USER GUIDE. Prelude. FFPE RNA Isolation Module PART NO. 1410

Transcription

1 USER GUIDE Prelude FFPE RNA Isolation Module PART NO. 1410

2 Patents, Licensing and Trademarks NuGEN Technologies, Inc. All rights reserved. The Encore, Ovation and Applause families of products and methods of their use are covered by several issued U.S. and International patents and pending applications ( NuGEN, Ovation, SPIA, Ribo-SPIA, Applause, Encore, Prelude, Mondrian and Imagine More From Less are trademarks or registered trademarks of NuGEN Technologies, Inc. Other marks appearing in these materials are marks of their respective owners. The purchase of this product conveys to the buyer the limited, non-exclusive, non-transferable right (without the right to modify, reverse engineer, resell, repackage or further sublicense) under these patent applications and any patents issuing from these patent applications to use this product and methods, accompanying this user guide, for research and development purposes solely in accordance with the intended use described and the written instructions provided in this user guide. No license to make or sell products by use of this product is granted to the buyer whether expressly, by implication, by estoppels or otherwise. In particular, the purchase of this product does not include or carry any right or license to use, develop or otherwise exploit this product commercially and no rights are conveyed to the buyer to use the product or components of the product for purposes including commercial services or clinical diagnostics. For information on purchasing a license to the NuGEN patents for uses other than in conjunction with this product or to use this product for purposes other than research, please contact NuGEN Technologies, Inc., 201 Industrial Road, Suite 310, San Carlos, CA Phone or ; FAX or Warranty NuGEN warrants that this product meets the performance standards described in the Company s product and technical literature for a period of six months from the date of purchase, provided that the product is handled and stored according to published instructions, and that the product is not altered or misused. If the product fails to meet these performance standards, NuGEN will replace the product free of charge or issue a credit for the purchase price. NuGEN s liability under this warranty shall not exceed the purchase price of the product. NuGEN shall assume no liability for direct, indirect, consequential or incidental damages arising from the use, results of use or inability to use its products. NuGEN reserves the right to change, alter or modify any product to enhance its performance and design. NuGEN s products are developed, designed and sold FOR RESEARCH USE ONLY. This product is not to be used for diagnostic or therapeutic purposes, nor is it to be administered to humans or animals. Except as expressly set forth herein, no right to modify, reverse engineer, distribute, offer to sell or sell NuGEN s product is conveyed or implied by buyer s purchase of this NuGEN product. The buyer agrees to use NuGEN products accompanying the product insert in accordance with the intended use and the written instructions provided.

3 Table of Contents Contents I. Introduction... 1 A. Background... 1 B. NuGEN s Purification Technology... 1 C. Performance Specifications... 3 D. Quality Control... 3 E. Storage and Stability... 3 F. Material Safety Data Sheet (MSDS)... 3 II. Kit Components... 4 A. Reagents and Supplies Provided... 4 B. Additional Equipment, Reagents and Labware... 5 III. Planning the Experiment... 6 A. FFPE Tissue Input Requirements... 6 B. Using RNase-free Techniques... 6 C. RNA Quality... 6 D. RNA Storage... 7 IV. Protocol... 8 A. Overview... 8 B. Deparaffinization... 9 C. Lysate Preparation D. Column Purification E. DNase Treatment F. Wash and Elute RNA G. Measuring RNA Concentration and Purity V. Technical Support VI. Appendix A. Frequently Asked Questions (FAQs) B. Update History... 16

