Agilent Gene Expression FFPE Workflow Quick Start Guide
Contents
1. 10 Gene Expression FFPE Workflow Quick Start Guide Procedure Step 5 Label with ULS 1 For a 4 pack microarray hybridization prepare a tube that contains 1 65 ug of cDNA in a final volume of 16 35 uL If the 1 65 ug of cDNA is in a volume greater than 16 35 uL use a vacuum concentrator to concentrate the sample until the volume is 16 35 uL 1 Mix the components in Table 6 on ice to prepare one Cy3 labeling master mix Table 6 Preparation of Labeling Master Mix for 4 pack using FFPE samples Components Per reaction pL Per 4 reactions pL including excess ULS Cy3 1 65 8 25 10x Labeling Solution 2 10 Final Volume of Labeling Master Mix 3 65 18 25 2 Add 3 65 uL of Labeling Master Mix to each tube that contains the cDNA for a total volume of 20 uL Mix well by gently pipetting up and down 3 Incubate tubes at 85 C for 30 minutes 4 Transfer samples to ice and incubate on ice for at least 3 minutes 5 Spin in a microcentrifuge for 1 minute at 6000 x g to drive contents off the walls and lid Store labeled cDNA on ice until excess dye is removed using the Agilent KREApure columns Gene Expression FFPE Workflow Quick Start Guide 11 Procedure Step 6 Remove non reacted Cy ULS 1 Resuspend Agilent KREApure column material by briefly mixing on a vortex mixer 2 Loosen cap 1 4 turn and snap off the bottom closure 3 Put the Agilent KREApure column in a microcentrifuge and spin for 1 minute at maximum spe2. Table 1 Required Equipment continued Description Vendor and part number Sterile nuclease free aerosol barrier pipette tips Vortex mixer Timer Nitrogen purge box for slide storage Required Reagents Table 2 Required Reagents Description Vendor and part number Genomic DNA ULS Labeling Kit Agilent p n 5190 0419 Gene Expression Hybridization Kit Agilent p n 5188 5242 Gene Expression Wash Buffer 1 Agilent p n 5188 5325 Gene Expression Wash Buffer 2 Agilent p n 5188 5326 100 Ethanol Amresco p n E193 TITANIUM Taq DNA Polymerase Clontech p n 639208 DNase RNase free distilled water Invitrogen p n 10977 015 QlAquick PCR Purification Qiagen p n 28106 Sulfolane Sigma p n 122209 Transplex Whole Transcriptome Amplification kit Sigma p n WTA1 50RXN Absolutely RNA FFPE Kit Stratagene p n 400809 Milli O water or equivalent Gene Expression FFPE Workflow Quick Start Guide Before you Begin Optional Equipment Reagents Table3 Optional Equipment Reagents Description Vendor and part number 2100 Bioanalyzer Agilent p n G2938A RNA 6000 Nano Assay Kit RNA Series II Kit Agilent p n 5067 1511 DNA 1000 Kit Agilent p n 5067 1505 Stabilization and Drying Solution Agilent p n 5185 5979 Slide box Corning p n 07201629 Acetonitrile Sigma p n 271004 1L Gene Expression FFPE Workflow Quick Start Guide 5 Procedure Procedure Step 1 Isolate RNA 1 Extract RNA from FFPE blocks Follow the instructions in the user ma
3. 24 C for 15 minutes 42 C for 2 hours 95 C for 5 minutes 4 C hold 9 Cool reaction on ice and briefly centrifuge PCR plate Gene Expression FFPE Workflow Quick Start Guide 7 Procedure Step 3 Amplify WTA Library 1 Immediately prior to use gently mix the components listed in Table 5 for the WTA Amplification Mix by adding in the order indicated and keep on ice Clontech Titanium Taq DNA Polymerase is an enzyme which needs to be kept on ice and added to the WTA Amplification Mix just before starting the reactions The Titanium Taq DNA Polymerase is purchased separately from Clontech Table 5 WTA Amplification Mix Components Per reaction pL Per 4 reactions pL including excess Nuclease free water 300 1500 WTA Amplification Master Mix 37 5 187 5 dNTP Mix 7 5 37 5 Clontech Titanium Taq DNA Pol 5 25 Final Volume of