E.Z.N.A.®Plant RNA Kit - Omega Bio-Tek
Contents
1. whichever applicable see Step 11 RB Buffer is recommended 1 Collect tissue in a 1 5 mL microcentrifuge tube not provided and freeze by dipping in liquid nitrogen with a pair of tweezers to fill the tube Grind the tissue using disposable pestles Note Disposable Kontes pestles work well and are available for purchase product no SSI 1014 39 amp SSI 1015 39 One can allow liquid nitrogen to evaporate and store the samples at 70 C for later use Do not allow samples to thaw Use disposable pestle only once Alternatively a small clean mortar and pestle can be used The above methods for disrupting plant tissue cannot be replaced with mechanical homogenizers 12 E Z N A Plant RNA Kit Difficult Samples Protocol Transfer up to 100 mg frozen ground plant tissue to a new 1 5 mL microcentrifuge tube Note We recommend starting with 50 mg of tissue at first If results obtained are satisfactory you may start increasing the amount of starting material Samples should not be allowed to thaw before the addition of RPL Buffer in Step 3 Immediately add 600 uL RPL Buffer Vortex at maximum speed to mix thoroughly Note RPL Buffer must be mixed with 2 mercaptoethanol before use Please see Page 6 for instructions Ensure that all the samples are completely suspended and that there are no clumps in the solution Clumps will result in low yields Add 140 uL SP Buffer Vortex to mix thoroughly Centrifuge at 10 000 x g for 10 minu2. Abs ratios lower Abs values Use TE buffer to dilute buffer or water ee RNA prior to spectrophotometric analysis 22 Ordering Information The following components are available for purchase separately Call Toll Free 1 800 832 8896 C HiBind E Z N A and MicroElute are registered trademarks of Omega Bio tek Inc Qiagen QlAvac and Vacman are all trademarks of their respective companies PCR is a patented process of Hoffman La Roche Use of the PCR process requires a license 23 Notes 24
3. Buffer Lyse Transfer sample to a Homogenizer Column Add ethanol and transfer sample to HiBind RNA Mini Column Wash 3X Dry Elute Kit Contents Bricon sso e Storage and Stability All of the E Z N A Plant RNA Kit components are guaranteed for at least 12 months from the date of purchase when stored at room temperature During shipment or storage in cool ambient conditions precipitates may form in RB Buffer Dissolve such deposits by warming the solution at 37 C and gently shaking 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 reagents 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 Columns Avoid touching the membrane with pipet tips e 2 mercaptoethanol is key in denaturing RNases and can be added to an aliquot of RB Buffer before use Add 20 uL 2 mercaptoethanol per 1 mL RB Buffer T
4. HiBind RNA Mini Column to a new 2 mL Collection Tube Add 700 uL RNA Wash Buffer II Note RNA Wash Buffer II must be diluted with ethanol before use Please see Page 6 for instructions Centrifuge at 10 000 x g for 30 seconds Discard the filtrate and reuse collection tube Add 500 uL RNA Wash Buffer II Centrifuge at 10 000 x g for 30 seconds Discard the filtrate and reuse collection tube Centrifuge at maximum speed for 1 minute 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 15 27 28 29 16 E Z N A Plant RNA Kit Difficult Samples Protocol Transfer the HiBind RNA Mini Column to a clean 1 5 mL microcentrifuge tube Add 50 100 uL DEPC Water Note Make sure to add water directly onto the HiBind RNA Mini Column matrix Centrifuge at maximum 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 Heat the DEPC Water to 65 C before adding to the column Let sit at room temperature for 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 P
5. of RNA from a variety of sources The key to this system is that it uses the reversible binding properties of the HiBind matrix a silica based material in combination with the speed of mini column spin technology Single or multiple samples can be processed quickly There is no need for phenol chloroform extractions and time consuming steps such as CsCl gradient ultra centrifugation and precipitation with isopropanol or LiCL are eliminated RNA purified using the E Z N A RNA purification system is ready for applications such as RT PCR Northern blotting poly A RNA mRNA purification nuclease protection and in vitro translation The E Z N A Plant RNA Kit can purify up to 100 ug plant RNA that is gt 200 nt Normally 10 100 mg plant tissue can be processed in a single experiment Lysis of cells or tissue occurs under denaturing conditions that inactivate RNases After the homogenization process samples are transferred to the HiBind RNA Mini Column to bind RNA Cellular debris and other contaminants are removed after three quick wash steps High quality RNA is eluted in sterile DEPC Water New In this Edition e The latest edition has been redesigned to enhance readability and protocol quality Binding Capacity e Each HiBind RNA Mini Column can bind approximately 100 ug RNA Using greater than 100 mg plant tissue is not recommended Illustrated Protocol Q N RJ Grind with liquid nitrogen Add RB
