E.Z.N.A.®miRNA Kit - Omega Bio-Tek
Contents
1. eukaryotic cells 1 x 10 bacterial cells 50 mg animal tissue or 100 mg plant tissue can be used in a single experiment RNA purified using the E Z N A Micro RNA method is ready for applications such as RT PCR Northern blotting and nuclease protection assays Overview The E Z N A Micro RNA Kit combines the reversible binding properties of HiBind matrix a new silica based material with the highly efficient lysis ability of RNA Solv to extract micro and large gt 200 nt RNA from a wide variety of starting materials A specially formulated high salt buffer system allows more than 100 yg of RNA to bind to the matrix Cells or tissues are first homogenized with RNA Solv Reagent that inactivates RNases After the addition of chloroform the homogenate is separated into aqueous and organic phases The aqueous phase which contains the RNA is adjusted with ethanol and loaded onto a HiBind RNA Mini Column that binds the large RNA The miRNA is filtered through the column and collected Large RNA subsequently can be eluted from the HiBind RNA Mini Column with DEPC Water The miRNA filtrate is loaded onto a HiBind Micro RNA Column so that proteins and other contaminates can be removed Purified miRNA is eluted with DEPC Water New in this Edition This manual has been edited for content and redesigned to enhance user readability Kit Contents R7034 00 R7034 01 R7034 02 Purifications 50 preps 200 preps 00 00 00 v2. Ca OMEGA Innovations in nucleic acid isolation bio tek Product Manual E Z N A miRNA Kit R7034 00 5 preps R7034 01 50 preps R7034 02 200 preps August 2011 For research use only Not intended for diagnostic testing E Z N A Micro RNA Kit Table of Contents Introduction and OVEFVIEW ssccssccsserecssscssecssecnsecseecseceneessees 2 Kit Contents Storage and Stability essecsecssecssecseecseers 3 Preparing Reagents essseesssseessseerssseeesseeessscesssseosssesssesssseseossss 4 Before BEGiNNingd ssssssscssscsscsssecsecssecssccssessecessesseessscsseeseecnseessess 5 Tissue Homogenization PLOtOCOIS ccsesssecsssneesseseesneeseeees 6 Micro RNA from Cells TiSSUeC cssssssssesssccsssseesessecscseeeseeseesees 7 Large RNA from the HiBind RNA Mini Column 11 DNase I Digestion Protocol ecssecsssssssecssssessessncssssseseeseesees 13 Troubleshooting Guide ssesssssssersssssssssseeeserssssssssrreseessnsssss 16 Orderin a E ERES 17 Manual Revision August 2011 024 OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview Introduction E Z N A Micro RNA Kit provides a rapid and easy method for the isolation of up to 50 ug of small and large size RNA from cultured eukaryotic cells or bacteria or from animal plant or fungal tissues Single or multiple samples can be simultaneously processed in less than 30 minutes Typically up to 1 x 10
3. HiBind Micro RNA Column HiBind RNA Mini Column 2 mL Collection Tubes RNA Wash Buffer Note RNA Solv Reagent contains guanidine thiocyanate and phenol handle this reagent with extra care Safety and risk phase R20 24 25 32 34 S13 26 36 37 39 45 Storage and Stability All components in E Z N A Micro RNA Kits except the RNA Solv Reagent should be stored between 22 25 C RNA Solv Reagent should be store between 2 8 C for long term storage Preparing Reagents Dilute RNA Wash Buffer II with 100 ethanol as follows and store at room temperature OoOo e 100 Ethanol To Be Added R7034 01 48 mL per bottle R7034 02 200 mL per bottle Important Notes Please take a few minutes to read this booklet in its entirety to become familiar with the procedures Prepare all materials required before starting to minimize RNA degradation Whenever working with RNA always wear gloves to minimize RNase contamination Use only clean RNase free disposable plastic pipette tips when using the supplied reagents Equilibrate samples and RNA Solv Reagent to room temperature before beginning this protocol All steps should be carried out at room temperature unless otherwise noted Work quickly but carefully e Prepare all materials required before starting the procedure to minimize RNA degradation e Carefully apply the sample or solution to the center of the HiBind RNA Mini and the HiBind Micro RNA Columns Avoid touch
