GEArrayTM Q Series KIT
Contents
1. 20 C GEArray Q Series User Manual 6 HI ADDITIONAL MATERIALS REQUIRED 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 The following labeling kits for cDNA Probe Synthesis are sold separately by SuperArray Product Cat No Reasons for Use Page RT Labeling Enzyme L 01 Experiments optimized using previous Q Series kits 8 TrueLabeling RT Enzyme L 02 Reduces high background and false positive signals 10 AmpoLabeling LPR Kit L 03N Amplifies signal intensities for limiting RNA or message 12 NOTE The AmpoLabeling LPR Kit contains an array specific reagent Please indicate the GEArray catalog number as well as catalog number L 03N when ordering For your convenience step by step instructions are provided for each protocol on the pages listed above If you already have RNase Inhibitor Promega Cat No N2511 and MMLV Reverse Transcriptase Promega Cat No M1701 or Applied Biosystems Cat No N808 0018 follow the procedure for the RT protocol The above table also describes the advantages of using each of our labeling kits Note to international customers The AmpoLabeling LPR Kit can be shipped internationally however one temperature sensitive reagent is excluded and must be ordered separately See the Protocol included in the labeling kit or on Page 12 of this manual At this time the RT Labeling Enzyme2. 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 Series membrane 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 Place the membrane between two plastic sheets or into a small plastic zip lock bag and smooth out any bubbles GEArray Q 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 softw
3. Data Analysis 17 V Troubleshooting Guide 18 LIMITED PRODUCT WARRANTY This product is intended for research purposes only and is not intended for drug or diagnostic purposes or for human use This warranty limits our liability to replace this product in the event the product fails to perform due to any manufacturing defect 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 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 GEARRAY 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 Series User Manual 3 I INTRODUCTION The advance
4. PROVIDED GEArray Q Series Trial Standard Bulk GEArray Q Series membranes in disposable tubes 2 4 12 GEArray Q Series Quick Protocol and grid card l l l BOX 1 cDNA Probe Synthesis Buffer A GEAprimer mix 20 ul 20nl 60u Buffer BN 5X GEAlabeling Buffer USA Customers Only 60 ul 6Oul 60ul OR 10X RT Buffer International Customers 100 ul 100 ul 100 ul RNase free H2O 1 ml 1 ml 1 ml Buffer C 10X Stop Solution 100 ul 100 ul 100 ul Buffer D 10X Denaturing Solution 100 ul 100 ul 100 ul Buffer E 2X Neutralization Solution 1 ml 1 ml 1 ml Hybridization and Detection GEAhyb Hybridization Solution 50 ml 50ml 50ml BOX 2 GEAblocking Solution Q 30 ml 30ml 30ml AP Streptavidin 20 ul 20 ul 20 nu 5X Buffer F 5X Washing Buffer 100ml 100ml 100 ml Buffer G AP Assay Buffer 100 ml 100 ml 100 ml CDP Star Substrate 5 ml S5Sml 16ml If using the AmpoLabeling LPR Kit to synthesize probe make sure to use the Buffer AF included with the labeling kit instead of the Buffer A included with the array kit Note for international customers When you first open BOX 1 the Nonrad GEArray Labeling Kit you need to convert the 10 X RT buffer to 5X Nonrad GEA Labeling Buffer Buffer BN To make 100 uL of Buffer BN combine 50 uL of 10 X 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 the entire box at
5. and the TrueLabeling RT Enzyme Kits may not be available in all international territories Please contact your distributor for more information You may also order and use your own MMLV Reverse Transcriptase and RNase Inhibitor as described above and follow the labeling protocol for the RT enzyme The following reagents are required but are not supplied by SuperArray cDNA Probe Synthesis Biotin 16 dUTP Roche Cat No 1 093 070 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 dehydrate 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 to 65 C if necessary to dissolve Store at room temperature 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 Series User Manual 7 IV GEArray Q SERIES ASSAY PROTOCOL Notes RNA Preparation Total RNA prepared using commercially available kits is suitable for GEArray Q Series 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 191
6. 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 The cDNA probe is now ready for hybridization B 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 Note We highly recommend checking the probe for dUTP incorporation before setting up the hybridization See the Troubleshooting Guide at the end of the Manual for details GEArray Q Series User Manual 12 A Probe Synthesis 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 before continuing with this section 3 GEArray AmpoLabeling LPR Protocol Catalog Number L 03N NOTE The AmpoLabeling LPR Kit contains an array specific reagent Please indicate the GEArray catalog number as well as catalog number L 03N when ordering International customers The RE component below is not included with the kit Please order MMLV reverse transcriptase Promega Cat No M1701 or Applied Biosystems Cat No N808 0018 and use in the place of RE You also need t
