RNA Clean-Up and Concentration Micro
Contents
1. Isolate RNA using a phenol guanidine based reagent such as Trizol or Tri Reagent according to manufacturer s instruction After the separation of the aqueous and organic phases collect the upper aqueous fraction containing the RNA into a new RNase free microcentrifuge tube not provided Note the volume Add one volume of 70 ethanol provided by the user to the fraction from step 1a Mix by vortexing for 10 seconds Note If the RNA sample being processed is highly enriched for small RNA such as microRNA siRNA Piwi interacting RNA etc increase the amount of ethanol added to the fraction from Step 1a In this case add one volume of 96 100 ethanol in Step 1b RNA isolated from bodily fluids serum plasma urine etc and exosomes are considered to belong to this category If the sample contains a heterogeneous mixture of a range of RNA sizes including large RNA follow the procedure as outlined in Step 1b without adjustments 2 Column Activation and Sample Binding to Column a b C d Assemble a column with one of the provided collection tubes Apply 500 uL of Column Activation Solution onto the column and centrifuge for 1 minute Discard the flowthrough Reassemble the spin column with its collection tube Apply up to 600 uL of the RNA sample with the ethanol from Step 1b onto the activated column and centrifuge for 1 minute Discard the flowthrough Reassemble the spin column with its collection tube I2. Prior to Use e A variable speed centrifuge should be used for maximum kit performance If a variable speed centrifuge is not available a fixed speed centrifuge can be used however reduced yields may be observed e Ensure that all solutions are at room temperature prior to use e Prepare a working concentration of the Wash Solution A by adding 90 mL of 96 100 ethanol provided by the user to the supplied bottle s containing the concentrated Wash Solution A This will give a final volume of 128 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added e Prepare an appropriate amount of Buffer RL by adding 10 uL of B mercaptoethanol provided by the user to each 1 mL of Buffer RL required B mercaptoethanol is toxic and should be dispensed in a fume hood It is recommended that no more than 45 ug of RNA be used per cleanup The maximum volume of RNA sample that can be processed is 200 uL It is important to work quickly during this procedure This kit purifies RNA with minimal amounts of DNA contamination However an optional protocol is provided in Appendix A for maximum removal of residual DNA that may affect sensitive downstream ap4plications such as quantitative PCR The procedure in Appendix A is to be carried out prior to performing the kit procedure below 1 Sample Preparation a Adjust the volume of the RNA sample to 100 uL by adding RNase free or DEPC treated water It is recommended
3. Fax 905 227 1061 BIOTEK ee CORPORATION Email techsupport norgenbiotek com 2 3430 Schmon Parkway N R F N Thorold ON Canada L2V 4Y6 x Phone 866 667 4362 e 905 227 8848 RNA Clean Up and Concentration Micro Elute Kit Product Insert Product 61000 Norgen s RNA Clean Up and Concentration Micro Elution Kit provides a rapid method for the purification cleanup and concentration of up to 45 ug of RNA isolated using different methods including phenol guanidine based protocols and from various upstream enzymatic reactions such as DNase treatment labeling and in vitro transcription The minimum recommended elution volume is 8 uL which enables the concentration of small amounts of all sizes of RNA from large mRNA and ribosomal RNA down to microRNA miRNA and small interfering RNA siRNA The RNA is preferentially purified from other reaction components such as proteins RNases and nucleotides without the use of phenol or chloroform The purified RNA is of the highest integrity and can be used in a number of downstream applications including end point or quantitative reverse transcription PCR Northern blotting RNase protection and primer extension expression array assays and next generation sequencing Norgen s Purification Technology Purification is based on spin column chromatography using Norgen s proprietary resin as the separation matrix The RNA is preferentially purified from other cellular components such as proteins wi
4. e handled with care Guanidinium salts form highly reactive compounds when combined with bleach thus care must be taken to properly dispose of any of these solutions Customer Supplied Reagents and Equipment You must have the following in order to use the RNA Clean up and Concentration Micro Kit For RNA Clean Up and Concentration from Enzymatic Reactions or Previously Isolated RNA e Benchtop microcentrifuge e mercaptoethanol e 96 100 ethanol e RNase free or DEPC treated water For RNA Clean up and Concentration from Aqueous Phase RNA fraction of Phenol Guanidine Based RNA Trizol or Tri Reagent Isolation Methods e Benchtop microcentrifuge e 70 ethanol Working with RNA RNases are very stable and robust enzymes that degrade RNA Autoclaving solutions and glassware is not always sufficient to actively remove these enzymes The first step when preparing to work with RNA is to create an RNase free environment The following precautions are recommended as your best defense against these enzymes e The RNA area should be located away from microbiological work stations e Clean disposable gloves should be worn at all times when handling reagents samples pipettes disposable tubes etc It is recommended that gloves are changed frequently to avoid contamination e There should be designated solutions tips tubes lab coats pipettes etc for RNA only e All RNA solutions should be prepared using at least 0 05 DEPC treated autoclaved wat