4. L polypropylene tube Note Unless the tube is pre cooled in liquid nitrogen the suspension will boil vigor ously and may cause loss of tissue e Once the liquid nitrogen has completely evaporated continue to Step 1 of the miRNA from Cells and Tissue Protocol on Page 7 Rotor stator Homogenizers Rotor stator homogenizers effectively homogenize most tissues The process usually takes less than a minute depending on the tissue Many rotor stator homogenizers operate with differently sized probes or generators that allow processing of small volumes in microcen trifuge tubes Such homogenizers are available from Tekmar Inc Cincinnati OH Tissuemizers BIOSPEC Products Bartlesville OK Tissue Tearor Craven Laboratories Austin TX Syringe Method High molecular weight DNA is responsible for the viscosity of cell lysates and can be shredded by passing the sample through a 19 21 gauge needle several times E Z N A Micro RNA Kit Protocol E Z N A Micro RNA Kit Protocol miRNA from Cells and Tissue Materials and Reagents to be Supplied by User 96 100 Ethanol Chloroform ACS Grade e RNase free filter pipette tips e 1 5 or 2 mL nuclease free microcentrifuge tubes e Microcentrifuge capable of 12 000 x g and 4 C e Ice Bucket Before Starting Prepare RNA Wash Buffer II according to the instructions in the Preparing Reagents section on Page 4 Prepare an Ice Bucket 1 Lyse ce
5. atrix Incubate at room temperature for 2 minutes Centrifuge at full speed for 1 minute Store eluted RNA at 70 C Note Any combination of the following steps can be used to help increase RNA yield Preheat the DEPC Water to 70 C before elution Increase the incubation time to 5 minutes Increase the elution volume Repeat the elution step with fresh DEPC Water this may increase the yield but decrease the concentration Repeat the elution step using the eluate from the first elution this may increase yield while maintaining elution volume E Z N A Large RNA Protocol E Z N A Micro RNA Kit Protocol Large RNA from HiBind RNA Mini Column This protocol is designed for isolation of RNA gt 200 nt If performing DNase digestion use the DNase Digestion Protocol found on Page 13 Materials and Reagents to be Supplied by User RNase free filter pipette tips 1 5 or 2 mL nuclease free microcentrifuge tubes Microcentrifuge capable of 12 000 x g Before Starting Prepare RNA Wash Buffer II according to the instructions in the Preparing Reagents section on Page 4 Important Notes All steps must be carried out at room temperature Work quickly but carefully 1 Complete Steps 1 12 of the Micro RNA Protocol protocol 2 Transfer the HiBind RNA Mini Column into a 2 mL Collection Tube provided 3 Add 500 uL RNA Wash Buffer I to the HiBind RNA Mini Column 4 Incubate at room tem
6. cells prior to extrac Degraded RNA tion unless they are lysed first Follow the protocol closely and work quickly Ensure not to introduce RNase during the procedure Check buffers for RNase contamination RNase contamination Ensure RNA Wash Buffer II has been diluted with ethanol as indicated in the Preparing Salt carry over Reagents section on Page 4 during elution 1X RNA Wash Buffer Il must be stored and used at room temperature Repeat the RNA Wash Buffer II wash step DNA e Digest with RNase free DNase and inactivate contamination at 75 C for 5 minutes DEPC Water is acidic and can dramatically lower As values Use TE buffer to dilute RNA prior to spectrophotometric analysis Problem in downstream applications RNA diluted in absorbance acidic buffer or ratios water 16 Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 DEPC Water 100 mL PRO32 RNA Wash Buffer 100 mL PRO30 RNA Wash Buffer II 20 mL PDR046 RNA Solv Reagent R6830 RNase free DNase Set E1091 2 mL DNase RNase free Microcentrifuge Tubes 500 pk 10 pk cs 391310790 HiBind E Z N A and MicroElute are registered trademarks of Omega Bio tek Inc Tissue Tearor and Tissuemizer are trademarks of their respective companies PCR is a patented process of Hoffman La Roche Use of the PCR process requires a license 17 Notes
7. e HiBind RNA Mini Column containing the sample into a 2 mL Collection Tube provided with this kit 13 10 11 12 13 14 15 14 E Z N A DNase I Digestion Protocol Add 250 uL RNA Wash Buffer to the HiBind RNA Mini Column Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Add 75 uL DNase digestion mixture directly onto the surface of the membrane of the HiBind RNA Mini Column Note Pipet the DNase directly onto the membrane DNA digestion will not be com plete if some of the mixture is retained on the wall of the HiBind RNA Mini Column Incubate at room temperature for 15 minutes Add 250 uL RNA Wash Buffer to the HiBind RNA Mini Column Incubate at room temperature for 2 minutes Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Add 500 uL RNA Wash Buffer II Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Note RNA Wash Buffer II must be diluted with ethanol before use Please see Page 5 for instructions Repeat Steps 12 14 for a second RNA Wash Buffer II wash step 16 17 18 19 20 E Z N A DNase I Digestion Protocol Centrifuge at full speed gt 12 000 x g for 2 minutes to completely dry the HiBind RNA Mini Column Note It is important to dry the HiBind RNA Mini Column matrix before elution Residual ethanol may interfere with dow