6. standard Northern assay Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 14 000 x g RNase free pipet tips and 1 5 mL microcentrifuge tubes 100 ethanol 70 ethanol e 2 mercaptoethanol Liquid nitrogen Optional Water bath incubator or heat block capable of 65 C Before Starting e Prepare RNA Wash Buffer Il and RB Buffer according to Preparing Reagents section on Page 6 1 Collect tissue in a 1 5 mL microcentrifuge tube not provided and freeze by dipping in liquid nitrogen with a pair of tweezers to fill the tube Grind the tissue using disposable pestles Note Disposable Kontes pestles work well and are available for purchase product no SSI 1014 39 amp SSI 1015 39 One can allow liquid nitrogen to evaporate and store the samples at 70 C for later use Do not allow samples to thaw Use disposable pestle only once Alternatively a small clean mortar and pestle can be used The above methods for disrupting plant tissue cannot be replaced with mechanical homogenizers 2 Transfer up to 100 mg frozen ground plant tissue to a new 1 5 mL microcentrifuge tube Note We recommend starting with 50 mg of tissue at first If results obtained are satisfactory you may start increasing the amount of starting material Samples should not be allowed to thaw before the addition of RB Buffer in Step 3 10 11 12 13 E Z N A Plant RNA Kit Standard Protocol Im
7. the Difficult Tissue Samples Protocol Pages 12 14 proceed with the following protocol User Supplied Material DNase Digestion Set 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 Freshly prepare DNase stock solution right before RNA isolation Standard DNase buffers are not compatible with on membrane DNase digestion The use of other buffers may affect the binding of RNA to the HiBind matrix and may reduce RNA yields and purity e All steps must be carried out at room temperature Work quickly but carefully 2 Insert the HiBind RNA Mini Column containing the sample into a 2 mL Collection Tube 19 10 11 12 13 14 15 20 E Z N A Plant RNA Kit 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 complete if some of the mixture is retained on the wall of the HiBind
8. Ca OMEGA Innovations in nucleic acid isolation bio tek Product Manual E Z N A Plant RNA Kit R6827 00 5 preps R6827 01 50 preps R6827 02 200 preps September 2012 For research use only Not intended for diagnostic testing E Z N A Plant RNA Kit Table of Contents ghd s 8 ako p arean eer nen eet re ee aiia Illustrated Protocol uccscccccesassscssessessosssesctoncesosusscncacosssussonsnsoecnssioases Kit Contents and StOLaG Gs sisssssisctsessicninsasnwninmainwanuend Important NOTES cits sisicensssaperrieriacaacieantuaneuannanuee Preparing REAGEMUS psssicssssisisninndisennninnarmisemasianmiaand Quantification of RNA seesssssessssoseeessesnssssssteeseessesssssessseessessss Plant RNA Standard Protocol sssssssssssssssesssssessesseseeseeeeseeenee Difficult Sample Types ProtoCoOl sssssessessessesseseesesssesserseese 12 Isolation from ArthropodS s ssessessessessesssesesrsereerssesesssesssss 17 Isolation from Fungal SamMpleS ssssessessessessesssesesssersessesse 18 DNase I Digestion Protocol ssessssserseeesrreesrssreerreeserssessess 19 Troubleshooting Guide ssesssesseessssssssssseeeseessssssssreeserssssssss 22 Order s een eee een enn oR a AEE 23 Manual Revision September 2012 02 OMEGA bio tek Innovations in nucleic acid isolation Introduction The E Z N A RNA family of products is an innovative system that radically simplifies the extraction and purification
9. RNA Mini Column Let sit at room temperature for 15 minutes Add 250 uL RNA Wash Buffer to the HiBind RNA Mini Column Let sit at room temperature for 2 minutes Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Add 600 uL RNA Wash Buffer II Note RNA Wash Buffer Il must be diluted with ethanol before use Please see Page 6 for instructions Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Repeat Steps 12 14 for a second RNA Wash Buffer II wash step 16 17 18 19 E Z N A Plant RNA Kit DNase I Digestion Protocol Centrifuge at maximum speed for 2 minutes to completely dry the HiBind RNA Mini Column matrix Note It is important to dry the HiBind RNA Mini Column matrix before elution Residual ethanol may interfere with downstream applications Place the column in a clean 1 5 mL microcentrifuge tube not supplied Add 50 100 uL DEPC Water Note Make sure to add water directly onto the HiBind RNA Mini Column matrix Centrifuge at maximum speed for 2 minutes and store eluted RNA at 70 C Note Any combination of the following steps can be used to help increase RNA yield Heat the DEPC Water to 65 C before adding to the column Let sit at room temperature for 5 minutes Increase the elution volume Repeat the elution step with fresh DEPC Water this may increase the yield but decrease the concentrati