Amp Master Mix 350 1750 Divide the library generated in Step 2 Prepare WTA Library 25 uL into 5 equivalent 5 uL aliquots in the wells of a PCR plate Add 70 uL of the WTA Amplification Mix Table 5 to each library aliquot and mix well by gently pipetting up and down The final reaction volume should be 75 uL Place PCR plate in thermal cycler and cycle as follows 95 C for 3 minutes 94 C for 20 seconds 65 C for 5 minutes for 22 cycles 4 C Hold Cool reaction on ice and briefly centrifuge PCR plate Gene Expression FFPE Workflow Quick Start Guide Procedure Make sure th
4. at the amplified library aliquots are purified and pooled before further analysis The purified and pooled amplified library can be stored at 20 C Gene Expression FFPE Workflow Quick Start Guide 9 Procedure Step 4 Purify WTA products using the QlAquick PCR Purification Kit All centrifuge steps are at 13 000 rpm approximately 17 900 x g ina conventional tabletop microcentrifuge The OlAquick kit is purchased separately from Qiagen For best results purify individual amplified aliquots separately then pool them together 1 Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix for example 375 uL Buffer PB to 75 uL PCR product 2 Put a QIAquick spin column in provided 2 mL collection tube 3 To bind DNA to the column apply the sample to the QIAquick column and spin in a centrifuge for 30 to 60 seconds 4 Discard the flow through Place the QIAquick column back into the same tube 5 To wash add 0 75 mL Buffer PE to the QIAquick column and centrifuge for 30 to 60 seconds 6 Discard flow through and place the QIAquick column back in the same tube Centrifuge the column for an additional 1 minute 7 Place QIAquick column in a clean 1 5 mL microcentrifuge tube 8 To elute the DNA add 50 uL nuclease free water to the center of the QIAquick membrane let the column stand for 1 minute and then spin the column in a centrifuge for 1 minute 9 Move to ice 10 Quantitate with NanoDrop using 1 5 uL of the eluted DNA
5. e e Gene Expression FFPE Workflow Quick Start Guide Before you Begin 3 Required Equipment 3 Required Reagents 4 Optional Equipment Reagents 5 Procedure 6 Step 1 Isolate RNA 6 Step 2 Prepare WTA Library 7 Step 3 Amplify WTA Library 8 Step 4 Purify WTA products using the QlAquick PCR Purification Kit 10 Step 5 Label with ULS 11 Step 6 Remove non reacted Cy ULS 12 Step 7 Prepare labeled cDNA for hybridization 13 What is Sigma WTA The TransPlex Whole Transcriptome Amplification WTA kit allows for rapid amplification of total RNA from formalin fixed paraffin embedded FFPE samples in less than 4 hours without 3 bias The WTA kit involves two steps First total RNA is reverse transcribed with a WTA Polymerase using non complementary primers composed of quasi random 3 end and a universal 5 end Then the resulting Omniplex cDNA library composed of overlapping 100 to 1000 base fragments is PCR amplified to produce microgram quantities of WTA products for downstream applications such as qPCR and microarray analyses Sigma makes two different WTA kits WTA1 and WTA2 This protocol uses the WTA1 kit ot j Agilent Technologies What is Sigma WTA ds cDNA Synthesis TransPlex Whole cDNA Library Generation Transcriptome Amplification Kit 30 min ULS Labeling Agilent gDNA ULS Labeling Kit Agilent KREApure Clean Up Agilent CGHb ock Figure 1 Various kits and steps involved in the analysis of FFPE samples with t
