GEArrayTM Q and S Series KITS
Contents
1. CDP Star is a registered trademark of Applera Corporation or its subsidiaries in the US and certain other countries2. B Hybridization Note To prevent leakage always tighten the caps of both your hybridization cylinders that come with your hybridization oven and our hybridization tubes containing the arrays Note An O ring is provided with the hybridization tube and should be placed around the end of the tube opposite the cap The O ring allows the tube to sit level inside your hybridization cylinder and insures that all volumes of solutions cover the membrane evenly 1 Pre hybridization Note This step can be performed during Probe Synthesis A p 9 11 OR 13 Before starting your experiment always record in your notebook the array bar code number which contains the array lot number Pre wet the array membrane by adding roughly 5 ml of deionized water to the hybridization tube Allow tube to sit inverted while preparing the GEAprehyb Warm the GEAhyb Hybridization Solution to 60 C and invert the bottle several times to allow complete dissolution of the buffer components Heat the sheared salmon sperm DNA at 100 C for 5 min and immediately chill on ice Prepare GEAprehyb Add the heat denatured salmon sperm DNA to the pre warmed GEAhyb Hybridization Solution to a final concentration of 100 ug ml You will need 3 ml of GEAprehyb for each array Keep the GEAprehyb solution at 60 C until needed Discard the deionized water from the hybridization tube Add 2 ml of the GEAprehyb solution and vortex the tube gently for a few seconds Be sure the
3. cap of the tube is screwed on hand tight Place the tube inside your hybridization cylinder Two GEArray Q or S Series hybridization tubes will fit inside a standard hybridization cylinder ID x L 35 x 150 mm Pre hybridize in your hybridization oven at 60 C for 1 to 2 hours with continuous agitation at 5 to 10 rpm 2 Hybridization Prepare GEAhyb Add the entire volume of denatured cDNA probe to 0 75 ml of pre warmed GEAprehyb Mix well and keep the GEAhyb at 60 C Discard the GEAprehyb from the hybridization tube Add the GEAhyb containing probe to the hybridization tube Hybridize overnight at 60 C with continuous agitation at 5 to 10 rpm GEArray Q and S Series User Manual 15 3 Washing Prepare excess Wash Solution 1 2X SSC 1 SDS 100 ml 20X SSC and 50 ml 20 SDS per liter Wash Solution 2 0 1X SSC 0 5 SDS 5 ml 20X SSC and 25 ml 20 SDS per liter You will need at least 10 ml of each per hybridization tube Warm both to 60 C Pour the GEAhyb solution from the hybridization tube into a clean micro centrifuge tube Store at 4 C until the end of the experiment in case another array needs to be probed Wash the membrane twice with 5 ml Wash Solution 1 at 60 C with 20 to 30 rpm agitation for 15 minutes each Vortex the tube gently with each wash Wash the membrane twice with 5 ml Wash Solution 2 at 60 C with 20 to 30 rpm agitation for 15 minutes each Vortex the tube gently with each w
4. on a sheet of plastic wrap and drop the 1 0 ml of CDP Star solution onto the membrane Note It is very important to have the membrane covered evenly with the substrate Blot the membrane on a piece of filter paper to remove excess CDP Star Solution Do not let the membrane completely dry out The membrane should be saturated and translucent without any solution dripping from it Place the membrane into a plastic sheet protector or into a small plastic zip lock bag and smooth out any bubbles GEArray Q and S Series User Manual 17 D Image and Data Acquisition and Analysis 1 Image Acquisition Note Remember the blank side of the array should face your film or camera Orient the array so that the bar code on the reverse side of the membrane is on the right and the name of the array on the reverse side is on the top as you face it See Figure 3 Expose the membrane to X ray film OR use a CCD camera and imaging station to acquire the image We recommend obtaining multiple exposures for various times Start with an initial exposure of 1 2 min on film and 5 10 min using a digital imaging system The visualization of high abundance messages will require shorter exposures lower abundance messages longer exposures If your CCD camera imaging software allows you visualize when the signals are saturated obtain the longest exposure without saturating any of the signals The CDP Star substrate reaches peak light emission between 2 4 hr and pe