10. RNA is stable for more than a year Integrity of RNA It is highly recommended that RNA quality be determined prior to beginning all downstream applications The quality of RNA can be best assessed by denaturing agarose gel electrophoresis with ethidium bromide staining The ribosomal RNA bands should appear as sharp clear bands on the gel The 28S band should appear to be double that of the 18S RNA band 23S and 16S if using bacteria If the ribosomal RNA bands in any given lane are not sharp and appear to be smeared towards the smaller sized RNA it is very likely that the RNA undergone degradation during the isolation handling or storage procedure Although RNA molecules less than 200 bases in length do not efficiently bind to the HiBind matrix a third RNA band the tRNA band may be visible when a large number of cells are used Expected Yields Sample yields from 100 mg starting tissue Yields obtained with the E Z N A Plant RNA Kit Arabidopsis 30 ug E Z N A Plant RNA Kit Standard Protocol E Z N A Plant RNA Kit Standard Protocol This protocol is suitable for most fresh or frozen tissue samples thereby allowing efficient recovery of RNA However due to the tremendous variation in water and polysaccharide content in plants sample size should be limited to lt 100 mg Best results are obtained with young leaves or needles The method outlined in this protocol will isolate a sufficient amount of RNA for tracks on a
11. ase digestion is not necessary for most downstream applications However certain sensitive RNA applications may require further DNA removal If an additional RNA removal step is required please continue to the DNase Digestion Protocol found on Page 19 See DNase Digestion Set Cat E1091 for more information If DNase digestion is not required proceed to Step 14 14 T5 16 IFA 18 19 20 21 22 23 10 Add 500 uL RNA Wash Buffer I Centrifuge at 10 000 x g for 30 seconds Discard the filtrate and the collection tube Transfer the HiBind RNA Mini Column to a new 2 mL Collection Tube Add 700 uL RNA Wash Buffer II Note RNA Wash Buffer Il must be diluted with ethanol before use Please see Page 6 for instructions Centrifuge at 10 000 x g for 30 seconds Discard the filtrate and reuse collection tube Add 500 uL RNA Wash Buffer II Centrifuge at 10 000 x g for 30 seconds Discard the filtrate and reuse collection tube 24 25 26 27 E Z N A Plant RNA Kit Standard Protocol Centrifuge the empty HiBind RNA Mini Column at maximum speed for 2 minutes to dry the column Note This step is critical for removal of trace ethanol that may interfere with downstream applications Transfer the HiBind RNA Mini Column to a clean 1 5 mL microcentrifuge tube Add 50 100 uL DEPC Water Note Make sure to add water directly onto the HiBind RNA Mini Column matri
12. his mixture can be stored for 1 month at room temperature Preparing Reagents Dilute RNA Wash Buffer II with 100 ethanol as follows and store at room temperature Kit oo 100 Ethanol to be Added R6827 02 200 mL Add 20 uL 2 mercaptoethanol per 1 mL RB Buffer This mixture can be stored for one month at room temperature Note Only prepare what is needed RB Buffer is required without the addition of 2 mercaptoethanol in the Difficult Sample Types protocol For Difficult Sample Types protocol Page 12 add 20 uL 2 mercaptoethanol per 1 mL RPL Buffer This mixture can be stored for one month at room temperature Quantification of RNA Quantification and Storage of RNA To determine the concentration and purity of RNA measure absorbance at 260 nm and 280 nm with a spectrophotometer One OD unit measured at 260 nm corresponds to 40 g mL RNA DEPC Water is slightly acidic and can dramatically lower absorbance values We suggest that you dilute the sample in a buffered solution TE for spectrophotometric analysis The A A ratio of pure nucleic acids is 2 0 while an A A ratio of 0 6 denotes pure protein A ratio of 1 8 2 0 corresponds to 90 100 pure nucleic acid Phenol has a maximum absorbance at 270 nm and can interfere with spectrophotometric analysis of DNA or RNA However the E Z N A Plant RNA Kit eliminates the use of phenol and avoids this problem Store RNA samples at 70 C in water Under these conditions
