22550 - Protocol (25 prep)
Contents
1. 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 Customer Supplied Reagents and Equipment You must have the following in order to use the Urine Exfoliated Cell RNA Purification Kit Benchtop microcentrifuge Swinging bucket centrifuge 96 100 ethanol B mercaptoethanol 50 mL conical tubes Microcentrifuge tubes Lysozyme containing TE Buffer o 3 mg mL lysozyme in TE Buffer Flow Chart Procedure for Purifying Urine RNA using Norgen s Urine Exfoliated Cell and Bacteria RNA Purification Kit Collect Urine Sample SPIN J Pellet exfoliated cells and bacteria Lyse cells using Buffer SK Add Ethanol Bind to column OA aoa SPIN A AL Wash twice with Wash Solution A SPIN D AAL Elute RNA with Elution Buffer E SPIN 1A Purified Total Urine RNA 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 recommend2. gt k 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 i Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 BIOTEK amp CORPORATION Email techsupport norgenbiotek com Urine Exfoliated Cell and Bacteria RNA Purification Kit Product Insert Product 22550 Norgen s Urine Exfoliated Cell and Bacteria RNA Purification Kit provides a rapid method for the isolation and purification of total RNA from exfoliated cells that have been shed into the urine from the urinary tract The kit is also designed for the rapid preparation of bacterial RNA from urine samples RNA biomarkers from exfoliated cells can be used as a non invasive tool for a number of diagnostic and research applications including the diagnosis and monitoring of bladder kidney or other urinary tract cancers Bacterial RNA can be isolated from both human urine samples and urine samples from animals in order to study the levels and types of bacteria that are present as well as to study the stage of bacterial pathogenesis through the use of RNA biomarkers The kit allows for the isolation of RNA from both Gram negative and Gram positive bacteria including E coli Proteus spp Klebsiella spp Enterobacter spp Serratia Spp Pseudomonas spp Clostridial ssp and Leptospirosis spp as well as Chlamydia trachomatis and Neisseria gonorrhoeae The kit purifies all sizes of RNA from large mRNA and ribosomal RNA down to microRNA miRNA and small interfering RNA siRNA The RN
3. through the column If needed spin at 14 000 x g 14 000 RPM for an additional minute 5 After the centrifugation in Step 4 pipette the flowthrough that is present in the collection tube back onto the top of the column Note Ensure Step 5 is performed in order to ensure maximum DNase activity and to obtain maximum yields of RNA in particular for small RNA species 6 Incubate the column assembly at 25 30 C for 15 minutes 7 Without any further centrifugation proceed directly to the second wash step of the Column Wash section Step 3c of protocol Troubleshooting Guide Problem Possible Cause Solution and Explanation Ensure that the appropriate amount of Buffer SK was added to the exfoliated cell pellet Incomplete lysis of cells Ensure that the appropriate amount of lysozyme containing TE buffer and Buffer SK are added to the bacterial pellet in order to completely lyse the cells Do not exceed the recommended amounts of 50 mL of Column has urine or 1 x 10 cells The amount of starting material Becomecloaaed may need to be decreased if the column shows clogging 99 below the recommended levels See also Clogged Column below ee ee was It is recommended that the Elution Buffer E supplied with ised this kit be used for maximum RNA recovery Ethanol was not Ensure that the appropriate amount of ethanol is added added to the lysate to the lysate before binding to the column ee Weds Ensure tha
4. 000 x g 14 000 RPM except where noted All centrifugation steps are performed at room temperature Notes Prior to Use e We recommend the use of Norgen s Urine Preservative when collecting urine samples which is designed for the preservation of nucleic acids and proteins in fresh urine samples at ambient temperatures The components of the Urine Preservative allow samples to be stored for over 2 years at room temperature with no detected degradation of urine DNA RNA or proteins Norgen s Urine Preservative is available in 2 convenient formats in a liquid format in Norgen s Urine Preservative Single Dose Ampules as well as in a dried format in Norgen s Urine Collection and Preservation Tubes Please see the Related Products table below 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 42 mL of 96 100 ethanol provided by the user to the supplied bottle containing the concentrated Wash Solution A This will give a final volume of 60 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 SK by adding 10 uL of B mercaptoethanol provided by the user to each 1 mL of B
5. A is preferentially purified from other cellular components such as proteins as well as from the contaminating species found in urine such as glucose and salts without the use of phenol or chloroform Typical yields of RNA will vary depending on the urine sample and the bacterial species if any present in the urine The purified RNA is of the highest integrity and can be used in a number of downstream applications including real time PCR reverse transcription PCR Northern blotting RNase protection and primer extension and expression array assays 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 without the use of phenol or chloroform The process involves first pelleting the exfoliated cells or bacteria that are present in the urine sample through the use of centrifugation followed by lysing the exfoliated cells or bacteria with the provided Buffer SK please see the flow chart on page 4 Ethanol is then added to the lysate and the solution is loaded onto a spin column 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 will be removed in the flowthrough or retained on the top of the resin The bound RNA is then washed twice with the provided Wash Solution A in orde