4 I. Introduction A. Background The Prelude FFPE RNA Isolation Module (Part No. 1410) provides a rapid method for isolating and purifying total RNA from formalin-fixed paraffin-embedded (FFPE) tissue samples. Using formalin to fix tissues leads to crosslinking of the RNA and proteins, eventually leading to RNA fragmentation. The Prelude FFPE RNA Isolation Module provides an optimized protocol that allows for the partial reversal of the formalin modifications, resulting in a high yield while maximizing RNA sample integrity. The kit allows the recovery of RNA throughout a range of sizes, making it ideal for the isolation of intact or degraded mrna, ribosomal RNA, smaller microrna (mirna) and small interfering RNA (sirna) from FFPE tissues. The purified RNA can be used in a wide variety of downstream applications including qpcr, microarray and next-generation sequencing (RNA-Seq). The Prelude FFPE RNA Isolation Module contains spin columns and reagents required to process FFPE tissue samples and is available in kits for isolating total RNA from 60 samples. B. NuGEN s Purification Technology The Prelude FFPE RNA Isolation Module protocol (Figure 1) is based on spin column chromatography, using NuGEN s proprietary resin as the separation matrix. First the FFPE tissue is deparaffinized, followed by a Proteinase K digestion. The resulting lysate is then loaded onto the spin column. NuGEN s resin preferentially binds the total RNA in the sample, allowing the removal of contaminants. An on-column DNase treatment allows any remaining genomic DNA to be removed. The bound RNA is then washed, and eluted from the column. Advantages: Fast and easy processing using rapid spin-column format High yields of total RNA Isolates total RNA, including large rrna and microrna No phenol or chloroform extractions 1 Prelude FFPE RNA Isolation Module

5 I. Introduction Figure 1. The Prelude FFPE RNA Purification System Workflow. FFPE Thin Sections or FFPE Tissue Block Deparaffinize Prepare Lysate Bind RNA to Column On-column DNase Treatment Wash and Elute RNA Sample Assess Purity and Concentration of RNA Sample 2 Prelude FFPE RNA Isolation Module

6 I. Introduction C. Performance Specifications Table 1. Column Performance Characteristics COLUMN PERFORMANCE CHARACTERISTICS Maximum Column Binding Capacity (RNA) Maximum Column Loading Volume Maximum Input 50 μg 650 μl 5 sections <20 μm thick, or 25 mg of unsectioned block D. Quality Control Each lot of the Prelude FFPE RNA Isolation Module is tested to meet performance specifications. E. Storage and Stability This product contains components with multiple storage conditions The Prelude FFPE RNA Isolation Module is shipped at room temperature. The package should be unpacked immediately upon receipt. The DNase I should be stored at 20 C in a freezer without a defrost cycle upon arrival. The Proteinase K should be aliquoted into single use portions upon first use and the aliquots stored at 20 C. Do not refreeze the Proteinase K aliquots. Discard any unused portion after use. All remaining components should be stored tightly sealed at room temperature. The DNase I has been tested to perform to specifications after as many as five freeze/ thaw cycles. Kits handled and stored according to the above guidelines will perform to specifications for at least 12 months, unopened. F. Material Safety Data Sheet (MSDS) An MSDS for this product is available on the NuGEN website at 3 Prelude FFPE RNA Isolation Module

7 II. Kit Components A. Reagents and Supplies Provided Table 2. Lysate Preparation Reagents COMPONENT PART NUMBER Digestion Buffer Binding Solution Proteinase K Table 3. Purification Components and Reagents COMPONENT PART NUMBER Wash Solution RNA Elution Solution Spin Columns Collection Tubes Elution Tubes Table 4. Other Reagents COMPONENT PART NUMBER Enzyme Incubation Buffer DNase Note: The reagents in the Prelude FFPE RNA Isolation Module may be similar to reagents in our other kits; however, unless the part numbers are identical, these reagents do not have exactly the same composition and, therefore, are not interchangeable. Do not exchange or replace one reagent with another as it will adversely affect performance. 4 Prelude FFPE RNA Isolation Module

8 II. Kit Components B. Additional Equipment, Reagents and Labware Required Materials Equipment Microcentrifuge for individual 1.5 ml and 0.5 ml tubes μl pipette, 2 20 μl pipette, μl pipette, μl pipette Vortexer Appropriate spectrophotometer and cuvettes, or Nanodrop UV-Vis Spectrophotometer Three heating blocks or dry bath incubators (set to 50 C, 55 C and 80 C) Reagents 100% Ethanol (Sigma-Aldrich, Cat. #E7023) Xylene, histological grade (Sigma-Aldrich, Cat. #534056) ß-mercaptoethanol (Sigma-Aldrich, Cat. #63689) Supplies and Labware Nuclease-free pipette tips 1.5 ml and 0.5 ml RNase-free microcentrifuge tubes Disposable gloves Kimwipes Ice bucket RNaseZap (Ambion, Cat. #AM9780) Optional Equipment Agilent 2100 Bioanalyzer or materials and equipment for electrophoretic analysis of RNA To Order: Ambion Inc., Sigma-Aldrich, Inc., 5 Prelude FFPE RNA Isolation Module