7. 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 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 Available for both radioactive and non radioactive detection No special equipment required Cost effective for routine use Free software for data analysis and reporting GEArray Q Series User Manual Figure 1 Overview of the GEArray Q series procedure Gene Expression Profiling NAR Total RNA with GEArray Q Series 222X W A Probe labeling with P dCTP or Biotin dUTP GEArray Probe labeling 1 2 hour Pre hybridization Probe denaturation Hybridization Hybridization Overnight 32D labeled cDNA probe Biotinylated cDNA probe X ray film or chemiluminescence detection Phosphoimager X ray film or CCD camera 7 i Raw image J Data extraction S Raw data Data analysis with GEArray Analyzer Data table and graphs Data analysis GEArray Q Series User Manual II MATERIALS
8. 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 Ax69 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 transcriptase 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 Series User Manual 19 3 Insufficient probe labeli
9. 1 1912 or 1913 gives good results Total RNA should be dissolved in RNase Free H2O 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 RNA 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 Series nylon membran
10. 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 LPR Labeling Reaction Note The temperature should not exceed 85 C at anytime during LPR d Prepare the LPR Cocktail For each Q 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 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 OQ Series kit Please do not substitute Buffer AF meant for other GEArray Q Series kits Check the Catalog Number on the tube before use Also please do not use any source of Buffer A for LPR 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 g Denature the p
11. I SuperArray Bioscience Corporation GEArray Q Series KIT A PATHWAY SPECIFIC GENE EXPRESSION PROFILING SYSTEM 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 Important note to customers outside the USA All international array kits are shipped without dry ice The temperature sensitive reagent Buffer BN must be prepared when you receive the kit DTT and dNTPs are not included in this package SuperArray Bioscience Corp 7320 Executive Way Suite 101 Frederick MD 21704 USA FOR RESEARCH USE ONLY Version 6 1 1 31 2003 GEArray Q Series User Manual 2 CONTENTS I Introduction 3 II Materials Provided 5 IHI Additional Materials Required 6 IV GEArray Q Series Assay Protocol 7 A Probe Synthesis 1 RT Labeling Enzyme 8 2 TrueLabeling RT Enzyme 10 3 AmpoLabeling LPR Kit 12 B Hybridization 14 C Chemiluminescent Detection 16 D Image and
12. NA 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 small 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 yl 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 tr
13. alyzer 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 normalization 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 GEArray Q 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 V TROUBLESHOOTING GUIDE AND FREQUENTLY ASKED QUESTIONS RNA quality is crucial to the success of the array It is 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
14. anscriptases 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 Buffer BN is included in the kit which is shipped on dry ice However if improperly stored if you need to make more for a particular reason or if you are an international customer 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 prepared 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 CDP Star is a registered trademark of Applera Corporation or its subsidiaries in the US and certain other countries
15. are 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 persists 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 for each cDNA Collect the data in the order outlined in Figure 3 left to right top to bottom to insure that the order will match that of our gene lists 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 An
16. 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 cap of the tube is screwed on hand tight Place the tube inside your hybridization cylinder Two GEArray Q 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 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 ce
17. e Inhibitor Promega Cat No N2511 and MMLV reverse transcriptase Promega Cat No M1701 or Applied Biosystems Cat No N808 0018 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 3 ul RNase free H2O to adjust the final volume to 10 ul Note Buffer A is unique for each GEArray Q Series kit Please do not substitute Buffer A from other GEArray QO Series kits Check the Catalog Number on the tube before use 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 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 16 ul Biotin 16 dUTP 2 ul 4 ul 8 ul RNase free H2O 2 ul 4 ul 8 ul RNase Inhibitor RI 1 ul 2 ul 4 ul Reverse Transcriptase RE 1 A 2 F 4 F Warm the RT Cocktail at 42 C for 1 min before proceeding to the next step GEArray Q Series User Manual 9 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 pipet
18. e array is supplied in a disposable hybridization tube One side of the membrane is printed with the cDNAs in the tetra spot format 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 serial number and barcode to allow tracking of the array during the course of your experiment Array side Arrayed 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 Wear gloves while performing all steps of this protocol GEArray Q Series User Manual A Probe Synthesis 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 before continuing with this section 1 GEArray RT Labeling Enzyme Catalog Number L 01 International customers At this time the RT Labeling Enzyme Kit may not be available in all international territories Please contact your distributor for more information Note You may also follow this protocol using other sources of RNas