6. Maximam numer Do not exceed the recommended amounts of 50 mL of of cells exceeds kit 6 POR urine or 1 x 10 cells specifications The urine sample that was applied to the column Clogged contained too many bacterial cells Reduce the amount Too many bacteria Column of urine used Clogging can be alleviated by increasing present in the urine Sae l the g force and or centrifuging for a longer period of time until the urine passes through the column High amounts of The lysate may be passed through a 25 gauge needle genomic DNA attached to a syringe 5 10 times in order to shear the present in sample genomic DNA prior to loading onto the column Centrifuge Ensure that the centrifuge remains at room temperature g throughout the procedure Temperatures below 20 C temperature too a f h h jow 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 Procedure net In order to maintain the integrity of the RNA it is performed quickly i important that the procedure be performed quickly enough Lysozyme used Ensure that the lysozyme being used with this kit is RNA is may not be RNAse RNase free in order to prevent possible problems with Degraded free RNA degradation Improper storage of the purified RNA For sh
7. ed 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 water 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 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 rom can be calculated using the formula RPM RCF 1 118 x 10 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 Protocol for Total RNA Purification from Exfoliated Cells in Urine All centrifugation steps are carried out in a benchtop microcentrifuge at 14
8. minutes in a swinging bucket centrifuge to pellet the cells Pour off the supernatant carefully so as not to disturb or dislodge the cell pellet b Resuspend the bacteria thoroughly in 100 uL of the lysozyme containing TE Buffer prepared by user by vortexing Incubate at room temperature for 10 minutes Note The length of the incubation step may be decreased to as little as 5 minutes if the urine bacteria of interest from which RNA is being isolated is known to be Gram negative c Add 300 uL of Buffer SK and vortex vigorously for at least 10 seconds Transfer the lysate to a microcentrifuge tube d Add 200 uL of 96 100 ethanol provided by the user to the lysate Mix by vortexing for 10 seconds 2 Binding to Column a Assemble a column with one of the provided collection tubes b Apply the lysate with the ethanol onto the column and centrifuge for 1 minute Note Ensure the entire lysate has passed through into the collection tube by inspecting the column If the entire lysate volume has not passed spin for an additional minute c Discard the flowthrough Reassemble the spin column with its collection tube Optional Step Norgen s Urine Exfoliated Cell and Bacteria RNA Purification Kit isolates total RNA with minimal amounts of genomic DNA contamination However an optional On Column DNA Removal Protocol is provided in Appendix A for maximum removal of residual DNA that may affect sensitive downstream applications I
9. ort term 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 The cells are old Older samples contain prematurely lysed cells which release RNase and can degrade RNA Fresh urine samples are recommended Problem Possible Cause Solution and Explanation RNA does not RNA was not washed twice with the provided Wash Traces of salt from the binding step may remain in the sample if the column is not washed twice with Wash Solution A Salt may interfere with downstream perform well Solution A applications and thus must be washed from the column in downstream Ensure that the dry spin under the Column Wash applications Ethanol carryover procedure is performed in order to remove traces of y ethanol prior to elution Ethanol is known to interfere with many downstream applications Perform RNAse free DNasel digestion on the RNA Genomic Large amounts of sample after elution to remove genomic DNA DNA starting material contamination Appendix A It is recommended that contamination used Norgen s RNase Free DNase Kit Product 25710 be used for this step Technical Support Contact our Technical Support Team between the hours of 8 30 and 5 30 Eastern 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 th
10. r to remove any remaining impurities and the purified total RNA is eluted with the Elution Buffer E The purified RNA is of the highest integrity and can be used in a number of downstream applications Specifications Kit Specifications Maximum Column Binding Capacity 50 ug Volume of Urine Processed 1 50 mL Maximum Input of Exfoliated Cells 1x 10 Size of RNA Purified All sizes including small RNA lt 200 nt 20 minutes for Exfoliated Cells 30 minutes for Bacteria For Exfoliated Cells 1 ug RNA per 1 x 10 cells Varies due to cell density sample For Bacteria 0 5 ug RNA per 1 x 10 cells Varies due to cell density of sample Time to Complete 10 Purifications Average Yield Advantages e Fast and easy processing using rapid spin column format Isolate total RNA from large rRNA down to microRNA miRNA RNA can be isolated and detected from as little as 100 exfoliated cells Isolate high quality total RNA from urine 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 1 year in their unopened containers Kit Components Component Product 22500 25 samples Buffer SK 15 mL Wash Solution A 18 mL Elution Buffer E 6 mL Micro Spin Columns 25 Collection Tubes 25 Elution tubes 1 7 mL 25 Product Insert