13. lant RNA Kit Arthropod Samples Protocol E Z N A Plant RNA Kit Arthropod Samples The exoskeleton of arthropods poses the same problems that occur when trying to isolate RNA from plant specimens Pigments and polysaccharides often co purify with nucleic acids and interfere with downstream applications Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 14 000 x g e RNase free pipet tips and 1 5 mL microcentrifuge tubes Water bath incubator or heat block capable of 65 C 100 ethanol 70 ethanol 2 mercaptoethanol Liquid nitrogen Before Starting Prepare RNA Wash Buffer Il and RB Buffer according to Preparing Reagents section on Page 6 Set water bath incubator or heat block to 65 C 1 Freeze and grind up to 100 mg arthropod tissue in liquid nitrogen Grind tissue completely to obtain a fine homogenous powder 2 Immediately add 500 uL RB Buffer Vortex at maximum speed to mix thoroughly Note RB Buffer must be mixed with 2 mercaptoethanol before use Please see Page 6 for instructions Ensure that all the samples are completely suspended and that there are no clumps in the solution Clumps will result in low yields 3 Proceed Step 4 of the Standard Protocol Page 9 17 E Z N A Plant RNA Kit Fungal Samples Protocol E Z N A Plant RNA Kit Fungal Samples The E Z N A Plant RNA Kit can also be used for fungal RNA isolation since many fungal sa
14. lving the RNA with RB Buffer has proven difficult Adjust the binding conditions by following either A or B below Note RB Buffer must be mixed with 2 mercaptoethanol before use Please see Page 6 for instructions A If RB Buffer was used in Step 10 add 250 uL RB Buffer and 350 uL 100 ethanol B If DEPC Water was used in Step 10 add 350 uL RB Buffer and 250 uL 100 ethanol Insert a HiBind RNA Mini Column into a 2 mL Collection Tube Transfer the entire sample including any precipitate that may have formed to the HiBind RNA Mini Column Centrifuge at 10 000 x g for 30 seconds Discard the filtrate and reuse the Collection Tube Optional This the starting point of the optional on membrane DNase Digestion Protocol Since the HiBind matrix of the RNA Mini Column eliminates most DNA DNase digestion is not necessary for most downstream applications However certain sensitive RNA applications may require further DNA removal If an additional RNA removal step is required please continue to the DNase Digestion Protocol found on Page 19 See DNase Digestion Set Cat E1091 for more information If DNase digestion is not required proceed to Step 16 14 16 17 18 19 20 21 22 23 24 25 26 E Z N A Plant RNA Kit Difficult Samples Protocol Add 500 uL RNA Wash Buffer I Centrifuge at 10 000 x g for 30 seconds Discard the filtrate and the collection tube Transfer the
15. mediately add 500 uL RB Buffer Vortex at maximum speed to mix thoroughly Note RB Buffer must be mixed with 2 mercaptoethanol before use Please see Page 6 for instructions Ensure that all the samples are completely suspended and that there are no clumps in the solution Clumps will result in low yields Insert a Homogenizer Column into a 2 mL Collection Tube Transfer the lysate to a Homogenizer Column Centrifuge at 14 000 x g for 5 minutes at room temperature Transfer cleared lysate to a new 1 5 mL microcentrifuge tube Do not disturb or transfer any of the insoluble pellet Measure the volume of the lysate Add 1 volume 70 ethanol Vortex at maximum speed for 20 seconds A precipitate may form at this point it will not interfere with DNA isolation Passing the mixture through a needle using a syringe or by pipetting up and down 10 15 times may break up the precipitates Insert a HiBind RNA Mini Column into a 2 mL Collection Tube Transfer 700 uL sample including any precipitates that may have formed to the HiBind RNA Mini Column Centrifuge at 12 000 x g for 1 minute at room temperature Discard filtrate and reuse the collection tube Repeat Steps 10 12 until all of the sample has been transferred to the column E Z N A Plant RNA Kit Standard Protocol Optional This the starting point of the optional on membrane DNase Digestion Protocol Since the HiBind matrix of the RNA Mini Column eliminates most DNA DN
