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1. speed for 5 minutes at room temperature to pellet the tissue c Without disturbing the pellet completely remove the supernatant and discard NOTE If rehydrated tissue does not form a compact pellet increase centrifugation time to 15 minutes Return to the procedure on page 2 to solubilize the tissue and complete sample preparation Package Insert 5 US Headquarters European Headquarters Affymetrix Inc Panomics Srl 3420 Central Expressway Via Sardegna 1 Santa Clara CA 95051 20060 Vignate Milano Italy Tel 1 888 362 2447 Tel 39 02 95 360 250 Direct 1 408 731 5000 Fax 39 02 95 360 992 Fax 1 408 731 5380 Email info_europe affymetrix com Email info affymetrix com Email order_europe affymetrix com Email orders_fremont affymetrix com Email techsupport_europe affymetrix com Email pqbhelp affymetrix com Asia Pacific Headquarters Affymetrix Singapore Pte Ltd No 7 Gul Circle 2M 01 08 Keppel Logistics Building Singapore 629563 Tel 65 6395 7200 Fax 65 6395 7300 Email info_asia affymetrix com Email order_asia affymetrix com Email techsupport_asia affymetrix com www affymetrix com Please visit our website for international distributor contact information For research use only Not for use in diagnostic procedures P N 13059 Rev D 100616 2010 Affymetrix Inc All rights reserved Affymetrix is a registered trademark of Affymetrix Inc QuantiGene is a registered trademark e2. Id Package Insert Affymetrix Revolutionize life QuantiGene Sample Processing Kit FFPE Tissues About Sample Processing Kits Sample Processing Kits are designed for use with both single plex and multiplex QuantiGene assays for quantification of RNA or DNA targets directly from a variety of sample types About this Kit This QuantiGene Sample Processing Kit for FFPE Tissue Homogenates contains reagents and instructions for the preparation of tissue homogenates from FFPE tissue sections for use in QuantiGene 2 0 and QuantiGene Plex 2 0 assays for RNA targets and QuantiGene Plex DNA assays for DNA targets For more information refer to the appropriate QuantiGene Reagent System User Manual Fr IMPORTANT For quantitating RNA targets we highly recommend the use of QuantiGene 2 0 Sample Assessment Kit to evaluate relative cell number and RNA quality of FFPE tissue homogenates For more information see the QuantiGene 2 0 Sample Assessment Kit Package Insert Contents and Storage Kit components have a shelf life of 12 months from the date of delivery Table 1 Kit contents and storage conditions Cat No 050107 Q50108 050109 Kit Size 10 Samples 25 Samples 100 Samples Storage Component Quantity Quantity Quantity Homogenizing Solution 10 mL 20 mL 75 mL 15 30 C Proteinase Kt 50 ug uL 36 uL 90 uL 360 uL 20 C A sample is defined as 25 100 mm x 50 60 um total thickness of FFPE tissue sections
3. h microfuge tube avoiding any residual paraffin and debris Repeat if necessary to completely remove debris NOTE Any residual paraffin will solidify at room temperature during centrifugation and may appear as a Solid residue above the homogenate It might be necessary to pierce the solid paraffin layer with a pipette tip in order to transfer the homogenate Discard the pipette tip if it becomes clogged 4 Use the homogenate immediately in a QuantiGene or QuantiGene Plex assay or store at 80 C for future use Determining Complete Tissue Homogenization We strongly recommend you validate your homogenate by doing the following Examine the homogenate It should be clear and non viscous Perform a serial dilution of the homogenate and run an appropriate QuantiGene or QuantiGene Plex assay with it Verify the expected fold change matches the observed fold change For example a 3 fold dilution should generate 3 fold changes 20 in the signal background subtracted of the targeted genes Clarifying Homogenates When using the QuantiGene Plex assay with Filter Plates it is very important that all extracellular debris is removed from the cell lysate Failure to remove particulates might result in clogged wells on the Filter Plate following the overnight hybridization step which could lower assay precision NOTE If using magnetic separation clarification of samples is rarely necessary Required Materials Table 4 Requi