6. ed minimum 16000 x g 4 Discard the cap and flow through and place the Agilent KREApure column back into the same collection tube Add 300 uL water to the column Spin for 1 minute at maximum speed minimum 16 000 x g Discard the flow through and collection tube Transfer the column to a clean 1 5 mL microcentrifuge tube Add the ULS labeled sample from Step 5 Label with ULS 20 uL for 4 pack to the center of the column membrane oon oO oI 10 Spin for 1 minute at maximum speed minimum 16 000 x g to collect the purified labeled sample in the collection tube 11 Quantitate the labeled cDNA with a NanoDrop using 1 5 uL of the eluted sample Make sure 18 5 uL of the ULS labeled sample remains in each tube 340 x pmol per uLdye ng per uLcDNA x 1000 nn Degree of labeling The optimal Degree of labeling is between 1 5 and 3 0 Because ULS labeling does not copy or amplify the yield after labeling will be the same as the input amount of cDNA 12 Gene Expression FFPE Workflow Quick Start Guide Procedure Step 7 Prepare labeled cDNA for hybridization 1 Prepare 100X Blocking Agent a Add nuclease free water to the vial of lyophilized 10X GE Blocking Agent depending on the Gene Expression Hyb Kit used Table 7 Nuclease free water volume Components Nuclease free Water uL For Agilent Gene Expression Hyb Kit 50 For Hi RPM Gene Express Hyb Kit 125 These volumes are 10 of the amount instructed w
7. he Agilent gene expression microarray workflow Gene Expression FFPE Workflow Quick Start Guide Before you Begin Before you Begin Make sure you have the material listed in this section Required Equipment Table 1 Required Equipment Description Vendor and part number Agilent Microarray Scanner Agilent p n G2565CA or G2565BA Hybridization Chamber stainless Agilent p n G2534A Hybridization Chamber gasket slides 1 microarray slide 5 slides box Agilent p n G2534 60003 2 microarrays slide 5 slides box Agilent p n G2534 60002 4 microarrays slide 5 slides box Agilent p n G2534 60011 8 microarrays slide 5 slides box Agilent p n G2534 60014 Hybridization oven temperature set at 65 C Agilent p n G2545A Hybridization oven rotator for Agilent Microarray Hybridization Agilent p n G2530 60029 Chambers Nuclease free 1 5 mL microfuge tubes Ambion p n 12400 or equivalent Magnetic stir bar x2 Corning p n 401435 or equivalent Magnetic stir plate x2 Corning p n 6795 410 or equivalent Microcentrifuge Eppendorf p n 5417R or equivalent NanoDrop ND 1000 UV VIS spectrophotometer NanoDrop p n ND 1000 or equivalent Slide staining dish with slide rack x3 Thermo Shandon p n 121 or equivalent Thermocycler with heated lid 96 well PCR plate or PCR tubes Clean forceps Ice bucket Pipetman micropipettors P 10 P 20 P 200 P 1000 or equivalent Powder free gloves Gene Expression FFPE Workflow Quick Start Guide 3 Before you Begin
8. is needed to eliminate background noise on the microarray 10 Dispense 100 uL of sample onto each 4x44K microarray 11 Hybridize at 65 C for 17 hours at 20 RPM 12 Use standard Agilent Gene Expression wash and scan conditions For details refer to the One color Microarray based Gene Expression Analysis Low Input Quick Amp Labeling Protocol p n G4140 90040 14 Gene Expression FFPE Workflow Quick Start Guide Procedure Microarray QC Metrics for FFPE samples These metrics are only appropriate for samples analyzed with Agilent Gene Expression microarrays by following the standard operational procedures provided in this FFPE Quick Start Guide These metrics are exported to a table in the Feature Extraction QC report The QC metrics can be used to assess the relative data quality across a set of microarrays in an experiment In some cases they can indicate potential processing errors that may have occurred or suggest that the data from particular microarrays might be compromised Many factors can influence the range of these metrics including the biological sample source quality of starting FFPE samples experimental processing scanner sensitivity and image processing The value guidelines presented below represent the thresholds that Agilent has observed when analyzing FFPE samples using this protocol Table9 QC metric thresholds for labeling FFPE Samples AnyColorPrentFeatNonUnifOL lt 1 gNegCtrlAveNetSig lt 100 gNegCtr