8. ing the membrane with pipet tips Starting Materials Although the binding capacity for the HiBind RNA Mini and the HiBind Micro RNA Columns are approximately 100 ug the maximum amount of starting material depends on the type of tissue being processed and its corresponding RNA content It is essential to begin with the correct amount of tissue to get optimal RNA yield and purity with the E Z N A Micro RNA Kit For the first time user we recommend to use less than 30 mg of tissue per sample Depending on the yield and purity obtained it may be possible to increase the starting material up to 100 mg maximum amount Tissue Homogenization Protocols Efficient sample disruption and homogenization is essential for successful RNA isolation Cell wall and plasma membrane disruption is necessary for the release of RNA from the sample and homogenization is necessary to reduce the viscosity of the lysates Homogenization shears genomic DNA and other high molecular weight cell components creating a homogenous lysate Incomplete homogenization can cause the HiBind RNA Mini Column to clog resulting in low or no yield Liquid Nitrogen Method Recommended Wear appropriate gloves and take great care when working with liquid nitrogen e Excise tissue and promptly freeze in a small volume of liquid nitrogen e Grind tissue with a ceramic mortar and pestle under approximately 10 mL liquid nitrogen e Pour the suspension into a pre cooled 15 m
9. lls or tissue with 1 mL RNA Solv Reagent Note 1 mL of RNA Solv Reagent is sufficient for the lysis of 1 x 10 cultured cells 30 50 mg animal tissue or 50 100 mg plant tissue A For culture cells grown in monolayer fibroblasts endothelial cells etc lyse the cells directly in the culture vessel as follows 1 Aspirate and discard the culture medium 2 Add 1mLRNA Solv Reagent directly to the cells making sure to cover the entire surface of the vessel to ensure complete lysis 3 Transfer the lysate to a clean 2 mL microcentrifuge tube not provided 4 Proceed to Step 2 Note This method is preferable to trypsinization followed by washing because it minimizes RNA degradation by nuclease contamination B For cells grown in suspension cultures Pellet cells at no greater than 1 500 rpm 400 x g for 5 minutes Discard the supernatant Add 1 mL RNA Solv Reagent Vortex or pipette up and down to lyse the cells Transfer the lysate to a clean 2 mL microcentrifuge tube not provided Proceed to Step 2 Or Ur Bie E Z N A Micro RNA Kit Protocol C For tissue samples determine the size of the samples and homogenize by using one of the methods discussed on Page 6 Unless using liquid nitrogen homogenize samples directly in 1 mL RNA Solv Reagent and proceed to Step 2 Incubate at room temperature for 3 minutes Add 0 2 mL chloroform per 1 mL RNA Solv Reagent Cap the tubes securely and vortex vigorously for 15
10. nstream applications Place the column in a clean 1 5 or 2 0 mL microcentrifuge tube not supplied Add 40 70 uL DEPC Water Note Make sure to add water directly onto the HiBind RNA Mini Column matrix Incubate at room temperature for 2 minutes Centrifuge at full speed for 1 minute and store eluted RNA at 70 C Note Any combination of the following steps can be used to help increase RNA yield Preheat the DEPC Water to 70 C before adding to the column Increase the incubation time to 5 minutes Increase the elution volume Repeat the elution step with fresh DEPC Water this may increase the yield but decrease the concentration e Repeat the elution step using the eluate from the first elution this may increase yield while maintaining elution volume 15 Troubleshooting Guide Please use this guide to troubleshoot any problems that may arise For further assistance please contact the technical support staff toll free at 800 832 8896 Repeat the elution step Preheat DEPC Water to 70 C prior to elution Increase the incubation time to 10 minutes Column is F Reduce the amount of starting material overloaded Completely homogenize sample Increase centrifugation time Reduce the amount of starting material RNA remains on Littleorno the column RNA eluted Clogged Incomplete column homogenization Quickly freeze starting material in liquid nitrogen Source Do not store tissue culture
11. p using the eluate from the first elution this may increase yield while maintaining elution volume E Z N A DNase I Digestion Protocol E Z N A Micro RNA Kit DNase I Digestion Protocol Since the HiBind matrix of the RNA Column eliminates most DNA DNase digestion is not necessary for most downstream applications However certain sensitive RNA applica tions may require further DNA removal See DNase Digestion Set Cat E1091 for more information After completing Steps 1 15 of the Animal Cell Protocol Pages 11 15 Steps 1 13 of the Animal Tissue Protocol Pages 17 19 or Steps 1 6 of the Vacuum Spin Protocol Pages 21 22 proceed with the following protocol User Supplied Material DNase I Digestion Set Cat E1091 1 For each HiBind RNA Mini Column prepare the DNase stock solution as follows E Z N A DNase Digestion Buffer 73 5 uL RNase free DNase 20 Kunitz uL 1 5 ul Total Volume 75 uL Important Notes DNase lis very sensitive and prone to physical denaturing Do not vortex the DNase mixture Mix gently by inverting the tube e Freshly prepare DNase stock solution right before RNA isolation Standard DNase buffers are not compatible with on membrane DNase diges tion Using other buffers may affect the binding of RNA to the HiBind matrix and may reduce RNA yields and purity All steps must be carried out at room temperature Work quickly but carefully 2 Insert th