5. A preparation should yield two sharp bands representing the 28S and the 18S ribosomal RNA rRNA The intensity of the 28S slower mobility band should also be roughly twice that of the 18S greater mobility band A smearing of either two bands or a decrease in the intensity ratio indicates RNA degradation Sometimes even intact RNA does not guarantee good array results because RNA samples may also be contaminated with enzyme inhibitors that can reduce labeling efficiency We recommend that first time users of our array kits check their probe labeling before hybridization See the Troubleshooting Guide Notes Description Of Membrane Appearance Each GEArray Q and S Series nylon membrane array is supplied in a disposable hybridization tube One side of the membrane is printed with the cDNAs in an 8x14 grid of tetra spots Q Series or a 16x up to 30 grid of single spots S Series visible initially as blue dots These dots will fade by the end of the procedure and this side of the membrane will then appear blank This side of the membrane should always face the inside of the hybridization tube and should face your film or CCD camera during image acquisition On the reverse side of the membrane every array has a unique barcode number to allow tracking of the array during the course of your experiment Before starting your experiment always record in your notebook the array bar code number which contains the array lot number Array side Arrayed
6. ARRAY TO MANUFACTURE COMMERCIAL PRODUCTS WITHOUT WRITTEN APPROVAL OF SUPERARRAY BIOSCIENCE CORPORATION NO OTHER LICENSE EXPRESSED IMPLIED OR BY ESTOPPEL IS GRANTED U S PATENTS MAY COVER CERTAIN ISOLATED DNA SEQUENCES INCLUDED ON THE GEARRAY PRESENTLY IT IS NOT CLEAR UNDER U S LAWS WHETHER COMMERCIAL USERS MUST OBTAIN LICENSES FROM THE OWNERS OF THE RIGHTS TO THESE U S PATENTS BEFORE USING GEARRAY GEArray Q and S Series User Manual 3 I Introduction The advancement of nucleic acid array technology has made it possible to analyze the expression of multiple genes in a single experiment However most commercial gene expression array products designed to include as many genes as possible produce vast amounts of data that is overwhelming and difficult to interpret In addition the cost of detection equipment and bioinformatics software is often prohibitive Based on our popular first generation of pathway specific gene expression arrays the GEArray Q and S Series provide additional features that allow researchers to characterize gene expression associated with a specific biological pathway in a more comprehensive and cost effective manner The GEArray Q and S Series are a result of combining superior array design with state of the art arraying and detection technologies They are easy to use sensitive economical and accessible for routine use in every research laboratory The GEArray Q Series gene expression array contai
7. J SuperArray Bioscience Corporation GEArray Q and S Series KITS PATHWAY SPECIFIC GENE EXPRESSION PROFILING SYSTEMS USER MANUAL For chemiluminescent detection ORDERING INFORMATION AND TECHNICAL SERVICE e TEL 1 888 503 3187 301 682 9200 e FAX 1 888 465 9859 301 682 7300 e ON LINE ORDER www superarray com e E MAIL order superarray net customer superarray net support superarray net Orders may be placed by fax e mail or from our website Each order should include the following information Caller s contact information Product name catalog number and quantity Purchase order number or credit card information Visa or MasterCard Shipping address Billing address For more information visit us at http www superarray com SuperArray Bioscience Corp 7320 Executive Way Suite 101 Frederick MD 21704 USA FOR RESEARCH USE ONLY Version 7 4 10 17 2003 GEArray Q and S Series User Manual 2 CONTENTS I Introduction 3 II Materials Provided 5 IHI Additional Materials Required 6 IV GEArray Q and S Series Assay Protocol 7 A Probe Synthesis THREE OPTIONS Option 1 RT Labeling Kit Protocol 8 Option 2 TrueLabeling RT Kit Protocol 10 Option 3 AmpoLabeling LPR Kit Protocol 12 B Hybridization 14 C Chemiluminescent Detection 16 D Image and Data Analysis 17 V Troubleshooting Guide 18 LIMITED PRODUCT WARRANTY FOR GEARRAY PRODUCTS This product is intended for research purposes only and is no
8. Option 2 Catalog Number L 02 TrueLabeling RT Kit Reduces high background and false positive signals Available to US Customers and Customers Outside the USA with a local distributor Option 3 Catalog Number L 03 AmpoLabeling LPR Kit Reduces high background and false positive signals Detects lower abundance messages Provides the most accurate representation of your RNA in gene expression profiles Available to US Customers and Customers Outside the USA with a local distributor Customers Outside the USA without a local distributor in a modified form Alternative Option Catalog Number L 04 RT Labeling Buffers Used with RNase Inhibitor and MMLV Reverse Transcriptase purchased from other suppliers Follows same protocol as Option 1 the RT Labeling Kit Available to US Customers and Customers Outside the USA with a local distributor Customers Outside the USA without a local distributor in a modified form C Miscellaneous Molecular Biology Reagents and Equipment The following reagents are required but are not supplied by SuperArray 1 cDNA Probe Synthesis Biotin 16 dUTP Roche Cat No 1 093 070 2 Hybridization and Washing Sheared Salmon Sperm DNA Invitrogen Life Technologies Cat No 15632 011 20X SSC Dissolve 175 3 g NaCl and 88 2 g sodium citrate dihydrate into 900 ml H20 Adjust pH to 7 0 with 1M HCl Dilute to 1 L with H20 Store at room temperature 20 SDS Dissolve 200 g sodium dodecyl sulfate in 1 L H20 Heat
