E.Z.N.A.®PF Micro RNA Kit
Contents
1. 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 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 Columns Avoid touching the membrane with pipet tips Starting Materials Although the binding capacity for 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 PF Micro RNA Kit For the first time user we recommend using 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 Homogenizat2. Transfer 700 uL of the mixture from Step 10 to the HiBind Micro RNA Column Centrifuge at 13 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Repeat Steps 12 13 until all the remaining sample has been transferred to the HiBind Micro RNA Column Add 500 uL 100 ethanol to the HiBind Micro RNA Column Centrifuge at 13 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Add 500 uL XD Binding Buffer to the HiBind Micro RNA Column Centrifuge at 13 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube 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 Centrifuge at 13 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Repeat Steps 19 20 for a second RNA Wash Buffer Il wash step Centrifuge at 13 000 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 23 24 25 26 10 E Z N A PF Micro RNA Kit Protocol Transfer the HiBind Micro RNA Column to a clean 1 5 or 2 mL microcentrifuge tube Add 15 30 uL DEPC Water Note Make sure to add DEPC Water directly onto the HiBind Micro RNA Column matrix Incubate at room temperature for 2 minutes Centrifuge at 13
3. 000 x g 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 10 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 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 p 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 cells prior to Degraded RNA extraction 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 100 ethanol
4. 10 eukaryotic cells 1 x 10 bacterial cells 50 mg animal tissue or 100 mg plant tissue can be used in a single experiment Overview The E Z N A PF Micro RNA Kit combines the reversible binding properties of HiBind matrix a silica based material with the unique lysis and binding procedure to extract micro and large gt 200 nt RNA from a wide variety of starting materials A specially formulated high salt lysis and binding buffer system allows more than 100 ug RNA to bind to the matrix Cells or tissues are first homogenized with NTL buffer that lyses the cells and inactivates RNases After adjusting condition with XD Binding Buffer the lysate is loaded to a filter column to remove cell debris and other contaminants The flow through liquid which contains the miRNA is mixed with ethanol and loaded onto a HiBind Micro RNA Column to bind the miRNA After a few quick wash steps the miRNA can be eluted from the HiBind Micro RNA Column with nuclease free water New In this Edition e The HiBind Micro RNA Column has been redesigned to increase DNA recovery by reducing the HiBind matrix retention volume Kit Contents Storage and Stability All of the E Z N A PF Micro RNA Kit components are guaranteed for at least 12 months from the date of purchase when stored at room temperature Preparing Reagents Dilute RNA Wash Buffer II with 100 ethanol as follows and store at room temperature x 100 Ethanol to be Added
5. Ca OMEGA Innovations in nucleic acid isolation Product Manual E Z N A PF Micro RNA Kit R7036 00 5 preps R7036 01 50 preps June 2013 For research use only Not intended for diagnostic testing E Z N A PF Micro RNA Kit Table of Contents Introduction and OVErVIEW scssssssccssesseccseecseesscessecseecneceees Kit Contents Storage and Stability ssessescseececsseeees Preparing Reagents esssesssssssssseeesssesesesssssseeesseeeecoesesesoosssseee Before BEGiNNind ssscssssssscssscssscssesscsncessecsecesscessecsecessesseesseeses Tissue Homogenization Protocol s sscsscsssesseeceesecnseess PP Micro RNA PROLOG Ol sscccotiscucsarecsenussteasstheccciessandacaserslsmncinien Troubleshooting Guid scsi cnc ee Ordering senare E T EARR Manual Revision June 2013 02 OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview Introduction Majority of the current commercial products for isolating miRNA involve organic extraction most commonly phenol based in the procedure E Z N A PF Micro RNA Kit uses an innovative buffer system that completely eliminate the use of phenol extraction This product 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
6. ant Add 0 5 mL Lysis Mixture Vortex or pipette up and down to lyse the cells Transfer the lysate to a clean 2 mL microcentrifuge tube Proceed to Step 2 Au PWN gt 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 0 5 mL Lysis Mixture and proceed to Step 2 D Bacterial cells Collect bacteria grown to log phase by centrifugation add 50 uL Lysozyme 15 mg mL in TE and incubate 10 minutes at 37 C Add 450 uL Lysis Mixture and vortex for 1 minute Proceed to Step 2 Incubate at room temperature for 3 5 minutes Add 250 uL XD Binding Buffer Cap the tubes securely and vortex vigorously for 15 seconds Incubate on ice for 10 minutes Centrifuge at 13 000 x g at 4 C for 15 minutes Insert a HiBind X Press Column into a 2 mL Collection Tube provided with this kit Transfer entire supernatant from Step 5 to the HiBind X Press Column Centrifuge at 13 000 x g at room temperature for 1 minute Measure the volume of the filtrate collected and transfer the filtrate to a new 2 mL microcentrifuge tube Add 1 2 volumes of ethanol to the filtrate Vortex 20 seconds to mix thoroughly 11 12 13 14 15 16 17 18 19 20 21 22 E Z N A PF Micro RNA Kit Protocol Insert a HiBind Micro RNA Column into a clean 2 mL Collection Tube provided with this kit
7. as indicated in the Salt carry over Preparing Reagents section on Page 4 during elution RNA Wash Buffer II must be stored and used at room temperature Repeat the RNA Wash Buffer II wash step DNA e Digest the RNA with RNase free DNase and contamination inactivate 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 11 Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 DEPC Water 100 mL PRO32 RNA Solv Reagent R6830 RNase free DNase Set E1091 2 mL DNase RNase free Microcentrifuge Tubes 500 pk 10 pk cs SSI 1310 00 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 12
8. ion shears genomic DNA and other high molecular weight cell components creating a homogenous lysate Incomplete homogenization can cause the HiBind Micro RNA 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 mL 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 microcentrifuge 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
9. needle several times E Z N A PF Micro RNA Kit Protocol E Z N A PF Micro RNA Kit Protocol Materials and Equipment to be Supplied by User 100 Ethanol 2 Mercaptoethanol e RNase free filter pipette tips e 1 5 or 2 mL nuclease free microcentrifuge tubes Microcentrifuge capable of 13 000 x g and 4 C 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 Prepare Lysis Mixture according to the following table per sample NTL Lysis Buffer miRNA Binding Enhancer 15 uL 2 Mercaptoethanol 1 Lyse cells or tissue with 0 5 mL Lysis Mixture see above preparation table following one of the steps below A For cultured 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 0 5 mL Lysis Mixture 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 4 Proceed to Step 2 Note This method is preferable to trypsinization followed by washing because it minimizes RNA degradation by nuclease contamination 10 E Z N A PF Micro RNA Kit Protocol B For cells grown in suspension cultures Pellet cells at no greater than 1 500 rpm 400 x g for 5 minutes Discard the supernat
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