9 III. Planning the Experiment A. FFPE Tissue Input Requirements Generally, FFPE tissue samples for RNA extraction should be taken from a previously unexposed portion of the tissue block. Oxidation can rapidly degrade RNA from exposed margins. Section the FFPE block into as many as five thin sections (<20 μm thick) using a microtome. Sections should be trimmed to minimize excess paraffin. Sections exceeding 20 μm thickness may not be efficiently digested in the Proteinase K step, resulting in lower yields of RNA. Alternatively, use up to 25 mg of unsectioned tissue from an FFPE block as input. Remove excess paraffin, then grind the tissue into fine powder after freezing with liquid nitrogen. B. Using RNase-free Techniques RNase contamination through reagents and work environment will lead to RNA degradation. Follow these guidelines to minimize contamination: Wear disposable gloves and change them frequently. Avoid touching surfaces or materials that could introduce RNases. Use reagents provided. Substitutions may introduce RNases. Clean and decontaminate work areas and instruments, including pipettes, with commercially available cleaning reagents, such as RNaseZap. Use only new RNase-free pipette tips and microcentrifuge tubes. Use a work area specifically designated for RNA work and do not use other high copy number materials in the same area. C. RNA Quality The Prelude FFPE RNA Isolation Module was designed and optimized for use with the highly crosslinked and degraded RNA found in FFPE tissue samples. A significant proportion of the degradation seen in FFPE RNA samples is a result of RNA shearing during the isolation process, due to the extensive, formalin-induced crosslinking present in such tissue samples. The Prelude FFPE RNA Isolation Module utilizes methods designed to reverse crosslinking to the extent possible, allowing the efficient isolation of FFPE RNA while minimizing additional shearing due to crosslinking. The quality of RNA obtained from FFPE tissues can be quite low based on traditional measures. Measures of RNA quality using rrna ratios, such as the Agilent 2100 Bioanalyzer RNA Integrity Number (RIN) and others, have not proven to be useful in predicting which FFPE RNA samples will be suitable for analysis. Some FFPE-derived RNA samples may be too severely compromised for use in gene expression analysis or other applications. 6 Prelude FFPE RNA Isolation Module

10 III. Planning the Experiment D. RNA Storage RNA samples prepared with the Prelude FFPE RNA Isolation Module must be stored at 80 C. To prevent degradation of your sample, avoid frequent freeze/thaw cycles. 7 Prelude FFPE RNA Isolation Module

11 IV. Protocol A. Overview The Prelude FFPE RNA Isolation Module protocol is performed in three stages: 1. Deparaffinization 0.5 hours 2. Lysate preparation 0.75 hours 3. Column purification 0.25 hours Total time to prepare FFPE RNA ~1.5 hours Protocol Notes All centrifugation steps are carried out in a benchtop microcentrifuge at a relative centrifugal force (RCF) of 16,100 X g at room temperature, except where specifically noted. Failure to perform the procedure with the appropriate RCF may lead to lower than expected yield or other performance issues. Do not assume the rotations per minute (RPM) setting on a centrifuge is interchangeable with RCF. Note that the relationship between RPM and RCF depends on the diameter of the rotor used. The RPM setting corresponding to a given RCF for a rotor of a given radius can be calculated using the formula: RPM = RCF (1.118 x 10-5 ) (r) where RPM = the number of revolutions per minute required to achieve the desired g-force; RCF = relative centrifugal force in units of g; and r = radius of the rotor in cm. After each column centrifugation step, all liquid should be cleared from the top of the column. If liquid remains on the column after centrifugation, check to make certain that the correct RCF has been used for the spin. Continue the centrifugation at the recommended RCF until all liquid has passed through the column. Ensure that all non-enzyme components are at room temperature prior to use. Enzymes should be stored at 20 C. During use, thaw enzymes and keep on ice unless otherwise noted. Upon first use, reconstitute the Proteinase K in the appropriate volume of molecular biology grade water as outlined in Table 5. Aliquot the reconstituted enzyme into single use portions and store at 20 C until needed. Prepare the Wash Solution by adding the appropriate volume of % ethanol to the concentrated Wash Solution stock as detailed in Table 8. Be sure to indicate on the Wash Solution bottle that the ethanol has been added. 8 Prelude FFPE RNA Isolation Module