9. ach array transfer 10 ul of the pre warmed RT Cocktail to the 10 ul Annealing Mixture Mix well but gently with a pipettor Continue incubation at 42 C for 90 min Stop the RT Reaction by adding 2 ul of Buffer C Note We highly recommend checking the probe for dUTP incorporation at this point before denaturing the probe and setting up the hybridization See the Troubleshooting Guide at the end of the Manual V 3 for details d Denature the probe Add 2 ul of Buffer D to each RT Reaction now containing labeled cDNA probe Incubate at 68 C for 20 min Add 25 ul of Buffer E and continue incubation at 68 C for another 10 min Note Buffer E may precipitate when thawed at room temperature We recommend warming Buffer E to 68 C and vortexing before use Alternatively the cDNA probe can be denatured by heating at 94 C for 5 min and quickly chilling on ice Note It is not necessary to remove unincorporated biotin 16 dUTP from the cDNA probe The total labeling reaction mixture can be used for hybridization directly The RT Labeling probe synthesis protocol is now complete The cDNA probe is now ready for hybridization B Please proceed to page 15 GEArray Q and S Series User Manual 10 OPTION 2 of 3 for Probe Synthesis TrueLabeling RT Kit Protocol Catalog Number L 02 Synthesis of cDNA probes with Biotin 16 dUTP Roche Cat No 1 093 070 Note If you plan to perform your hybridization immediately after probe syn
10. all amount of plasmid RNA may still be converted to cDNA and labeled by non specific priming in the RT reaction Therefore it is possible to get hybridization signals on pUC18 spots E The MMLYV reverse transcriptase from Promega is 200 Units ul Do I need to dilute it to 50 Units pl before use No Using 5 to 10 fold more reverse transcriptase during Probe Synthesis will not impair your results However the concentration of the reverse transcriptase should not be below 50 Units l F Can I use a reverse transcriptase from another vendor Yes the GEArray labeling buffer works well with reverse transcriptases from many other sources Make sure that the concentration of your reverse transcriptase is 50 Units l or higher G How do I prepare buffer BN International customers must convert the 10X RT Buffer included in this kit to Buffer BN If the Buffer BN included with the Probe Synthesis Kits is stored improperly domestic customers will need to obtain from us either Buffer BN or 10X RT Buffer in order to regenerate Buffer BN Use the following recipe For every 50 ul 10X RT Buffer add 1 ul of 1M DTT and 50 ul of 10X dNTP mix 5 mM dATP 5 mM dCTP 5 mM dGTP and 500 uM dTTP Mix well Store the buffer BN at 20 C If you have additional questions please check our website www superarray com for a more complete listing of Frequently Asked Questions FAQs or call our technical support representatives at 1 888 503 3187 or 301 682 9200
11. ash GEArray Q and S Series User Manual 16 C Chemiluminescent Detection Note All of the following detection steps are performed at room temperature Note GEAblocking Solution Q and 5X Buffer F may cloud during storage at 4 C Warm the solutions to 37 C and invert the bottles several times to allow any precipitate to completely dissolve Allow the solutions to sit at room temperature until needed 1 Blocking the Array After discarding the last wash immediately add 2 ml GEAblocking Solution Q Incubate for 40 min with continuous agitation at 20 to 30 rpm Binding of alkaline phosphatase conjugated streptavidin AP Prepare Binding Buffer Dilute 5X Buffer F five fold to prepare excess 1X Buffer F Dilute AP 1 7 500 into 1X Buffer F to obtain your binding buffer We suggest dispensing volumes of AP no smaller than 2 ul You will also need 16 ml of 1X Buffer F per tube for washing 3 Discard the GEAblocking Solution Q from the tube Add 2 ml Binding Buffer and incubate for 10 min with continuous but gentle 5 10 rpm agitation Washing Wash the membrane four times with 4 ml 1X Buffer F for 5 min with gentle agitation Vortex the tube gently after each addition of fresh 1X Buffer F Rinse or wash twice with 3 ml Buffer G Detection Add 1 0 ml CDP Star chemiluminescent substrate to the hybridization tube Incubate at room temperature for 2 to 5 min Alternatively place the GEArray Q or S Series membrane