11. rough email at techsupport norgenbiotek com Related Products Product RNase Free DNase Kit 25710 Total RNA Purification Kit 17200 Urine Collection and Preservation Tubes 50 cc 1 tube 18111 Urine Collection and Preservation Tubes 50 cc 50 tubes 18113 Urine Collection and Preservation Tubes 15 cc 1 tube 18120 Urine Collection and Preservation Tubes 15 cc 50 tubes 18122 Urine Collection and Preservation Tubes 5 cc 1 tube 18116 Urine Collection and Preservation Tubes 5 cc 50 tubes 18118 Urine Preservative Single Dose 1 tube 18124 Urine Preservative Single Dose 50 tubes 18126 1kb RNA Ladder 15003 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 2014 Norgen Biotek Corp P122550 1 M14 10
12. stored at 20 C for a few days It is recommended that samples be placed at 70 C for long term storage Appendix A Protocol for Optional On Column DNA Removal Norgen s Urine Exfoliated Cell and Bacteria RNA Purification Kit isolates total RNA with minimal amounts of genomic DNA contamination However an optional protocol is provided below for maximum removal of residual DNA that may affect sensitive downstream applications It is recommended that Norgen s RNase Free DNase Kit Product 25710 be used for this step 1 For every on column reaction to be performed prepare a mix of 15 uL of DNase I and 100 uL of Enzyme Incubation Buffer A using Norgen s RNase Free DNase Kit Product 25710 Mix gently by inverting the tube a few times DO NOT VORTEX Note If using an alternative DNase I prepare a working stock of 0 25 Kunitz unit uL RNase free DNase solution according to the manufacturer s instructions A 100 uL aliquot is required for each column to be treated 2 Perform the RNA isolation procedure up to and including Binding to Column Steps 1 and 2 of protocol 3 Apply 400 uL of Wash Solution A to the column and centrifuge for 2 minute Discard the flowthrough Reassemble the spin column with its collection tube 4 Apply 100 uL of the RNase free DNase solution prepared in Step 1 to the column and centrifuge at 14 000 x g 14 000 RPM for 1 minute Note Ensure that the entire DNase solution passes
13. t 42 mL of 96 100 ethanol is added to the Poor RNA Solution A supplied Wash Solution A prior to use Recovery Low cell density in the sample The cell number in different urine samples vary While individuals with various diseases have gt 1000 exfoliated cells per mL of urine a healthy male may have a number much lower than the 1000 cells per mL limit It is possible that the total RNA isolated is not visible when resolved on an agarose gel or detected by spectrophotometry In such cases a larger input volume may be used Alternatively a more sensitive method such as BioAnalyzer or RT PCR may be used for detection There is very little or no bacteria in the urine The expected amount of bacteria in a urine sample is very little A healthy individual usually has lt 10 000 CFU mL therefore it is possible that the urine sample has very little bacteria present The isolated RNA may not be visible when resolved on a gel In such cases a larger input volume may be used Alternatively a more sensitive method such as BioAnalyzer or PCR amplification may be used for detection Problem Possible Cause Solution and Explanation Ensure that the appropriate amount of Buffer SK was aal added to the exfoliated cell pellet Insufficient aoa or Ensure that the appropriate amount of lysozyme containing TE buffer and Buffer SK are added to the bacterial pellet in order to completely lyse the cells
14. t is recommended that Norgen s RNase Free DNase Kit Product 25710 be used for this step This step should be performed at this point in the protocol 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 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 50 uL of Elution Buffer E to the column c Centrifuge for 2 minutes at 200 x g 2 000 RPM followed by a 1 minute spin at 14 000 x g 14 000 RPM Note the volume eluted from the column If the entire 50 uL has not been eluted spin the column at 14 000 x g 14 000 RPM for 1 additional minute Note A smaller volume of Elution Buffer E may be used in order to obtain a more concentrated sample A minimum volume of 20 uL is recommended 5 Storage of RNA The purified RNA sample may be
15. uffer SK required B mercaptoethanol is toxic and should be dispensed in a fume hood e Cell pellets can be stored at 70 C for later use or used directly in the procedure e Frozen pellets should be stored for no longer than 2 weeks to ensure that the integrity of the RNA is not compromised e It is important to work quickly during this procedure e The maximum input of urine per column is 50 mL or 1 x 10 exfoliated cells 1 A Lysate Preparation for Exfoliated cells a Transfer 30 mL of urine to a 50 mL conical tube Centrifuge the samples in a swinging bucket centrifuge at 650 x g for 5 minutes The maximum input of urine is 50 mL or 1 x 10 cells per column Pour off the supernatant carefully so as not to disturb or dislodge the cell pellet Note For samples less than 1 5 mL transfer urine to a micro centrifuge tube and centrifuge at 250 x g 2 000 RPM for 5 minutes to pellet the cells Pour off the supernatant carefully so as not to disturb or dislodge the cell pellet b Add 350 uL of Buffer SK to the pellet Lyse cells by vortexing for 15 seconds Ensure that the entire pellet is completely dissolved before proceeding to the next step Transfer the lysate to an RNase free microcentrifuge tube c Add 200 uL of 96 100 ethanol provided by the user to the lysate Mix by vortexing for 10 seconds B Lysate Preparation for Bacteria a Transfer 20 30 mL of urine to a 50 mL conical tube and centrifuge at 3 000 x g for 5
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