5. eal for concentrating RNA purified from exosomes plasma serum urine and other bodily fluids and any RNA samples initially purified in large volumes e Efficient RNA cleanup from enzymatic reactions labeling DNase treatment and in vitro transcription Cleanup of RNA isolated using different methods including phenol chloroform extractions Fast and easy processing using rapid spin column format Suitable for all sizes of RNA from large rRNA down to microRNA miRNA No phenol or chloroform extractions Storage Conditions and Product Stability All solutions should be kept tightly sealed and stored at room temperature These reagents should remain stable for at least 2 years in their unopened container Kit Components Component Product 61000 50 preps Buffer RL 40 mL Wash Solution A 38 mL Elution Solution A 6 mL Column Activation Solution 30 mL Micro Elute RNA Spin Columns 50 Collection Tubes 50 Elution tubes 1 7 mL 50 Product Insert 1 Precautions and Disclaimers This kit is designed for research purposes only It is not intended for human or diagnostic use Ensure that a suitable lab coat disposable gloves and protective goggles are worn when working with chemicals For more information please consult the appropriate Material Safety Data Sheets MSDSs These are available as convenient PDF files online at www norgenbiotek com The Buffer RL contains guanidinium salts and should b
6. eased if the column shows clogging below the recommended levels See also Clogged Column below alee was It is recommended that the Elution Solution A supplied Poor RNA with this kit be used for maximum RNA recovery Recovery used Ethanol was not Ensure that the appropriate amount of ethanol is added added to the lysate to the lysate before binding to the column Priano wasna Ensure that 90 mL of 96 100 ethanol is added to the added to the Wash A supplied Wash Solution A prior to use Solution A High amounts of Ensure that no more than 45 ug of RNA is used as the RNA in the input input for each column High amounts of The lysate may be passed through a 25 gauge needle Clogged genomic DNA attached to a syringe 5 10 times in order to shear the Column present in sample genomic DNA prior to loading onto the column Centrifig Ensure that the centrifuge remains at room temperature g throughout the procedure Temperatures below 15 C temperature too ee aw may cause precipitates to form that can cause the columns to clog RNases may be introduced during the use of the kit RNase Ensure proper procedures are followed when working contamination with RNA Please refer to Working with RNA at the beginning of this user guide RNA is ee fee Degraded licen hear In order to maintain the integrity of the RNA it is Eh q y important that the procedure be performed quickly Improper storage of the purified RNA For short ter
7. er or molecular biology grade nuclease free water e Clean all surfaces with commercially available RNase decontamination solutions e When working with purified RNA samples ensure that they remain on ice during downstream applications Flow Chart Procedure for Purifying RNA using Norgen s RNA Clean up and Concentration Micro Kit RNA Sample Add Buffer RL and Ethanol Vortex to mix Bind to activated column SPIN Wash three times with Wash Solution A SPIN lt b al lt a la Elute RNA with Elution Solution A SPIN lt Purified Total RNA Procedures All centrifugation steps are carried out in a benchtop microcentrifuge Various speeds are required for different steps so please check your microcentrifuge specifications to ensure that it is capable of the proper speeds All centrifugation steps are performed at room temperature The correct rpm can be calculated using the formula RPM RCF 1 118 x 105 r where RCF required gravitational acceleration relative centrifugal force in units of g r radius of the rotor in cm and RPM the number of revolutions per minute required to achieve the necessary g force A Protocol for RNA Clean Up and Concentration from Enzymatic Reactions or Previously Isolated RNA All centrifugation steps are carried out in a benchtop microcentrifuge at 14 000 x g 14 000 RPM except where noted All centrifugation steps are performed at room temperature Notes