4. on Re dissolve by incubating at 37 C followed by gentle swirling To dewax FFPE samples 1 2 Measure and transfer tissue A Measure the length L and width W of the tissue and calculate the cross sectional area L x W in square millimeters mm2 B Using a clean scalpel completely scrape the tissue from the slide s and transfer it to a microcentrifuge tube Avoid transferring excess paraffin De wax the sample FH IMPORTANT Add 100 ethanol according to the label to concentrated Dewaxing Solution before use Add 1 mL Dewaxing Solution and vortex for 30 seconds Incubate for 5 minutes at room temperature B Spin the sample in a microcentrifuge at maximum speed for 5 minutes at room temperature to pellet the tissue c Without disturbing the pellet remove the supernatant and discard Add 1 mL 70 ethanol and vortex for 30 seconds Incubate for 2 minutes at room temperature NOTE Prepare 70 ethanol fresh weekly from 100 ethanol and nuclease free water E Spin the sample in a microcentrifuge at maximum speed for 5 minutes at room temperature to pellet the tissue F Without disturbing the pellet remove the supernatant and discard Repeat step 2 one or more times until paraffin is visibly reduced It is not necessary to completely remove the paraffin Rinse tissue to remove all traces of ethanol A Gently add 1 mL nuclease free water Do not mix B Spin in a microcentrifuge at maximum
5. r scalpel scrape the slide to completely remove the FFPE section and transfer it to a 1 5 mL microfuge tube Avoid transferring excess paraffin r IMPORTANT For best results ensure adequate sample input by combining the equivalent of 50 60 um thick x 25 100 mm area of tissue For example if sections are 10 um in thickness combine 5 6 sections into a single microfuge tube NOTE H amp E stained slides are compatible with QuantiGene and QuantiGene Plex assays NOTE Optional See Dewaxing of FFPE Samples Optional on page 4 for dewaxing of samples 2 Solubilize the tissue A Using the volumes specified in the tables below add Homogenizing Solution and Proteinase K to the tissue For tissue sections 50 60 um combined total thickness Table 3 Tissue input for preparing homogenates Tissue Area mm Homogenizing Solution pL Proteinase K Volume pL 25 100 300 3 100 225 600 6 gt 225 900 9 B If quantitating DNA targets incubate the samples at 65 C for 30 minutes to 1 hour Briefly vortex the samples then return them to 65 C overnight 16 20 hours cC If quantitating RNA targets incubate the samples at 65 C for 6 hours For every hour of incubation vortex samples for 1 minute at maximum speed Package Insert 3 3 Centrifuge the samples in a microfuge at maximal speed for 5 minutes at room temperature to pellet the cellular debris then transfer the homogenate to a fres
6. red materials for clarifying lysates Item Source 0 45 um cellulose nitrate filter Affymetrix P N PC5512 or Whatman P N 7700 3307 plate 96 well polypropylene plate Fisher P N 07 201 156 Corning 3371 collection plate Adhesive plate seal Major laboratory supplier Microplate centrifuge Eppendorf 5804R and rotor A 2 DWP or equivalent Procedure To clarify homogenates 1 Determine the number of wells to use on the cellulose nitrate filter plate based on the number of samples and volume prepared for each sample Seal the wells that will not be used with an adhesive plate seal Fi IMPORTANT Do not add more than 300 pL well 2 Add the samples to the 0 45 um cellulose nitrate filter plate Place cellulose nitrate plate with samples on top of the collection plate 4 Spin the nitrate plate collection plate assembly in the microplate centrifuge at 1 444 x g for 2 5 minutes at room temperature If the sample has not filtered through the cellulose plate spin an additional 2 3 minutes 5 Use lysates immediately in a QuantiGene or QuantiGene Plex assay or seal the plate with an adhesive seal and store at 80 C for later use w 4 Package Insert Dewaxing of FFPE Samples Optional Table 5 Required materials for dewaxing Item Source Concentrated Dewaxing Solution Affymetrix P N QS0508 Add 100 ethanol before use Procedure NOTE Precipitates may form in the De Waxing Soluti
7. t Place on ice during use We recommend storage at 20 C in an enzyme storage box for example NEB Cool Box New Eng land Biolabs P N T04005 NEVER store at 80 C Safety Warnings and Precautions All chemicals should be considered potentially hazardous We recommend that this product and its components be handled by those trained in laboratory techniques and be used according to the principles of good laboratory practice What this Package Insert Covers This package insert provides recommendations and step by step procedures for the following a Materials Required but not Supplied Preparing FFPE Tissue Homogenates a Determining Complete Tissue Homogenization Clarifying Homogenates a Dewaxing of FFPE Samples Optional 2 Package Insert Materials Required but not Supplied Table 2 Required materials not supplied Item Source RNase Zap if quantifying RNA Ambion P N AM0780 Disposable razor blades or scalpels Major laboratory supplier Preparing FFPE Tissue Homogenates Before You Start If you are planning to quantitate RNA targets treat all surfaces with RNase Zap according to the manufacturer s instructions Procedure To prepare tissue homogenates from FFPE tissue sections 1 Measure and transfer the tissue For FFPE tissue sections A Measure the length L and width W of the tissue and calculate the cross sectional area L x W in square millimeters mm B Using a clean razor blade o
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