12. following recipe Combine 50 uL of 10X RT Buffer 1 uL of 1 M DTT and 50 uL of dNTP mix 5 mM each dATP dCTP and dGTP and 0 5 mM dTTP Mix well Store at 20 C Note There is no need to treat total RNA with DNase a Prepare the Annealing Mixture For each total RNA sample combine the following in a sterile PCR tube Total RNA 1 0 5 0 ug Buffer A CLEAR tube 3 ul RNase free H2O Adjust the final volume to 10 ul Note Buffer A is unique for each GEArray Q and S Series kit Please do not substitute Buffer A from other GEArray Kits Check the Catalog Number on the tube before use Also please do not use any source of Buffer AF in the RT Labeling Kit Protocol Mix the contents well but gently with a pipettor followed by brief centrifugation Place the mixture in a pre heated heat block or thermal cycler at 70 C for 3 min Cool to 42 C and keep at that temperature for 2 min Note A pre programmed PCR thermal cycler is recommended GEArray Q and S Series User Manual 9 b Prepare the RT Cocktail This mixture can be prepared while the Annealing Mixture is incubating at 70 C RT Cocktail 1 array 2 arrays 4 arrays Buffer BN 4 ul Sul 16ul Biotin 16 dUTP 2u 4l 8 ul RNase free H2O 2 ul 4 ul 8 ul RNase Inhibitor RI 1 ul 2 ul 4 ul Reverse Transcriptase RE Tul 2p 4ul Warm the RT Cocktail at 42 C for 1 min before proceeding to the next step c RT Reaction For e
13. for the AmpoLabeling LPR Kit Protocol NOTE fusing another kit to amplify your RNA be sure to obtain instructions for generating labeled probe from our website www superatray com answer php ID R3 e Linear Polymerase Reaction LPR For each array add 30 ul of the LPR Cocktail to each RT Reaction and mix well but gently with a pipettor Program the thermal cycler for LPR as follows 85 C 5 min 30 cycles of 85 C 1 min 50 C 1 min 72 C 1 min then 72 C 5 min The cycle number can be reduced when using larger amounts of total RNA and or when specifically interested in more abundant messages We do not recommend increasing the number of cycles f Immediately stop LPR by adding 5 ul of Buffer C and chilling on ice Note We highly recommend checking the probe for dUTP incorporation at this point before denaturing the probe and setting up the hybridization See the Troubleshooting Guide at the end of the Manual V 3 for details g Denature the probe Denature the labeled cDNA probe by heating the tube containing the LPR at 94 C for 2 min and quickly chilling on ice Note It is not necessary to remove unincorporated biotin 16 dUTP from the cDNA probe The total labeling reaction mixture can be used for hybridization directly The AmpoLabeling LPR probe synthesis protocol is now complete The cDNA probe is now ready for hybridization B Please proceed to page 15 GEArray Q and S Series User Manual 14
14. ing buffer for Original and Q Series Kits are interchangeable 4 Improper washing in 0 1x SSC solution Excessive washing of the membrane in high stringency buffer 0 1x SSC solution may strip off the hybridized probes Washing time should not exceed 15 minutes 5 Loss of labeled probe during column purification Column purification can result in a low yield of labeled probe We recommend adding the complete Probe Synthesis reaction directly into the hybridization solution 6 Improper hybridization temperature Check the actual temperature inside your hybridization oven with a thermometer The temperature reading on your hybridization oven could be several degrees off calibration B HIGH BACKGROUND Other than the quality of RNA sample the following factors may cause high background 1 Pre hybridization step incomplete Be sure to denature your sheared salmon sperm DNA completely before making your pre hybridization solution 2 Inaccurate AP streptavidin dilution Since the AP streptavidin is dissolved in a glycerol containing solution special caution should be taken when pipeting the small volume of AP streptavidin We suggest diluting no less than 2 ul of AP streptavidin into 15 ml of buffer a 1 7 500 dilution The final working AP streptavidin dilution can also be increased to 1 12 000 3 Improper incubation time with AP streptavidin Incubation can be reduced to 10 min or less 4 Improper washing temperature conditions Be su
15. iptase AE 1 F 2 k 4 i Warm the RT Cocktail at 42 C for 1 min before proceeding to the next step GEArray Q and S Series User Manual 11 c RT Reaction For each array transfer 10 ul of the pre warmed RT Cocktail to the 10 ul Annealing Mixture Mix well but gently with a pipettor and incubate at 42 C for 90 min Stop the RT Reaction by adding 2 ul of Buffer C Note We highly recommend checking the probe for dUTP incorporation at this point before denaturing the probe and setting up the hybridization See the Troubleshooting Guide at the end of the Manual V 3 for details d Denature the probe Add 2 ul of Buffer D to each RT Reaction now containing labeled cDNA probe Incubate at 68 C for 20 min Add 25 ul of Buffer E and continue incubation at 68 C for another 10 min Note Buffer E may precipitate when thawed at room temperature We recommend warming Buffer E to 68 C and vortexing before use Alternatively the cDNA probe can be denatured by heating at 94 C for 5 min and quickly chilling on ice Note It is not necessary to remove unincorporated biotin 16 dUTP from the cDNA probe The total labeling reaction mixture can be used for hybridization directly The TrueLabeling RT probe synthesis protocol is now complete The cDNA probe is now ready for hybridization B Please proceed to page 15 GEArray Q and S Series User Manual 12 OPTION 3 of 3 for Probe Synthesis AmpoLabeling LPR Kit Protoco
