inch by three inch glass slide Molecular capabilities Able to accurately detect activate and read Cy3 and Cy5 fluorescent molecules Table 7 below contains a partial list of microarray scanner products that are compatible for use with the Human Microarray For more information about the products listed below refer to each respective company s Web site Table 7 Compatible Microarray Scanners Manufacturer Product Name and Description Molecular Devices GenePix 4000 A B Genomic Solutions Inc GeneTAC 2000 Perkin Elmer Inc ScanArray 5000 TECAN LS 200 300 400 Agilent Technology DNA Microarray Scanner G2565B 22 Human Microarray User Manual v3 1 Using Human Microarray Se Check Control Probe Data Human Microarray contains 1 082 built in control probes for performance monitoring of the hybridization process They are used to confirm or deny whether the experiment was completed successfully Details about these probes are explained below Additional information about the control probes is included on the Product Support CD and on our Web site at www eurogentec com Human Microarray Control Probes Orientation Settings and Controls Orientation Grid Alignment Mark OGAM gt Probe ID Number PH_c_0000089 gt Purpose To define the orientation of the scanned image gt OGAM is a Cy3 Cy5 labeled 60 mer oligonucleotide probe designed from an alien sequence that does not cros
26. ire labeled target plus the microarray set up into an closable chambered box that is humidified by 2X SSPE buffer in the 42 C oven for 14 to 16 hours Ensure that the appropriate humidity level is maintained inside the oven during this 14 to 16 hour period Figure 3 below provides an example of this Human Microarray User Manual v3 1 17 Using Human Microarray Figure 3 below provides an illustration of Step 4C completing the hybridization protocol using the glass cover slide method and specifically placing the Human Microarray into the chambered box Place the hybridized microarray slide on top of the filled chambers inside the box and close the box Figure 3 Step 4C gt aRNA or cDNA Hybridization Glass Slide Inside Chamber Box Chambered box The Hinged 100 Place Storage amp Freezer Polypropylene Box from USA Scientific has been used with much success to complete this step The small approximately inch x 1 2 inch chambers within the box are filled about 34 full of buffer then the microarrays are laid on top of the chambers The box is then closed and placed inside the oven For information about this product or other USA Scientific products access their Web site at www usascientific com 18 Human Microarray User Manual v3 1 Using Human Microarray ED Wash the Hybridized Microarray IMPORTANT Washed and dried microarrays should be scanned within a couple of hours NOTE
27. ith the No Cuvettes Spectrophotometer from NanoDrop RNA Sample Amounts Generally the amount needed of quality RNA is 10 20 ug for each labeling reaction If you have an ample supply of RNA samples you have the choice of using a protocol that either amplifies or does not amplify the RNA sample If you have a limited amount of RNA samples it is recommended that you use a protocol that includes a linear amplification of the RNA samples 8 Human Microarray User Manual v3 1 Using Human Microarray Eee Pre Hybridize the Microarray General Instructions Meet Human Microrray requires a pre hybridization step prior to O hybridization of the labeled target The pre hybridization step reduces background signals and increases the performance of the microarray Complete the pre hybridization step by carefully following the instructions below D 2 3 4 5 Pre hybridization solution 5X SSPE 0 1 SDS and 1 BSA Warm the pre hybridization solution to 42 C Carefully and slowly fully submerge the Human Microarray in an abundant amount of pre hybridization solution for two hours at 42 C Transfer the slide s to room temperature distilled water for two minutes Spin dry the slide s for two minutes It is recommended that you use the slides in the hybridization protocol within one hour of completing the pre hybridization process Instructions Using the Round Cap Tube Included Follow the steps be
