RNAlater® Tissue Collection: RNA Stabilization Solution User Guide
Contents
1. and plasma however the procedure is not presented here see the Ambion RiboPure Blood Kit Cat no AM1928 protocol for detailed instructions RNAlater Tissue Collection RNA Stabilization Solution Protocol 5 Storage in RNAlater Solution Yeast Bacteria Pellet up to 3 x 108 cells centrifuge at 12 000 x g for 2 min Remove supernatant and immediately resuspend the pellet in 0 5 1 mL of RNAIater Solution Yeast cells can be stored in RNAlJater Solution for up to 8 hr at 25 C or up to a week at 4 C For long term storage incubate the cells in RNAlater Solution for 1 hr Repellet the cells centrifuge at gt 12 000 x g for 5 min remove supernatant flash freeze and store at 80 C RNAlater Solution is bacteriostatic although bacteria do not grow in it the cells remain intact E coli stored in RNAlater Solution for 1 month at 4 C are intact and yield undegraded RNA Storage in RNAlater Solution If refrigeration is available Storage at 80 C Storage at 80 C is recommended for archival samples and will provide optimal preservation Samples can be stored at 80 C indefinitely RN Alater Solution will freeze at 80 C To prepare samples for storage at 80 C first incubate the samples in RNAlater Solution overnight at 4 C to allow thorough penetration of the tissue then transfer to 80 C To expedite thawing of the samples we recommend removing the tissue or pelleting cells f2. equal mass amounts of each tissue using the indicated methods RNA 5 yg was run on denaturing agarose and stained with ethidium bromide N uN uw BUD o oS in DNA can be isolated from RNA later Solution stored samples For more information go to www lifetechnologies com support Proteins are also preserved in RNAlater Solution RNAlater Solution will denature proteins therefore protein obtained from samples stored in it will be suitable for applications such as Western blotting or 2D gel electrophoresis but not for applications that require native protein Guidelines for use of RNAlater Solution e Use RNAlater Solution with fresh tissue only do not freeze tissues before immersion in RNAlater Solution e Before immersion in RNAlater Solution cut large tissue samples to 9 5 cm in any single dimension e Place the fresh tissue in 5 10 volumes of RNAlater Solution RNAlater Tissue Collection RNA Stabilization Solution Protocol Animal tissue Plant tissue Tissue culture cells Blood and plasma Guidelines for use of RNAlater Solution e Most samples in RNAlater Solution can be stored at room temperature for 1 week without compromising RNA quality or at 20 C or 80 C indefinitely e Do not freeze samples in RNAlater Solution immediately store at 4 C overnight to allow the solution to thoroughly penetrate the tissue remove supernatant then move to 20 C or 80 C for lon
3. mouse tissues stored in RNAlater Solution as shown The top panel is an ethidium bromide stained denaturing agarose gel the bottom panel shows a Northern blot of the same gel 37 C for day RT for week 4 C for month 28S rRNA 4 8 kb 18S rRNA 1 8 kb wee cso ouo one GAPDH 1 4 kb Liver Spleen Kidn Liver Spleen Kidney Liver Spleen Kidney Figure 2 mRNA Profiles of Mouse Tissues Stored in RNAlater Solution Mouse tissues were stored in RNAlater Solution for 1 or 4 weeks at 4 C RNA was isolated from each tissue and analyzed using the Ambion RPA III Kit The data demonstrate the stability of expression profiles in tissue stored in RNAlater Solution g s Kidney Liver Spleen gs of Protected Probe Iwk 4wk Iwk 4wk Iwk 4wk Full length Fragments Probe c myc c myc p53 p53 a e Egr Ezri ras cyclophilin a oe cyclophilin o ae E a RNAlater Tissue Collection RNA Stabilization Solution Protocol Product information Product information Storage and stability Sample types compatible with RNAlater Solution Compatible RNA isolation methods RNAlater Tissue Collection RNA Stabilization Solution Protocol e Store RNAlater Solution at room temperature e If any precipitation of RNAlater Solution is seen heat it to 37 C and agitate to redissolve it e Ifthe crystals do not go into solution at 37 C loosen the cap
4. vacuum pressure to pass lysates through glass fiber filters RNAlater Tissue Collection RNA Stabilization Solution Protocol Quality control Quality control One step disruption extraction solutions When using one step RNA isolation products such as TRI Reagent Solution or RNAWIZ Reagent available only in Japan on RNAlater Solution preserved samples the aqueous phase will occasionally appear cloudy this will not adversely affect RNA recovery or quality With RNAWIZ Reagent there may be a problem getting the aqueous phase to mix with isopropanol at the precipitation step because of RNAlater Solution carryover If this occurs add a mixture of 50 water 50 isopropanol until the solution becomes clear and the two phases mix The amount of water isopropanol required will depend on how much RNAlater Solution was carried over if the sample was mostly RNAlater Solution as much as an equal volume may be needed RNAlater Solution undergoes quality assurance testing to verify that its composition is invariant from lot to lot Appendix A Safety information Chemical safety AN WARNING GENERAL CHEMICAL HANDLING To minimize hazards ensure laboratory personnel read and practice the general safety guidelines for chemical usage storage and waste provided below and consult the relevant SDS for specific precautions and instructions e Read and understand the Safety Data Sheets SDSs provided by the chemical manufactu