16. l Catalog Number L 03 Synthesis of cDNA probes with Biotin 16 dUTP Roche Cat No 1 093 070 Note If you plan to perform your hybridization immediately after probe synthesis start the pre hybridization B step 1 p 15 before continuing with this section Note In order for LPR to work properly your RNA samples must be free of fragmented genomic DNA We do not recommend treating your sample with DNase because the fragmented genomic DNA may increase the background Instead take care to remove genomic DNA intact from your sample during your isolation protocol Customers outside the USA without a local distributor The RE and RI components below are not included with the kit Please order MMLV reverse transcriptase and RNasin Ribonuclease Inhibitor Promega Cat Nos M1701 and N2511 and use in the place of RE and RI respectively You also need to convert 10X RT Buffer into Buffer BN Combine 50 uL of 10X RT Buffer 1 uL 1 M DTT and 50 uL dNTP mix 5 mM each dATP dCTP and dGTP and 0 5 mM dTTP Mix well Store at 20 C RT Reaction a Prepare the Annealing Mixture For each total RNA sample combine the following into a sterile PCR tube Total RNA 0 5 5 0 ug Buffer P l ul RNase free H2O Adjust the final volume to 10 ul We have successfully synthesized labeled probe with as little as 0 1 ug total RNA Mix the contents gently with a pipettor followed by brief centrifugation Place the mixture in a thermal cycler at 70 C f
17. ml Buffer G AP Assay Buffer 100 ml 100ml 100ml CDP Star Substrate 5 ml 5 ml 16 ml If using the RT Labeling Kit L 01 or the TrueLabeling RT Kit L 02 protocols for Probe Synthesis make sure to use Buffer A If using the AmpoLabeling LPR Kit L 03 protocol for Probe Synthesis make sure to use Buffer AF NOTE If using another kit to amplify your RNA be sure to obtain instructions for generating labeled probe from our website www superatray com answer php ID R3 Please check your Probe Synthesis Kit and its User Manual for other materials mentioned in this GEAtrray User Manual and required for the protocols within Note for Customers outside the USA As of September 15 2003 all other buffers required for probe synthesis are sold separately from you GEArray kit Please also order either the AmpoLabeling LPR Kit Catalog Number L 03 OR the RT Labeling Buffers Catalog Number L 04 GEArray Q and S Series User Manual HI ADDITIONAL MATERIALS REQUIRED A Software The GEArrayAnalyzer software can be downloaded from www superarray com free of charge If you prefer please call us and a CD can be sent to you Cat No GA001 B Probe Synthesis Kit Choose one of the following options available from SuperArray Option 1 Catalog Number L 01 RT Labeling Kit Used for experiments optimized with older GEArray kits Available to US Customers and Customers Outside the USA with a local distributor
18. ns up to 96 and the GEArray S Series contains up to 450 cDNA fragments from genes associated with a specific biological pathway Gene specific cDNA fragments are printed on a 3 8 x 4 8 cm nylon membrane with an advanced non contact printing technology Each cDNA fragment on the Q Series is printed with our tetra spot format which provides a signal in an easily identifiable pattern The cDNA fragments on the S Series are printed in single spots to accommodate an increased number of genes in a grid of 16 columns and up to 30 rows The catalog number and a unique bar coded serial number are printed on the reverse side of both the Q and S Series membranes for easy tracking and documentation The GEArray Q and S Series are designed for one time use only Each array membrane is packaged in a disposable hybridization tube Multiple arrays are included in each kit allowing for a parallel and comparative study of multiple samples Finally the GEArray analysis software is available free of charge for GEArray users and can be downloaded from our website at www superarray com Features of the GEArray Q and S Series Easy to use with minimal hands on time Comprehensive collection of genes in a specific pathway Proprietary arraying detection technology offers high sensitivity Tetra spot format for reliable signal detection and identification Q Series Only Available for both radioactive and non radioactive detection No special equipment required C