28. low to complete the pre hybridization process using the 25 ml Round Cap Tube that holds the Human Microarray NOTE While the round cap tube is included with the Human Microarray product the pre hybridization solution itself is not D 2 Pre warm 25 ml of pre hybridization solution 5X SSPE 0 1 SDS and 1 BSA to 42 C Carefully remove the arrays from the round cap tube Be sure to remember the original location and direction of the arrays in the tube see Figure 1 Human Microarray User Manual v3 1 9 Using Human Microarray 3 Pour 25 ml of the pre warmed pre hybridization buffer solution into the same round cap tube 4 Carefully and slowly insert the slides back into the tube in exactly the same location and position as they were originally Wiese Wu Try to insert the slides into the correct position the first time 0 Avoid inserting and removing the slides more than once from the pre hybridization buffer solution 5 After the slides have been inserted properly into the pre hybridization buffer screw the cap onto the tube so that it is tightly sealed invert the tube gently two times and place the tube upright on a stable and flat surface in an oven at 42 C for two hours NOTE It is recommended that the tube be placed on a rack or other stable apparatus to prevent it from accidentally tipping over during this process 6 After two hours transfer the slides to the distilled water for two minutes 7 Spin
29. mpanies products refer to information provided by the company Table 6 below lists the setting for using the GenePix 4100 For a list of scanners that are compatible with the Human Microarray please refer to Table 6 below NOTE The performance of each scanner may differ Therefore to ensure best results it is recommended that the scanner be adjusted based on standard microarray calibration parameters Turn on and warm up the scanner for the duration according to manufacture instructions for the scanner Use the gal file and Gene List provided with this product or refer to our Web site at www eurogentec com Table 6 Scanner Settings Using GenePix 4100 from Molecular Devices Wavelength 635 nm 532 nm PMT 680 V 640 V Minimum diameter 80 Maximum diameter 180 CPI Threshold 100 NOTE For lower versions of GenePix software adjust the property parameter to 142 8 um manually to obtain best results 20 Human Microarray User Manual v3 1 Using Human Microarray Figure 4 below provides a visual example of the Human Microarray glass slide with spotted probe region 80 10 um 142 Bum E gt e 142 Bum Figure 4 Human Microarray Glass Slide with Spotted Probe Region Human Microarray User Manual v3 1 21 Using Human Microarray Human Microarray Scanner Specifications Select and use a microarray scanner that meets the specifications below Microarray Scanner Specifications Format capab
30. nearly from the lowest concentration unit in order to observe the saturation level 28 Human Microarray User Manual v3 1 Table 8 below provides the ID number of the Cy3 amp Cy5 Using Human Microarray Intensity Ladder C3_IL amp C5 IL controls and detailed information about their content C3_IL PH_c_0000002 PH_c_0000003 PH_c_0000004 PH_c_0000005 PH_c_0000006 PH_c_0000007 PH_c_0000008 GAM PH_c_0000009 C5_IL PH_c_0000010 PH_c_0000011 PH_c_0000012 PH_c_0000013 PH_c_0000014 PH_c_0000015 PH_c_0000016 PH_c_0000017 Human Microarray User Manual v3 1 Table 8 C3_IL amp C5_IL Probe Information Probe Concentration Unit 0 39 0 78 1 56 3 125 6 25 12 50 25 00 50 00 29 Using Human Microarray Figures 7 and 8 below provide plotted and graphed examples of the results of Cy3 and Cy5 Intensity Ladder controls Cy3 Intensity Ladder C3 IL ID PH_c_0000002 PH c 0000002 1 Take the average PH c 0000002 of the replicates PH c 0000002 p F532 Median PH c 0000002 gt PH_c_0000002 22413 e E PH_c_0000003 25734 C BE er 0000003 PH_c_0000004 60404 PH_c_0000003 PH c 0000005 65535 PH c 0000003 PH c 0000006 65535 abs PH c 0000007 65535 PH c 0000004 PH c 0000008 65535 PH c 0000004 PH c 0000009 65535 PH c 0000004 PH c 0000004 PH c 0000004 PH c 0000004 PH c 0000005 PH c 0000005 PH c 0000005 PH c 0000005 PH c 0000005 PH c 0000005 PH c 0000006 PH c 0000006 PH c 0000006 P