12 IV. Protocol Add 1% ß-Mercaptoethanol to the Binding Solution. ß-Mercaptoethanol is toxic and should be dispensed in a fume hood. Note: ß-Mercaptoethanol is hazardous and should be handled in accordance with appropriate chemical safety standards and the manufacturer s recommendations. An MSDS may be consulted for specific details. The maximum recommended FFPE tissue input is five thin sections of <20 μm thickness, or 25 mg of unsectioned FFPE block. It is important to obtain the tissue sample from the interior of an FFPE block in order to avoid collecting RNA damaged by oxidation in the exposed margins. It is important to work efficiently during this procedure. Use a good quality, nuclease-free water prepared without DEPC. We do not recommend the use of DEPC-treated water with this protocol. Components and reagents from other NuGEN products should not be interchanged with the components supplied with this product. B. Deparaffinization Note: Instructions for deparaffinization are provided for a single sample. 1. Obtain xylene and 100% ethanol from room temperature storage. Do not exceed the recommended amount of tissue. Using too much tissue can overload the column, leading to clogging and sample loss. 2. Section the FFPE block into as many as five thin sections (<20 μm thick) using a microtome. Remove excess paraffin. Note: Alternatively, use up to 25 mg of unsectioned tissue from an FFPE block. Remove excess paraffin, then grind the tissue into fine powder after freezing with liquid nitrogen. 3. Transfer the prepared tissue sample into a clean, labeled, RNase-free 1.5 ml microcentrifuge tube. 4. Add 1 ml of xylene to the sample, mix by vortexing and spin. Note: Xylene is hazardous and should be handled in accordance with appropriate chemical safety standards and the manufacturer s recommendations. An MSDS may be consulted for specific details. 5. Incubate at 50 C for 5 minutes. 6. Centrifuge the sample at 16,100 X g for 2 minutes. 7. Carefully remove the xylene without disturbing the pellet. 8. Add 1 ml of 100% ethanol from a fresh stock. Mix by vortexing. 9. Centrifuge the sample at 16,100 X g for 2 minutes. 10. Carefully remove the ethanol without disturbing the pellet. 9 Prelude FFPE RNA Isolation Module

13 IV. Protocol 11. Repeat steps 8 10 once. 12. Air dry the pellet for up to 1 hour at room temperature. Ensure that all residual ethanol has evaporated. 13. Inspect each tube carefully to be sure that all the ethanol has evaporated. It is critical that residual ethanol be removed prior to Lysate Preparation. 14. Continue immediately with the Lysate Preparation protocol. C. Lysate Preparation 1. Obtain the Proteinase K from 20 C storage and the Digestion Buffer, Binding Solution, ß-Mercaptoethanol and 100% ethanol from room temperature storage. Note: Upon first use, you must reconstitute the Proteinase K by adding the appropriate volume of nuclease-free water as shown in Table 5. Table 5. Proteinase K Reconstitution 60 SAMPLES Proteinase K provided (mg) 14 Volume of Nuclease-free Water to add (µl) 700 The Proteinase K solution should be aliquoted in single use portions and stored at 20 C in a freezer without a defrost cycle. 2. Make a master mix by combining the Digestion Buffer and Proteinase K in an appropriately sized capped tube, according to the volumes shown in Table 6. Table 6. Digestion Master Mix (volumes listed are for a single sample) DIGESTION BUFFER PROTEINASE K 300 µl 10 µl 3. Add 310 μl of Digestion Master Mix to the sample and mix by vortexing. Do not exceed 15 minutes of incubation at 80 C as this may degrade the RNA. 4. Incubate at 55 C for 15 minutes, followed by 80 C for 15 minutes. Vortex to mix occasionally. 5. Make a master mix by combining the Binding Buffer and ß-Mercaptoethanol in an appropriately sized capped tube, according to the volumes shown in Table Prelude FFPE RNA Isolation Module