19. or 3 min Cool to 37 C and keep at that temperature for 10 min b Prepare the RT Cocktail This mixture can be prepared while the Annealing Mixture is incubating at 37 C RT Cocktail 1 array 2 arrays 4 arrays Buffer BN 4ul yul 16 ul RNase free H20 4ul Byul 16 ul RNase Inhibitor RI lull 2ul 4u Reverse Transcriptase RE lul 2ul Au Warm the RT Cocktail at 37 C for 1 min before proceeding to the next step c RT Reaction For each array transfer 10 ul of the RT Cocktail to the 10 ul Annealing Mixture Mix well but gently with a pipettor and continue incubation at 37 C for 25 min Heat at 85 C for 5 min to hydrolyze the RNA and to inactivate the reverse transcriptase Hold the finished RT Reaction on ice until the next step GEArray Q and S Series User Manual 13 LPR Labeling Reaction Note The temperature should not exceed 85 C at anytime during LPR d Prepare the LPR Cocktail For each Q or S Series kit mix the following in a sterile PCR tube LPR Cocktail l array 2 arrays 4 arrays Buffer L 18 ul 36 ul 72 ul Buffer AF BLUE tube 9 ul 18 ul 36 ul Biotin 16 dUTP 2 ul 4 ul 8 ul DNA Polymerase LE 1 ul 2 ul 4 ul Note Buffer AF is unique for each GEArray Q and S Series kit Please do not substitute Buffer AF meant for other GEArray Kits Check the Catalog Number on the tube before use Also please do not use any source of Buffer A
20. ost effective for routine use Free software for data analysis and reporting GEArray Q and S Series User Manual Figure 1 Overview of the GEArray Q and S Series procedure Note The spotting pattern on the S series membrane is a 16x18 to 16x30 grid of single spots Gene Expression Profiling with GEArray Q Series Q2n Y 7W Probe labeling with 97P dCTP l GEArray or Biotin dUTP Pre hybridization Probe denaturation Hybridization 32P labeled cDNA probe Biotinylated cDNA probe X ray film or chemiluminescence detection Phosphoimager X ray film or CCD camera Raw image J Data extraction N Raw data 4 Data analysis with GEArray Analyzer j Data table and graphs man _ Total RNA Probe labeling 1 2 hour Hybridization Overnight Detection 2 3 hours Data analysis GEArray Q and S Series User Manual 5 II MATERIALS PROVIDED GEArray Q or S Series Trial Standard Bulk GEArray Q or S Series membranes in disposable tubes 2 4 12 BOX 1 cDNA Probe Synthesis Buffer A GEAprimer mix in CLEAR tube 20 ul 20 ul 3x20 ul Buffer AF GEAprimer mix 40 ul 40 ul 3x40 ul For AmpoLabeling LPR Kit in BLUE tube Hybridization and Detection GEAhyb Hybridization Solution 50 ml 50 ml 50 ml BOX 2 GEAblocking Solution Q 30 ml 30 ml 30 ml AP Streptavidin 20 ul 20 ul 20 ul 5X Buffer F 5X Washing Buffer 100 ml 100 ml 100
21. re to use enough Wash Solution 2 during the low stringency washing step Also be sure to wash the GEArray Q or S Series membrane at 60 C with agitation at 20 30 rpm GEArray Q and S Series User Manual 20 5 Biotin contamination in containers or buffers Milk contains a high amount of biotin Never use any lab ware that has been used for Western blotting 6 Improper membrane incubation Always use enough solution to completely cover each membrane Never let the membranes dry out at any stage in the procedure 7 Over exposure Try multiple exposures for various times 8 Too much total RNA Reduce the amount of total RNA used for Probe Synthesis C How do I prevent leakage of the hybridization tube that you provide Put the tube in a regular hybridization cylinder not provided Close the cap of this cylinder and our hybridization tube cap hand tight An airtight cylinder can prevent the hybridization tube from leaking We also suggest using DuraSeal a sealing film from Diversified Biotech Cat No DS1 150 etc It seals the hybridization tubes very well which is especially important for users of the Radioactive Detection method D Why do I get a hybridization signal on the negative control pUC18 spot A positive signal on the pUC18 negative control occurs most often when the RNA sample used for Probe Synthesis came from a plasmid transfected cell line Although we use a gene specific primer mix for the labeling reaction a sm
22. rmalization options It generates data tables and other graphical reports including bar charts scatter plots and pseudo color plots Please refer to the GEArray Analyzer User Manual for more detailed instructions Each GEArray Q Series membrane is spotted with a negative control of pUC18 DNA blanks and housekeeping genes including B actin GAPDH cyclophilin A and ribosomal protein L13a All raw signal intensities should be corrected for background by subtracting the signal intensity of a negative control or blank All signal intensities should also be normalized to that of a housekeeping gene These corrected normalized signals can then be used to estimate the relative abundance of particular transcripts Note At the current time there is no pUCI8 DNA control on the S Series membranes Use a blank for your background subtraction The same housekeeping genes ARE included on both Q and S Series membranes Otherwise perform the array analysis as described GEArray Q and S Series User Manual 18 Order in which numbered Reverse side has printed bar genes and resulting data code on right side edge are and should be placed Array side faces the detector Figure 3 Data extraction sequence for GEArray Q Series Note for S Series users Collect data from the grid of single spots from left to right and top to bottom V TROUBLESHOOTING GUIDE AND FREQUENTLY ASKED QUESTIONS RNA quality is crucial to the success of the array It i