9. ith the Hyb Kit b Mix briefly on a vortex mixer and leave at room temperature for 60 minutes to reconstitute sample before use or storage c Cross out 10X on the label on the blocking agent vial and write 100X 2 Bring the Agilent CGHblock supplied with the ULS Labeling Kit to room temperature for use in step 9 Agilent CGHBlock contains components that cannot be heated to 95 C 3 Equilibrate water baths or heat blocks to 95 C 4 Mix the components below to prepare the Hyb Master Mix Table 8 Preparation of Hybridization Master Mix Components Per reaction pL Per 4 reactions pL including excess Nuclease free water 8 4 42 100X GE Blocking Agent 1 1 5 5 2x Hi RPM GE Hyb Buffer 55 275 Final Volume of Hyb Master Mix 64 5 322 5 Gene Expression FFPE Workflow Quick Start Guide 13 Procedure 5 Add 64 5 uL of the Hyb Master Mix to the 18 5 uL of ULS labeled cDNA from step 11 on page 12 for a total volume of 83 uL 6 Incubate at 95 C for 3 minutes then place on ice Spin samples briefly in a microcentrifuge to drive contents off the walls and lid 8 Make sure that the Agilent CGHblock is completely equilibrated to room temperature before you continue 9 Add 27 uL of Agilent CGHblock to each sample for a final volume of 110 uL and store at room temperature Make sure the samples are kept at room temperature until you dispense them onto the arrays The addition of Agilent CGHBlock to the hybridization
10. lAveBGSubSig 10 to 5 gNegCtrlSDevBGSubSig lt 10 gSpatialDetrendRMSFit lt 15 gNonCntrlMedCVProcSig Oto 8 Gene Expression FFPE Workflow Quick Start Guide 15 www agilent com In This Book The Quick Start Guide presents overview instructions to process FFPE RNA samples These instructions are based on the Gene Expression Microarray Analysis of Archival FFPE Samples Application Note p n 5990 3917EN Agilent Technologies Inc 2010 Version 1 0 August 2010 G4112 90000 Revision Al Ee Agilent Technologies
11. nual for the Stratagene Absolutely RNA FFPE Kit cat 400809 at http www stratagene com manuals 400809 pdf For the Proteinase K Digestion step of the Stratagene Absolutely RNA FFPE manual incubate the tubes at 55 C for 18 hours instead of 3 hours 6 Gene Expression FFPE Workflow Quick Start Guide Procedure Step 2 Prepare WTA Library 1 Thaw the WTA Library Synthesis Buffer and WTA Library Stabilization on ice and mix on a vortex mixer If either solution has a precipitate briefly heat at 37 C and mix the tube s on a vortex mixer until the precipitate is gone 2 Add nuclease free water to 25 to 300 ng of FFPE extracted RNA to get a total volume of 19 uL use tubes strips plates that will fit in a PCR thermal cycler 3 Prepare the following library preparation mix Table4 WTA Library Preparation Mix Components Per reaction pL Per 4 reactions pL including excess WTA Library Synthesis Buffer 2 5 12 5 WTA Library Stabilization Solution 2 5 12 5 Final Volume of Library Prep Master Mix 5 25 4 Add 5 uL of the Library Preparation Mix Table 4 to FFPE extracted RNA for a total volume of 24 uL 5 Mix samples well by pipetting and incubate at 70 C for 5 minutes 6 Cool reaction on ice and briefly centrifuge liquid to bottom of PCR plate 7 Add 1 uL of Library Synthesis Enzyme to each sample for a total volume of 25 uL and mix well by pipetting 8 Place PCR plate in thermal cycler and incubate as follows
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Agilent Gene Expression FFPE Workflow Quick Start Guide