14 IV. Protocol Table 7. Binding Master Mix (volumes listed are for a single sample) BINDING BUFFER ß-MERCAPTOETHANOL 297 µl 3 µl 6. Add 300 μl of Binding Master Mix. Vortex to mix. 7. Add 600 μl of 100% ethanol. Vortex to mix. D. Column Purification 1. Upon first use, prepared the Wash Solution by adding % ethanol as outlined in Table 8. Refer to the label on the bottle for specifics and be sure to note ethanol addition using the check box provided on the Wash Solution. Table 8. Wash Solution Preparation 60 SAMPLES Volume of ethanol to add (ml) 63 Total volume (ml) 90 Obtain and label a spin column and place it into a collection tube. Obtain the DNase I from 20 C storage and the Wash Solution, Enzyme Incubation Buffer, and RNA Elution Solution from room temperature storage. 2. Load one half (600 μl) of the lysate mixture onto the column. If liquid remains on the column after a centrifugation step, check to make certain that the correct RCF has been used for the spin. Continue the centrifugation at the recommended RCF until all liquid has passed through the column. Make sure the appropriate volume of 100% ethanol has been added to the Wash Solution prior to first use. 3. Centrifuge for 1 minute at 16,100 X g in a microcentrifuge. 4. Discard the flow-through and replace the column in the same collection tube. 5. Load the remaining lysate mixture onto the column. 6. Centrifuge for 1 minute at 16,100 X g. 7. Discard the flow-through and replace the column in the same collection tube. 8. Apply 400 μl of Wash Solution to the column. 9. Centrifuge for 2 minutes at 16,100 X g. 10. Discard the flow-through and replace the column in the same collection tube. 11. Continue immediately with the DNase Treatment protocol. 11 Prelude FFPE RNA Isolation Module

15 IV. Protocol E. DNase Treatment 1. Make a master mix by combining the Enzyme Incubation Buffer and DNase I in an appropriately sized capped tube, according to the volumes shown in Table 9. Table 9. DNase I Master Mix (volumes listed are for a single sample) ENZYME INCUBATION BUFFER DNase I 96 µl 4 µl 2. Apply 100 μl of DNase I Master Mix to the column. 3. Centrifuge at 16,100 X g for 1 minute. 4. Pipet the flow-through in the collection tube back onto the column. Do not centrifuge. Note: In order to ensure maximum DNase I activity, it is important to carry out both passages of the DNase I Master Mix described in steps 2 4 above. Failure to do so may result in suboptimal yields, especially of smaller RNA fractions. 5. Incubate at room temperature for 15 minutes. 6. Continue immediately with the Wash and Elute RNA protocol. F. Wash and Elute RNA 1. Add 400 μl of Wash Solution to the column. 2. Centrifuge for 1 minute at 16,100 X g. 3. Discard the flow-through and replace the column in the same collection tube. 4. Repeat steps 1 3 once more. 5. Centrifuge the column for an additional 2 minutes at 16,100 X g to remove all residual Wash Solution and dry the resin. Note: The resin will not dry completely unless the flow-through is discarded before this additional centrifugation. 6. Place the column into a clean, labeled elution tube. 7. Add 50 μl of the provided Elution Solution to the column. 8. Centrifuge the column for 2 minutes at 200 X g, followed by 1 minute at 16,100 X g. Note the volume eluted from the column. Note: A second elution step may be carried out by repeating steps 6 8. This will maximize RNA recovery. Use a second elution tube in order to minimize dilution of the more concentrated first elution. 12 Prelude FFPE RNA Isolation Module