19. ment 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 Series provides 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 Series is a result of combining superior array design with state of the art arraying and detection technologies It is easy to use sensitive economical and accessible for routine use in every research laboratory The GEArray Q Series cDNA expression array contains up to 96 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 is printed with our tetra spot format which provides a signal in an easily identifiable pattern The catalog number and a unique bar coded serial number are printed on the reverse side of the membrane for easy tracking and documentation The GEArray Q Series is designed
20. meter 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 pipetting 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 sure to use enough Wash Solution 2 during the low stringency washing step Also be sure to wash the GEArray Q series membrane at 60 C with agitation at 20 30 rpm 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 GEArray Q Series User Manual 20 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 R
21. ng Many factors including the two mentioned above can result in inadequate labeling of the probe We highly recommend checking biotinylated probe labeling efficiency in the following manner a b Add 2 ul of the completed Probe Synthesis reaction to 38 ul of 1X agarose gel loading buffer 20 fold dilution 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 Separately spot 1 ul of each diluted sample onto a HyBond nylon membrane e g Amersham Pharmacia Cat No RPN303B Allow the membrane to air dry for at least 10 minutes Follow the Chemiluminescent Detection protocol in the GEArray Original Series User Manual for Chemiluminescent Detection If the total biotin deoxyuridylate incorporation is detectable at dilutions of 1000 fold or higher the probe should yield good array signals 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 thermo
22. ntrifuge 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 wash GEArray Q Series User Manual 16 C Chemiluminescent Detection Note All 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 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
23. o convert 10X RT Buffer to 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 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 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 to 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 for 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 Bul 16 ul RNase free H20 4ul yul 16 ul RNase Inhibitor RI lul 2ul 4u Reverse Transcriptase RE lul 2u 4ul Warm the RT Cocktail at 37 C for 1 min before proceeding to the next step GEArray Q Series User Manual 13 c RT Reaction For each array transfer 10 ul of the RT
24. re information 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 3 ul RNase free H2O to adjust the final volume to 10 ul Note Buffer A is unique for each GEArray Q Series kit Please do not substitute Buffer A from other GEArray Q Series kits Check the Catalog Number on the tube before use 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 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 8 ul RNase Inhibitor RI lul 2ul 4ul Reverse Transcriptase AE 1 i 2 T 4 i Warm the RT Cocktail at 42 C for 1 min before proceeding to the next step GEArray Q 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 d Denature the probe Stop the RT Reaction by adding 2 ul of Buffer C Add 2 ul of Buffer D to each RT Reaction now containing labeled cDNA probe Incubate
25. robe Denature the labeled cDNA probe by heating the tube containing the LPR at 94 C for 2 min and quickly chilling on ice The cDNA probe is now ready for hybridization B 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 Note We highly recommend checking the probe for dUTP incorporation before setting up the hybridization See the Troubleshooting Guide at the end of the Manual for details GEArray Q Series User Manual 14 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 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
26. tor Continue incubation at 42 C for 90 min d Denature the probe Stop the RT Reaction by adding 2 ul of Buffer C 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 The cDNA probe is now ready for hybridization B 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 Note We highly recommend checking the probe for dUTP incorporation before setting up the hybridization See the Troubleshooting Guide at the end of the Manual for details GEArray Q Series User Manual 10 A Probe Synthesis 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 before continuing with this section 2 GEArray TrueLabeling RT Enzyme TL RT Protocol Catalog Number L 02 International customers At this time the TrueLabeling RT Enzyme Kit may not be available in all international territories Please contact your distributor for mo
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