5. USER GUIDE ambion RNA vy Life vocrnatoges RNAlater Tissue Collection RNA Stabilization Solution Catalog Number AM7020 AM7021 AM7022 AM7023 AM7024 IMPORTANT Before using this product read and understand the Safety Information appendix in this document Product description 6 66 c cece eee 1 Product information 0 cee cece eee eee 3 Guidelines for use of RNAlater Solution 4 Storage in RNAlater Solution 0 00 0e cece eens 6 RNA isolation from samples in RNAlater Solution 7 Quality control iiss cc cee ee ea seen eae brine saw e new 9 Appendix A Safety information 0000008 9 Documentation and support 0 cece eee eee 11 Product description RNAlater Tissue Collection RNA Stabilization Solution is an aqueous tissue storage reagent that rapidly permeates most tissues to stabilize and protect RNA in fresh specimens It eliminates the need to immediately process or freeze samples the specimen can simply be submerged in RNAlater Solution and stored for analysis at a later date Samples in RNAlater Solution can be stored for extended periods under conditions where RNA degradation would normally take place rapidly Figures 1 and 2 Tissues can be stored indefinitely in RNAlater Solution at 20 C or below oe technologies Product description Figure 1 RNA from Tissue Stored in RNAlater Solution RNA was extracted from
6. active or biohazardous materials may require special handling and disposal limitations may apply A WARNING Depending on the samples used on the instrument the surface may be considered a biohazard Use appropriate decontamination methods when working with biohazards A WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following RNAlater Tissue Collection RNA Stabilization Solution Protocol Documentation and support e U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at www cdc gov biosafety e Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 www access gpo gov nara cfr waisidx_01 29cfr1910a_01 h
7. and heat the solution at a higher temperature up to 65 C for 30 minutes mixing periodically Once the crystals have dissolved store the solution in smaller aliquots in case crystals re form after cooling Warming the solution in this way does not affect performance of RNAlater Solution RNAlIater Solution can be used for RNA preservation with most tissues cultured cells bacteria and yeast It may not be effective in tissues that are poorly penetrated by the solution such as waxy plant tissue and bone RNAlater Solution has been extensively tested with animal tissues including brain heart kidney spleen liver testis skeletal muscle fat lung and thymus It has also been proven effective for RNA preservation in E coli Drosophila tissue culture cells white blood cells and some plant tissues RNAlIater RNA Stabilization Solution is compatible with one step RNA isolation methods as well as methods that use glass binding acid phenol extraction or oligo dT selection of mRNA see Figure 3 Guidelines for use of RNAlater Solution Isolating genomic DNA from RNAlater Solution stored samples Isolating protein from RNAlater Solution stored samples Figure 3 RNA isolated from tissue stored in RNAlater Solution using different methods Whole mouse hearts left lane of each set and livers right lane of each set were dissected and stored in RNAlater Solution for 3 days at 4 C RNA was isolated from
8. ars slightly degraded marginally acceptable for Northern analysis but still of sufficient quality for nuclease protection assays or RT PCR analysis Storage at 37 C RNA isolated from samples stored at 37 C is intact after a 24 hour incubation but is partially degraded after 3 days RNA isolation from samples in RNAlater Solution Remove RNAlater Solution from samples RNase inactivation is reversible do not rinse RNAlater Solution from samples before using Blot tissues with a wipe or pellet cells to remove excess RNAlater Solution Tissue Retrieve tissue from RNAlater Solution with sterile forceps quickly blot away excess RNAlater Solution with an absorbent lab wipe or paper towel and then submerge the sample in RNA isolation lysis solution Homogenize tissue promptly after placing it in lysis denaturation solution RNAlater Tissue Collection RNA Stabilization Solution Protocol 7 RNA isolation from samples in RNAlater Solution Tips for RNA isolation Cells There are two options for isolating RNA from cells stored in RNAlater Solution The preferred method is to remove the solution from the cells prior to extraction Alternatively cells in RNAlater Solution can be used directly for RNA extraction Because of the greater volume that the cells are in this method generally requires additional lysis solution e Removal of RNAlater Solution prior to extraction Because of the density of RNAlJa
9. d questions FAQs e Search for user documents SDSs vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents e Obtain information about customer training e Download software updates and patches Limited Product Life Technologies Corporation and or its affiliate s warrant their Warranty products as set forth in the Life Technologies General Terms and Conditions of Sale found on Life Technologies website at www lifetechnologies com termsandconditions If you have any questions please contact Life Technologies at www lifetechnologies com For Research Use Only Not for use in diagnostic procedures The information in this guide is subject to change without notice DISCLAIMER LIFE TECHNOLOGIES CORPORATION AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT TO THE EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF Important Licensing Information This product may be covered by one or more Limited Use Label Lic
10. enses By use of this product you accept the terms and conditions of all applicable Limited Use Label Licenses 2014 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified Part Number 7020M Rev G 15Jul2014 Headquarters 5791 Van Allen Way Carlsbad CA 92008 USA Phone 1 760 603 7200 Toll Free in USA 800 955 6288 ld For support visit www appliedbiosystems com support technologies www lifetechnologies com