8. f the volume of the RNA sample is greater than 600 uL repeat Steps 2d and 2e until all the remaining RNA sample has passed through the column 3 Column Wash a Apply 400 uL of Wash Solution A to the column and centrifuge for 1 minute Note Ensure the entire Wash Solution A has passed through into the collection tube by inspecting the column If the entire wash volume has not passed spin for an additional minute Discard the flowthrough and reassemble the spin column with its collection tube Repeat steps 3a and 3b to wash the column a second time e Wash column a third time by adding another 400 uL of Wash Solution A and centrifuging for 1 minute f Discard the flowthrough and reassemble the spin column with its collection tube g Spin the column for 2 minutes in order to thoroughly dry the resin Discard the collection tube a9 4 RNA Elution a Place the column into a fresh 1 7 mL Elution tube provided with the kit b Add 8 15 uL of Elution Solution A to the column Note For maximum concentrations of RNA use the 8 uL elution volume For maximum recovery of RNA the 15 uL volume is recommended c Centrifuge for 2 minutes at 200 x g 2 000 RPM followed by 1 minute at 14 000 x g 14 000 RPM Note the volume eluted from the column If the entire volume has not been eluted spin the column at 14 000 x g 14 000 RPM for 1 additional minute Note For maximum RNA recovery it is recommended that a second elution be perf
9. m storage RNA samples may be stored at 20 C for a few days It is recommended that samples be stored at 70 C for longer term storage Problem Possible Cause Solution and Explanation RNA does not RNA was not washed three times with the provided Wash Solution A Traces of salt from the binding step may remain in the sample if the column is not washed three times with the Wash Solution A Salt may interfere with downstream applications and thus must be washed from the column perform well in downstream Ensure that the dry spin under the Column Wash applications Ethanol procedure is performed in order to remove traces of pr A ethanol prior to elution Ethanol is known to interfere with many downstream applications DNA or Large amounts of Perform RNAse free DNasel digestion on the RNA Genomic sta ina matetial sample prior to starting the protocol as outlined in DNA 9 Appendix A Itis recommended that Norgen s RNase contamination used Free DNase Kit Product 25710 be used for this step Related Products Product RNA Clean Up and Concentration Micro Kit 23600 RNase Free DNase I Kit 25710 Total RNA Purification Kit 17200 CleanAll DNA RNA Clean Up and Concentration Micro Kit 23800 100b RNA Ladder 15002 1kb RNA Ladder 15003 Technical Support Contact our Technical Support Team between the hours of 8 30 and 5 30 Easter
10. n Standard Time at 905 227 8848 or Toll Free at 1 866 667 4362 Technical support can also be obtained from our website www norgenbiotek com or through ort norgenbiotek com email at techsu 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 905 227 8848 Fax 905 227 1061 Toll Free in North America 1 866 667 4362 2015 Norgen Biotek Corp P161000 1
11. o the activated column and centrifuge for 1 minute Discard the flowthrough Reassemble the spin column with its collection tube If the volume of the RNA sample is greater than 600 pL repeat Steps 2d and 2e until all the remaining RNA sample has passed through the column 3 Column Wash a Apply 400 uL of Wash Solution A to the column and centrifuge for 1 minute Note Ensure the entire Wash Solution A has passed through into the collection tube by inspecting the column If the entire wash volume has not passed spin for an additional minute b Discard the flowthrough and reassemble the spin column with its collection tube c Repeat steps 3a and 3b to wash the column a second time d Wash column a third time by adding another 400 uL of Wash Solution A and centrifuging for 1 minute e Discard the flowthrough and reassemble the spin column with its collection tube f Spin the column for 2 minutes in order to thoroughly dry the resin Discard the collection tube 4 RNA Elution a Place the column into a fresh 1 7 mL Elution tube provided with the kit b Add 8 15 uL of Elution Solution A to the column Note For maximum concentrations of RNA the 8 elution uL volume may be used For maximum recovery of RNA the 15 uL volume is recommended c Centrifuge for 2 minutes at 200 x g 2 000 RPM followed by 1 minute at 14 000 x g 14 000 RPM Note the volume eluted from the column If the entire volume has not been eluted spin the c