23. rsists for several days Therefore there is ample time to collect the image but it should be performed as soon as possible If X ray film is used to record the image use a scanner to convert the image into a grayscale TIFF file Be sure to save this file If using a CCD camera to obtain the image you may also save the image as a grayscale TIFF file Alternatively you may use the imaging station s software to save the image and convert it into numerical data 2 Data Acquisition If using a grayscale TIFF image convert the image of tetra spots into numerical data using the free ScanAlyze software developed by Dr Michael Eisen and featured as a hyperlink on our website www superarray com The software saves the numerical data as a tabular file recognizable by Microsoft Excel If using your imaging station s software to convert the image into numerical data be sure to collect data from the entire tetra spot Q Series or single spot S Series for each cDNA Collect the data from left to right and top to bottom to insure that the order will match that of our gene lists Q Series can also refer to Figure 3 Save the raw data as a Microsoft Excel file 3 Data Analysis SuperArray has free data analysis software GEArray Analyzer available on our website www superarray com GEArray Analyzer matches your raw data table with the gene list for the particular GEArray It also provides you with a list of background subtraction and data no
24. s important to check your RNA quality The ratio of the 260 nm and 280 nm absorbance readings should be greater than 1 8 Also a good RNA prep should produce two sharp bands on an ethidium bromide stained agarose gel representing the 28S and the 18S ribosomal RNA rRNA The 28S band should be roughly 2 times more intense than the 18S band A smearing of either of the two bands a decrease in the 28S to 18S intensity ratio or a ratio of Ax6o A280 less than 1 8 indicates degradation of the RNA sample In which case perform your RNA preparation again before proceeding with the array analysis However sometimes even intact RNA does not guarantee a good result because the RNA sample may still contain inhibitors that can reduce labeling efficiency We also recommend that you check the probe labeling before starting the hybridization See below A WEAK OR NO SIGNALS Other than the quality of RNA sample the following factors may also impair your results 1 Low RNA sample concentration If the RNA sample is too dilute it may not perform well in the assay The RNA concentration should be at least 0 5 mg ml 2 Inhibited reverse transcriptase a Total RNA should be dissolved in RNase free dH2O or TE Buffer 10mM Tris HCl lmM EDTA Be sure that the EDTA concentration does not exceed 1 mM a high EDTA concentration inhibits the reverse transcriptase b Solutions that contain SDS as well as unautoclaved DEPC water will inhibit the reverse transcripta
25. se c If CsCl or LiCl were used in the process of total RNA purification trace amounts of Cs or Li will inhibit the reverse transcriptase GEArray Q and S Series User Manual 19 3 Insufficient probe labeling Many factors including poor RNA quality and contaminants can result in inadequate labeling of the probe We highly recommend checking biotinylated probe labeling efficiency in the following manner a Add 1 ul of the completed but non denatured Probe Synthesis reaction to 19 ul of 1X agarose gel loading buffer to yield a 20 fold dilution b Perform 4 fold serial dilutions by mixing 3 ul aliquots of each successive dilution with 9 ul of 1X agarose gel loading buffer to yield 80 320 1280 and 5120 fold dilutions c Separately spot 1 ul of each diluted sample onto a 3 8 cm x 4 8 cm piece of HyBond nylon membrane e g Amersham Pharmacia Cat No RPN303B Allow the membrane to air dry for at least 10 minutes d Follow the Chemiluminescent Detection protocol in the GEArray Q Series User Manual for Chemiluminescent Detection You may even put the membrane in a previously used but clean GEArray Hybridization tube e Ifthe total biotin deoxyuridylate incorporation is detectable at dilutions of 1000 fold or higher 500 fold or higher for TrueLabeling RT the probe should yield good array signals f Ifyou require more reagents order an Original GEArray Box 2 Catalog Number 9905300 Note For these purposes the GEAblock
26. t intended for diagnostic purposes or for human use Most of the cDNA fragments spotted on the GEArray membrane products are generated utilizing computer algorithm designed gene specific primers and RT PCR Their identities are then verified by using a nested PCR technique but not by automated sequencing The nature of gene array technology in general including all GEArray product lines necessitates the validation of any array result by an independent method such as Northern blotting or RT PCR This warranty limits our liability to replace this product in the event the product fails to perform due to a physical manufacturing defect but not due to an inability to validate by an independent method any results obtained using this product SuperArray Bioscience Corporation makes no other warranties of any kind expressed or implied including without limitation warranties of merchantability or fitness for a particular purpose SuperArray Bioscience Corporation shall not be liable for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product Warranties are subject to change at any time without notice NOTICE TO PURCHASER THE PURCHASE OF GEARRAY PRODUCTS INCLUDES A LIMITED NONEXCLUSIVE LICENSE TO USE THE MEMBRANES FOR RESEARCH USE ONLY THIS LICENSE DOES NOT GRANT RIGHTS TO USE THE MEMBRANES FOR REPRODUCTION OF GEARRAY FILTERS TO MODIFY GEARRAY FILTERS FOR RESALE OR TO USE GE