12. perature for 5 minutes 5 Centrifuge at 10 000 x g for 1 minute 6 Discard the filtrate and reuse the Collection Tube 11 10 11 12 13 14 T5 12 E Z N A Large RNA Protocol Add 500 uL RNA Wash Buffer II to the HiBind RNA Mini Column Note RNA Wash Buffer II must be diluted with ethanol before use Please see Page 4 for instructions Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Repeat Steps 7 9 for a second RNA Wash Buffer II wash step Centrifuge at full speed gt 12 000 x g for 2 minutes to completely dry the HiBind RNA Mini Column Note It is important to dry the HiBind RNA Mini Column matrix before elution Residual ethanol may interfere with downstream applications Transfer the HiBind RNA Mini Column to a clean 1 5 or 2 mL microcentrifuge tube not supplied Add 40 70 uL of DEPC Water Note Make sure to add water directly onto the HiBind RNA Mini Column matrix Incubate at room temperature for 2 minutes Centrifuge at full speed for 1 minute Store eluted RNA at 70 C Note Any combination of the following steps can be used to help increase RNA yield Preheat the DEPC Water to 70 C before adding to the column e Increase the incubation time to 5 minutes Increase the elution volume Repeat the elution step with fresh DEPC Water this may increase the yield but decrease the concentration e Repeat the elution ste
13. seconds Incubate on ice for 10 minutes Centrifuge at 12 000 x g at 4 C for 15 minutes Note The mixture separates into a lower phenol chloroform phase an inter phase and an upper aqueous phase RNA remains entirely in the upper aqueous phase Transfer no more than 80 of the upper aqueous phase to a new 2 mL microcentrifuge tube not provided Add 0 33 volumes of ethanol Vortex to mix thoroughly Note A precipitate may form after the addition of ethanol Removal of Large RNA gt 200 nt 10 11 Insert a HiBind RNA Mini Column into a 2 mL Collection Tube provided with this kit Transfer 700 uL of the mixture from Step 7 to the HiBind RNA Mini Column Centrifuge at 10 000 x g at room temperature for 1 minute Transfer the filtrate into a new 2 mL microcentrifuge tube not provided for small RNA isolation E Z N A Micro RNA Kit Protocol 12 Repeat Steps 9 11 until all the remaining sample has been transferred to the column Note Each transfer of the filtrate Step 11 should be to the same 2 mL microcentrifuge tube for one combined filtrate sample Note To isolate large size total RNA gt 200 nt continue to the Large RNA from HiBind RNA Mini Column Protocol in the next section on Page 11 For purification of miRNA continue to Step 13 below If both miRNA and large RNA gt 200 nt are required both protocols need to be completed in a single day Hold the HiBind RNA Mini Column at 4 C until
14. the miRNA Purification protocol below has been completed Purification of Micro RNA 13 Measure the volume of the filtrate collected at the end of Step 12 14 Add 0 65 volumes of ethanol to the filtrate Vortex to mix thoroughly 15 Insert a HiBind Micro RNA Column into a clean 2 mL Collection Tube provided with this kit 16 Transfer the mixture from Step 14 to the HiBind Micro RNA Column 17 Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube 18 Repeat Steps 16 17 until all the remaining sample has been transferred to the HiBind Micro RNA Column 19 Add 500 uL RNA Wash Buffer II to the HiBind Micro RNA Column Note RNA Wash Buffer II must be diluted with ethanol before use Please see Page 4 for instructions 20 Centrifuge at 10 000 x g for 1 minute Discard the liquid and reuse the Collection Tube 21 22 23 24 25 26 10 E Z N A Micro RNA Kit Protocol Repeat Steps 19 20 for a second RNA Wash Buffer II wash step Centrifuge at full speed gt 12 000 x g for 2 minutes to completely dry the HiBind matrix Note It is important to dry the HiBind Micro RNA matrix before elution Residual ethanol may interfere with downstream applications Transfer the HiBind Micro RNA Column to a clean 1 5 or 2 0 mL microcentrifuge tube not provided Add 15 30 uL DEPC Water Note Make sure to add water directly onto the HiBind Micro RNA Column m
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