12. olumn at 14 000 x g 14 000 RPM for 1 additional minute Note For maximum RNA recovery it is recommended that a second elution be performed into a separate microcentrifuge tube Repeat Steps 4b and 4c 5 Storage of RNA The purified RNA sample may be stored at 20 C for a few days It is recommended that samples be placed at 70 C for long term storage B Protocol for RNA Clean up and Concentration from Aqueous Phase RNA fraction of Phenol Guanidine Based RNA Trizol or Tri Reagent Isolation Methods All centrifugation steps are carried out in a benchtop microcentrifuge at 14 000 x g 14 000 RPM except where noted All centrifugation steps are performed at room temperature Notes Prior to Use A variable speed centrifuge should be used for maximum kit performance If a variable speed centrifuge is not available a fixed speed centrifuge can be used however reduced yields may be observed Ensure that all solutions are at room temperature prior to use Prepare a working concentration of the Wash Solution A by adding 90 mL of 96 100 ethanol provided by the user to the supplied bottle s containing the concentrated Wash Solution A This will give a final volume of 128 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added It is recommended that no more than 45 ug of RNA to be used per cleanup It is important to work quickly during this procedure 1 Sample Preparation a
13. ormed into a separate microcentrifuge tube Repeat Steps 4b and 4c 5 Storage of RNA The purified RNA sample may be stored at 20 C for a few days It is recommended that samples be placed at 70 C for long term storage Appendix A Optional DNA Removal in Solution Followed by RNA Clean Up and Concentration Norgen s RNA Clean Up and Concentration Micro Elute Kit purifies RNA with minimal amounts of DNA contamination An optional protocol is provided below for maximum removal of residual DNA that may affect sensitive downstream applications such quantitative PCR It is recommended that Norgen s RNase Free DNase Kit Product 25710 be used for this step This procedure is to be performed prior to starting the kit protocol 1 Adjust the volume of the RNA sample to be treated to 50 uL with RNase free water 2 Add 45 uL of the provided Enzyme Incubation Buffer 3 Add 5 uL of Norgen s DNase to the RNA sample Gently mix by pipeting the sample up and down using a pipette Do not vortex 4 Incubate at 25 to 30 C for 15 minutes 5 Proceed directly to Protocol A Protocol for RNA Clean up and Concentration from Enzymatic Reactions or Previously Isolated DNA Troubleshooting Guide Problem Possible Cause Solution and Explanation Do not exceed the recommended amounts of starting materials The amount of starting material may need to Column has if th h loggi h become clogged be decr
14. that no more than 45 ug of RNA be used for each column Note If an input volume between 100 and 200 uL is used adjust the sample volume to 200 uL maximum allowable with RNase free or DEPC treated water In this case use the volumes indicated in bold in the bracket in Steps 1b and 1c b Add 250 uL or 500 uL of Buffer RL to the RNA sample Mix by vortexing Cc Add 200 uL or 400 uL of 96 100 ethanol provided by the user to the mixture from Step 1b Mix by vortexing for 10 seconds Note If the sample being processed is highly enriched for small RNA such as microRNA siRNA Piwi interacting RNA etc increase the amount of 96 100 ethanol added to the mixture from Step 1b In this case add 350 uL or 700 uL for 200 uL original input volume of 96 100 ethanol in Step 1c RNA isolated from bodily fluids serum plasma urine etc and exosomes are considered to belong to this category If the sample contains a heterogeneous mixture of a range of RNA sizes including large RNA follow the procedure as outlined in Step 1c without adjustments 2 Column Activation and Sample Binding to Column a b C d e f Assemble a column with one of the provided collection tubes Apply 500 uL of Column Activation Solution onto the column and centrifuge for 1 minute Discard the flowthrough Reassemble the spin column with its collection tube Apply up to 600 uL of the RNA sample with the ethanol from Step 1c ont
15. thout the use of phenol or chloroform The process involves first mixing the RNA samples or enzymatic reactions containing RNA with Buffer RL please see the flow chart on page 4 Ethanol is then added and the mixture is loaded onto an activated spin column specifically designed for small elution volumes Norgen s resin binds RNA in a manner that depends on ionic concentrations Thus only the RNA will bind to the column while the contaminating proteins or nucleotides will be removed in the flowthrough The bound RNA is then washed three times with the provided Wash Solution A in order to remove any remaining impurities The purified RNA is then eluted using 8 15 uL of Elution Solution A The purified RNA is of the highest integrity and can be used in a number of downstream applications Norgen s RNA Clean Up and Concentration Micro Elution Kit purifies RNA with minimal amounts of DNA contamination An optional protocol is provided in Appendix A for maximum removal of residual DNA that may affect sensitive downstream applications such quantitative PCR Specifications Kit Specifications Maximum Column Binding Capacity 45 ug Size of RNA Purified All sizes including small RNA lt 200 nt Maximum Amount of Starting Material 45 ug of RNA Minimum Elution Volume 8 uL Time to Complete 10 Purifications 20 minutes Average Recovery gt 90 Advantages e Concentration of small amounts of RNA into 8 uL e Id
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