27. thesis start the pre hybridization B step 1 p 15 before continuing with this section International customers At this time the TrueLabeling RT Kit is only available in international territories with local distributors Other international customers should use Option 3 or the Alternation Option See the Additional Materials Required Section Note There is no need to treat total RNA with DNase a Prepare the Annealing Mixture For each total RNA sample combine the following in a sterile PCR tube Total RNA 2 5 5 0 ug Buffer A CLEAR tube 3 ul RNase free H2O Adjust the final volume to 10 ul Note Buffer A is unique for each GEArray Q and S Series kit Please do not substitute Buffer A from other GEArray Kits Check the Catalog Number on the tube before use Also please do not use any source of Buffer AF for the TrueLabeling RT Kit Protocol Mix the contents gently with a pipettor followed by brief centrifugation Place the mixture in a pre heated heat block or thermal cycler at 70 C for 3 min Cool to 42 C and keep at that temperature for 2 min Note A pre programmed PCR thermal cycler is recommended b Prepare the RT Cocktail This mixture can be prepared while the Annealing Mixture is incubating at 70 C RT Cocktail 1 array 2 arrays 4 arrays Buffer BN 4ul 8ul 16 ul Biotin 16 dUTP 2ul 4ul 8 ul RNase free H20 2ul 4ul ul RNase Inhibitor RI lul 2ul 4ul Reverse Transcr
28. to 65 C if necessary to dissolve Store at room temperature 3 You will also need the following laboratory equipment Thermal cycler or at least two water baths or hot blocks Hybridization oven and cylinders or other means to agitate at high temperature X ray film Kodak X OMAT and Scanner OR CCD Camera and its software GEArray Q and S Series User Manual 7 IV GEArray Q AND S SERIES ASSAY PROTOCOL Notes RNA Preparation Total RNA prepared using commercially available kits is suitable for GEArray analysis Total RNA prepared by Trizol Reagent Invitrogen Life Technologies Cat No 15596 018 RNeasy mini kits Qiagen Cat No 74104 or RNAqueous Ambion Cat No 1911 1912 or 1913 gives good results Total RNA should be dissolved in RNase Free HO It is not necessary to prepare poly A RNA however the quality of the RNA is crucial for the success of the assay It is also not necessary to treat RNA samples with DNase In fact DNase should not be used if you are synthesizing probe with the AmpoLabeling LPR Kit It is important to check the absorbance reading of your RNA sample not only to determine the concentration but also to assess its purity from proteins and free nucleotides The ratio of the absorbance reading at 260 nm to the reading at 280 nm should be greater than 1 8 In addition and if possible the integrity of the RNA preparation should be checked by ethidium bromide stained agarose gel electrophoresis A quality RN
29. with Reverse side Printed with cDNA spots Faces the inside information Faces the outside of the hybridization tube of the hybridization tube GEArray Q Series Figure 2 The two sides of the GEArray Q Series membrane Note for S Series users The spotting pattern on your membrane is a 16x18 to 16x 30 grid of single spots Note Wear gloves while performing all steps of this protocol GEArray Q and S Series User Manual 8 OPTION 1 of 3 for Probe Synthesis RT Labeling Kit Protocol Catalog Number L 01 Synthesis of cDNA probes with Biotin 16 dUTP Roche Cat No 1 093 070 Note If you plan to perform your hybridization immediately after probe synthesis start the pre hybridization B step 1 p 15 before continuing with this section International customers At this time the RT Labeling Kit is only available in international territories with local distributors Other international customers may follow the notes below Note Any customer may also follow this protocol using other sources of RNase Inhibitor Promega Catalog Number N2511 and MMLV reverse transcriptase Promega Catalog Number M1701 together with SuperArray s RT Labeling Buffers Catalog Number L 04 sold separately and available to customers inside and outside the USA International customers without a local distributor will also need to convert 10X RT Buffer from the RT Labeling Buffers L 04 into Buffer BN for Chemiluminescent Detection using the
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