11. g term storage Note We offer RNAlater ICE Cat no AM7030 to recover tissues that have already been frozen RNAlater ICE renders frozen tissues pliant enough for homogenization while maintaining the low temperatures needed to protect the RNA from degradation RNAlIater Solution does not disrupt the structure of tissues thus tissue that has been equilibrated in RNAlater Solution can be removed from the solution sectioned into smaller pieces and returned to RNAlater Solution if desired Small organs such as mouse liver kidney and spleen can be stored whole in RNAlater Solution Plant tissues that have natural barriers to diffusion such as waxy coatings on leaves will often require disruption to allow RNAlater Solution access to the tissue However many plant tissues can simply be submerged in RNAlater Solution whole we have successfully isolated intact RNA from tobacco leaf explants entire Arabidopsis and alfalfa seedlings and from potato shoot tips Pellet cells according to the protocols followed by your laboratory Remove supernatant and then add 5 10 volumes RNAlater Solution The cells can be washed in PBS before resuspending in RNAlater Solution if desired White blood cells can be effectively preserved in RNAlater Solution when separated from the red blood cells and sera and treated as tissue culture cells RNAlater Solution can also be added to small volumes of anticoagulated whole blood sera
12. rer before you store handle or work with any chemicals or hazardous materials To obtain SDSs see the Documentation and Support section in this document e Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing e Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood RNAlater Tissue Collection RNA Stabilization Solution Protocol 9 Quality control Biological hazard safety 10 Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS Handle chemical wastes in a fume hood Ensure use of primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage After emptying a waste container seal it with the cap provided Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure that the waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radio
13. rom the RNAlater Solution before freezing at 80 C Samples can subsequently be thawed at room temperature and refrozen without significantly affecting the amount or the integrity of the recoverable RNA Storage at 20 C Storage at 20 C can also be used for archival samples Samples will not freeze at 20 C but crystals may form this will not affect subsequent RNA isolation Samples can be stored at 20 C indefinitely To prepare samples for storage at 20 C first incubate the samples in RNAlater Solution overnight at 4 C to allow thorough penetration of the tissue then transfer to 20 C Samples can subsequently be thawed at room temperature and refrozen without affecting the amount or the integrity of the recoverable RNA RNAlater Tissue Collection RNA Stabilization Solution Protocol If refrigeration is not available RNA isolation from samples in RNAlater Solution Storage at 4 C Most samples can be stored in RNAlater Solution at 4 C for up to 1 month without significant RNA degradation Place samples in the coolest environment available If ambient temperature is above 25 C incubate the samples in RNAlater Solution on ice for a few hours if possible before storing at ambient temperature Storage at 25 C room temperature Most samples can be stored at 25 C in RNAlater Solution for up to 1 week without significant loss of RNA quality After 2 weeks at 25 C RNA generally appe
14. ter Solution greater centrifugal forces are required to pellet cells from RNAIater Solution than from normal media Generally cells become much less fragile when stored in RNAlater Solution and can be centrifuged at high speed without lysis Most cell types can be centrifuged at 5000 x g without damage to the cells Since different cell types vary in their ability to withstand centrifugal forces we recommend testing the centrifugal speed with an expendable sample Alternatively dilute the RNAlater Solution by adding an equal volume of ice cold PBS or other buffered solution immediately before centrifugation to reduce the density of the solution then centrifuge at normal speeds e RNA extraction from cells in RNAlater Solution One step phenol based disruption extraction solutions such as Ambion TRI Reagent Solution or RNAwiz Reagent available only in Japan can be used to purify RNA from cells suspended in RNAlater Solution This can be done by adding ten volumes of the one step solution to the cell mixture and proceeding normally When RNAwiz Reagent is used in this way it may be necessary to dilute the aqueous phase before the RNA precipitation step See below for more information Glass fiber based extraction Lysates from RNAlater Solution treated samples often require more force to pass through glass fiber filters than lysates from untreated samples Therefore it may be necessary to use centrifugation instead of
15. tml e Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials e Additional information about biohazard guidelines is available at www cdc gov In the EU Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization WHO Laboratory Biosafety Manual third edition found at www who int csr resources publications biosafety WHO_CDS_CSR_LYO_2004_11 en Documentation and support Obtaining SDSs Obtaining Certificates of Analysis Safety Data Sheets SDSs are available from www lifetechnologies com suppotrt Note For the SDSs of chemicals not distributed by Thermo Fisher Scientific contact the chemical manufacturer The Certificate of Analysis provides detailed quality control and product qualification information for each product Certificates of Analysis are available on our website Go to www lifetechnologies com support and search for the Certificate of Analysis by product lot number which is printed on the box Obtaining Support For the latest services and support information for all locations go to www lifetechnologies com support RNAlater Tissue Collection RNA Stabilization Solution Protocol 11 At the website you can e Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities e Search through frequently aske
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