16 IV. Protocol 9. The purified RNA should be stored at 80 C. G. Measuring RNA Concentration and Purity 1. Mix the RNA sample gently prior to taking the spectrophotometry readings. 2. Measure the absorbance at 260, 280 and 320 nm. You may need to make a 1:20 dilution of the cdna in water prior to measuring the absorbance. 3. Blank the spectrophotometer using the appropriate buffer (RNA Elution Solution or RNA Elution Solution diluted in water, etc.) It is important to blank with the same buffer that the RNA samples will be in for spectrophotometry. 4. Purity: Subtract the A320 value from both A260 and A280 values. The adjusted (A260 A320) / (A280 A320) ratio should be > Yield: Assume 1 A260 unit = 40 µg/ml. To calculate: (A260 A320 of diluted sample) X (dilution factor) X 50 (concentration in µg/ml of a 1 A260 unit solution) X 0.03 (final volume in ml) = total yield in micrograms Note: Alternatively, you may measure the concentration and purity of cdna with a Nanodrop using the RNA setting, or by manually setting 1 A260 unit = 40 µg/ml as the constant. 6. The RNA sample should be stored at 80 C. 13 Prelude FFPE RNA Isolation Module

17 V. Technical Support For help with any of our products, please contact NuGEN Technical Support at (direct) or , option 2 (toll-free, U.S. only). Send faxes to (toll-free) or techserv@nugen.com. In Europe contact NuGEN at +31(0) (Phone) or +31(0) (Fax) or europe@nugen.com. In all other locations, contact your NuGEN distributor for technical support. 14 Prelude FFPE RNA Isolation Module

18 VI. Appendix A. Frequently Asked Questions (FAQs) Q1. What is the expected yield from the Prelude FFPE RNA Isolation Module? Yield depends on tissue cellularity and can vary greatly from sample to sample. The column binding capacity is 50 μg. Q2. Does the Prelude FFPE RNA Isolation Module efficiently isolate small RNAs such as micrornas? Yes. Total RNA including micrornas will be efficiently isolated. If you wish to maximize microrna recovery, it is essential to carry out the DNase treatment protocol. Q3. With which NuGEN kits is the Prelude FFPE Isolation Module compatible? The extracted, purified RNA can be used directly with the Ovation FFPE WTA System (Part No. 3403), the Ovation RNA-Seq FFPE System (Part No. 7150) and the Ovation Human FFPE RNA-Seq System (Part Nos and 0341). Q4. Is the Prelude FFPE RNA Isolation Module compatible with other manufacturer s kits? The output is purified RNA and is therefore compatible with other manufacturer s reagents and assays. If you have questions regarding a specific application, please contact NuGEN Technical Support. Q5. Can the Prelude FFPE RNA Isolation Module be used with non-ffpe RNA? The kit has been optimized for use with FFPE tissue and is not recommended for use with non-ffpe samples. Q6. Can the Prelude FFPE RNA Isolation Module be used with microtome cut sections thicker than 20 μm? We do not recommended using sections thicker than 20 μm as doing so can result in incomplete Proteinase K digestion and deparaffinization. If it is necessary to work with sections thicker than 20 μm, we recommend using a liquid nitrogen based homogenizer. FFPE tissue samples up to 25 mg may be processed in this manner. Q7. Can genomic DNA be isolated with the Prelude FFPE RNA Isolation Module? No. The kit is designed to isolate total RNA, not DNA. 15 Prelude FFPE RNA Isolation Module

19 VI. Appendix B. Update History This document, the Prelude FFPE RNA Isolation Module user guide (M01240 v3) includes the following updates: Description Section Pages Removed references to 12 and 50 samples Throughout Throughout Removed references to Part Nos and Throughout Throughout Updated web address Throughout Throughout NuGEN Technologies, Inc. Headquarters USA 201 Industrial Road, Suite 310 San Carlos, CA USA Toll Free Tel: Toll Free Fax: Europe P.O. Box AC Leek The Netherlands Tel: Fax: For our international distributors contact information, visit our website NuGEN Technologies, Inc. All rights reserved. The Encore, Ovation and Applause families of products and methods of their use are covered by several issued U.S. and International patents and pending applications ( NuGEN, Ovation, SPIA, Ribo-SPIA, Applause, Encore, Prelude, Mondrian and Imagine More From Less are trademarks or registered trademarks of NuGEN Technologies, Inc. Other marks appearing in these materials are marks of their respective owners. For research use only. M01240 v3