16. mples posses similar cellular attributes to plant specimens Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 14 000 x g RNase free pipet tips and 1 5 mL microcentrifuge tubes Water bath incubator or heat block capable of 65 C 100 ethanol 70 ethanol 2 mercaptoethanol Liquid nitrogen Before Starting 18 Prepare RNA Wash Buffer Il and RB Buffer according to Preparing Reagents section on Page 6 Set water bath incubator or heat block to 65 C Freeze and grind up to 30 mg fungal tissue in liquid nitrogen Grind tissue completely to obtain a fine homogenous powder Immediately add 500 uL RB Buffer Vortex at maximum speed to mix thoroughly Note RB Buffer must be mixed with 2 mercaptoethanol before use Please see Page 5 for instructions Ensure that all the samples are completely suspended and that there are no clumps in the solution Clumps will result in low yields Proceed Step 4 of the Standard Protocol Page 9 E Z N A Plant RNA Kit DNase I Digestion Protocol E Z N A Total RNA Kit DNase I Digestion Protocol Since the HiBind matrix of the RNA Mini Column eliminates most DNA DNase digestion is not necessary for most downstream applications However certain sensitive RNA applications may require further DNA removal See DNase Digestion Set Cat E1091 for further information After completing Steps 1 13 of the Standard Protocol Pages 8 9 or Steps 1 15 of
17. on e Repeat the elution step using the eluate from the first elution this may increase yield while maintaining elution volume 21 Troubleshooting Guide Please use this guide to toubleshoot any problems that may arise For further assistance please contact the technical support staff toll free at 1 800 832 8896 Possible Problems and Suggestions RNA remains on the Repeat elution step column Heat DEPC Water to 65 C prior to elution Column is overloaded Reduce quantity of starting material Completely homogenize sample Little or no RNA eluted Clogged Incomplete ar Increase centrifugation time column homogenization Reduce amount of starting material Freeze starting material quickly in liquid Starting sample nitrogen problems Follow protocol closely and work quickly Degraded RNA Ensure not to introduce RNase during the RNase contamination Procedure Check buffers for RNase contamination Ensure RNA Wash Buffer II has been diluted with 4 volumes 100 ethanol as indicated on Problem in i Salt carryover during bottle downstream z elution RNA Wash Buffer II must be stored and used at applications room temperature Repeat wash with RNA Wash Buffer Il Cause Solution DNA eee Perform the optional DNase Digestion Pale DNA contamination contamination Protocol on Page 19 Problem Cause Solution _ __ 4 DEPC Water is acidic and can dramatically F RNA diluted in acidic s Low
18. tes Transfer cleared lysate to a new 1 5 mL microcentrifuge tube Do not disturb or transfer any of the insoluble pellet Measure the volume of the lysate Add 1 volume isopropanol Vortex to mix thoroughly Note In most cases 600 ul cleared lysate can easily be removed Add 600 uL isopropanol Depending on the sample the volume of the cleared lysate will vary This step removes much of the polysaccharide content and improves spin column performance by increasing RNA binding capacity and therefore yield in the steps that follow Incubation is not required after the addition of isopropanol Immediately centrifuge at 10 000 x g for 2 minutes Longer centrifugation does not improve yield Carefully aspirate or decant the supernatant Do not disturb the RNA pellet Invert the microcentrifuge tube on a paper towel for 1 minute to allow any residual liquid to drain Drying the pellet is not necessary 13 10 11 12 13 14 15 E Z N A Plant RNA Kit Difficult Samples Protocol Add 100 uL RB Buffer or sterile DEPC Water heated to 65 C Vortex at maximum speed to resuspend the pellet A brief incubation at 65 C may be necessary to effectively dissolve the RNA Important Do not add 2 mercaptoethanol to RB Buffer for this step RB Buffer is recommended for dissolving the RNA pellet especially when degradation was found after elution RB Buffer contains a strong RNase inhibitor DEPC Water should only be used when disso
19. x Centrifuge at maximum 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 Heat the DEPC Water to 65 C before adding to the column Let sit at room temperature for 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 11 E Z N A Plant RNA Kit Difficult Samples Protocol E Z N A Plant RNA Kit Difficult Sample Types In certain plant samples RNA isolation can be difficult due to their large amount of polysaccharides and phenolic compounds This protocol involves a simple and rapid precipitation that will remove much of these compounds Use this protocol when the standard protocol Page 8 results in low RNA yields Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 14 000 x g RNase free pipet tips and 1 5 mL microcentrifuge tubes Water bath incubator or heat block capable of 65 C 100 ethanol 70 ethanol e 2 mercaptoethanol Liquid nitrogen Before Starting Prepare RNA Wash Buffer Il RB Buffer and RPL Buffer according to Preparing Reagents section on Page 6 Set water bath incubator or heat block to 65 C Heat RB Buffer or DEPC Water to 65 C
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