31. r microarray manufacturing Human Microarray Genome Content Each microarray contains 32 050 oligonucleotides 30 968 human genome probes and 1082 experimental control probes Each oligonucleotide probe is designed to hybridize to a specific target gene described in the current public domain contents such as UniGene Cancer Genome Anatomy Project CGAP BioCarta Kyoto Encyclopedia of Genes and Genomes KEGG and validated by the Human Genome Sequencing Project HGSP Table 1 below provides an example of the contents of a human genome that can be studied using the Human Microarray Human Microarray User Manual v3 1 Getting Started Table 1 Human Genome Content Probe Type Number of Probes UniGene and RefSeq based 30 968 total UniGene build 175 based and or RefSeq based with Entrez 28 703 Gene ID including CGAP Cancer Genome Anatomy Project BioCarts and KEGG Kyoto Encyclopedia of Genes and Genomes pathways UniGene build 163 based with Gene ID and experimentally 2265 selected Human Microarray is guaranteed to print 95 or more of the total probe content NOTE Detailed gene lists gene annotations and probe sequences can be found on the Product Support CD that accompanied this product or on our Web site at www eurogentec com Human Microarray Control Features There are 1 082 control probes built into the Human Microarray that monitor the sample guality and hybridization process These control
32. s F532 Median F532 Mean 65535 59594 Stdev 0 3917 Figure 6 Example of Grid Alignment Marks GAM Human Microarray User Manual v3 1 27 Using Human Microarray Cy3 or Cy5 Intensity Ladder C3_IL or C5 IL gt Probe ID Numbers Cy3 Intensity Ladder PH_c_0000002 to PH_c_0000009 Cy5 Intensity Ladder PH c 0000010 to PH_c_0000017 gt Purpose To assist in adjusting the scanner settings gt Cy3 and Cy5 Intensity Ladder probes are a Cy3 or Cy5 labeled 60 mer oligonucleotide probes designed from an alien sequence that do not cross hybridize with human targets Eight dilutions and three to six replications of each dilution of these features can be excited respectively by a 532 nm laser or a 635 nm laser without hybridization gt Instructions 1 Convert scanned data into Excel format 2 Sort data by column name ID 3 Copy rows of data with Probe ID numbers above 4 Obtain the average of the 3 6 replicates in the F532 Medium and F635 Medium columns NOTE Occasionally some spots will be missing or misaligned so the intensity results can be removed for a more accurate average This is noted in Figure 8 with an asterisk Expected Outcome When the suggested scanner settings that are listed in Step 6 are used saturated or near saturated signal should be observed for probes C3 IL PH_c_0000008 and C5 IL PH c 0000016 Scanner settings should be adjusted so that the signal increases li
33. s hybridize with human targets It is located on top of the upper left corner of the print region and serves as an orientation landmark for template registration as different scanners may have different image orientation output formats Figure 6 below illustrates this Human Microarray User Manual v3 1 23 Using Human Microarray Corner Grid Alignment Marks CGAM gt Probe ID Number PH_c_0000072 gt Purpose To define the corners of the scanned image and to assist with image analysis gt CGAMs are mixtures of Cy3 Cy5 labeled 60 mer oligo designed from an alien sequence that does not cross hybridize with human targets CGAMs are located at the four corners of the image and serve as the orientation landmarks for auto or manual grid alignment under Cy3 532 nm or Cy5 635 nm scanning channel Figure 5 below illustrates this gt Instructions 1 Obtain the average and standard deviation of the results in these categories F532 Medium and F532 Mean Figure 5 below provides an illustration of the OGAM and CGAM positions 24 Human Microarray User Manual v3 1 Using Human Microarray e CGAM OGAM Phalanx Biot wi 0412310923 Figure 5 Human Microarray OGAM and CGAM Positions Human Microarray User Manual v3 1 25 Using Human Microarray Grid Alignment Mark GAM gt Probe ID Number PH_c_0000001 gt Purpose To assist in adjusting the scanner settings To monitor the scanner performan
34. s is not performing to the standard we promised we are willing to replace the product or credit the product purchase price Eurogentec accepts liability of ONLY the purchase price of its products and has no other liabilities Human Microarray User Manual v3 1 iii Contact Information Headquarters Continental Europe Eurogentec s a LIEGE Science Park Rue Bois SaintJean 5 B 4102 Seraing Belgium Ph 32 4 372 74 00 FAX 32 4 372 75 00 E mail info eurogentec com Web site www eurogentec com United Kingdom amp Northern Ireland Eurogentec Ltd P C House 2 South Street Hythe Southampton Hampshire SO45 6EB United Kingdom Ph 44 0 1794 511 411 FAX 44 0 1794 522 417 E mail info eurogentec com Web site www eurogentec com iv Human Microarray User Manual v3 1 User Guide and Technical Support Feedback Electronic version of this manual is available on the enclosed Product Support CD and online at www eurogentec com To reach technical support by telephone call Europe 32 372 76 65 We welcome your feedback regarding our products and this manual Please contact us at feedback eurogentec com All comments are welcome Human Microarray User Manual v3 1 V Trademarks and Copyrights Human Microarray and Microarray are trademarks of Eurogentec in the United States and in other countries All trademarks and copyrights used in this manual belong to their respective
35. s of a dye swap experiment performed using the Intrinsic Target Quality Control ITOC Using Human Microarray F635 Medium m 5532 Medium c 0000065 c 0000064 c 0000063 c 0000062 c 0000061 c 0000060 c 0000059 c 0000058 F635 Medium E F532 Medium PH c 0000065 PH c 0000064 PH c 0000063 PH c 0000062 c 0000057 c 0000056 c 0000055 c 0000054 PH c 0000061 PH c 0000060 PH c 0000059 PH c 0000058 c 0000053 c 0000052 c 0000051 c 0000050 PH c 0000057 PH c 0000056 PH c 0000055 PH c 0000054 c 0000049 c 0000048 c 0000047 c 0000046 PH c 0000053 PH c 0000052 PH c 0000051 PH c 0000050 c 0000045 c 0000044 c 0000043 c 0000042 ITQC 0501010115 U U U VU PH c 0000049 PH c 0000048 PH c 0000047 _c_0000046 c 0000041 c 0000040 c 0000039 c 0000038 EE z Te JE a H Intrinsic Target Quality Control ITQC III UD U U U _c_0000045 c_0000044 _c_0000043 _c_0000042 0 9000 8000 7000 6000 5000 4000 3000 2000 1000 _c_0000041 PH_c_0000040 PH_c_0000039 PH_c_0000038 Mbs ki at 0 9000 8000 7000 6000 5000 3000 2000 1000 4000 Dye Swap Experiment Sample A Cy5 Sample B Cy3 Example results of Dye Swap Experiment using Intrinsic
36. ug ul or Invitrogen Cot 1 DNA 2 5 10 ug l or Invitrogen Poly A 2 5 10 ug ul 4 5 6 7 Spin down the mixture for five minutes at maximum speed to eliminate to potential debris Transfer the mixture to a PCR tube Set a Denature program in the PCR machine as follows 95 C Five minutes 60 C Hold Place the hybridization mixture in the PCR machine and run the Denature program Keep tubes either on the PCR hot plate at 60 C or on a dry bath at 55 C Place the Probe Printed Region Guide included on the table and place the Human Microarray slide with the printed side up on top of the Guide being sure to align it properly with the Guide s markings see Figure 2 16 Human Microarray User Manual v3 1 Probe Printed Region Guide plastic underlay Human Microarray glass slide on top of niaatin MQuida Using Human Microarray Probe Printed Region Guide 50mm 1 96 WW 29 0 a o gt o co m Oo E O 75mm 2 97 s 66 0 ww Figure 2 Human Microarray Glass Slide with Probe Printed Region Guide Plastic Underlay 8 Pipette the hybridization mixture onto the spotted region of Human Microarray being sure to avoid creating any bubbles 9 Carefully place the glass cover slide over the spotted area in an even manner being sure to avoid creating any bubbles 10 Place the ent
37. ur specific needs Stratagene Ambion Invitrogen and other reagent companies offer many different RNA isolation products For more information you can visit each company s Web site Once the RNA samples are isolated you must confirm the quantity and quality of the samples Similarly many different protocols are available and you should choose a solution that is suitable for your needs For faster and more automated RNA analysis you may want to consider the No Cuvettes Spectrophotometer from NanoDrop or the 2100 Bioanalyzer from Agilent Technologies For more information visit each company s Web site ESP Label the Target IMPORTANT O For best results it is recommended that you use one of the commercially available labeling kits that has been tested for use with the Human Microarray please refer to Tables 2 and 3 in the section titled Step 4B Prepare Hybridization Human Microarray User Manual v3 1 7 Using Human Microarray General Guidelines for Target Labeling There are many commercially available labeling kits for microarray analysis Select a labeling kit or labeling method that is most suitable for your specific needs If you use a labeling kit that is not listed in Tables 2 nor 3 in the Step 4B Prepare Hybridization section it is recommended that you validate the method to test and determine its compatibility with the Human Microarray You may want to confirm the quality of the labeled target w
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