User Manual
Contents
1. Adding more may not affect the RNA yields EXCEPT if more Elution Buffer was added Eluting RNA in a higher volume of Elution Buffer will result in diluting your RNA 9 What if forgot to do a dry spin after my third wash e Your RNA elution will be contaminated with traces of the Wash Solution This may dilute the RNA yield in elution Also it may interfere with your downstream applications Re isolate the eluted RNA using the same procedure as you initially isolated the RNA from urine but using your elution as your input 10 Why did my samples show very low RNA yields e Some urine samples contain very little RNA This varies from individual to individual based on numerous variables In order to increase the yield the amount of urine input could be increased 11 Why does my RNA not perform well in downstream applications e f a different Elution Buffer was used other than the one provided in the kit the buffer should be checked for any components that may interfere with the application Common components that are known to interfere are high salts including EDTA detergents and other denaturants Check the compatibility of your elution buffer with the intended use 12 What if the solutions did not flow through the column e The centrifugation speed was too low Check the centrifuge to ensure that it is capable of generating a sufficient centrifugal force that is required to move the liquid phase through the resin You may also spin an ad2. final volume of 90 mL The bottle label contains a box to check to indicate that the ethanol has been added The use of B mercaptoethanol in lysis is highly recommended to isolate RNA for sensitive downstream applications Add 10 uL of B mercaptoethanol provided by the user to each 1 mL of RNA Lysis Solution required B mercaptoethanol is toxic and should be dispensed in a fume hood If precipitates are present in the RNA Lysis Solution it is highly recommended to warm up the RNA Lysis Solution at 60 C for 20 minutes and mix well until the solution becomes clear again It is Important to work quickly during this procedure Detailed Procedure 1 Aliquot a 5 mL urine sample into a 15 mL conical tube Add 5 mL of RNA Lysis Solution directly to the urine Lyse cells by vortexing for 15 seconds Note RNA Lysis Solution contains resin and must be mixed well before every pipeting 2 Add 5 mL of 96 100 ethanol provided by the user to the lysate Mix by vortexing for 10 seconds 3 Centrifuge for 5 minutes at 2 000 x g Discard the supernatant 4 Add 500 uL RNA Wash Solution mix well by pipeting and then transfer the entire contents into a Mini Filter Spin column and centrifuge for 1 minute at 14 000 x g 14 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube 5 Apply 500 uL of RNA Wash Solution to the column and centrifuge for 1 minute at 14 000 x g 14 000 RPM Discard the flowthrough and
3. reported immediately The kit contents are for laboratory use only and they must be stored in the laboratory and must not be used for purposes other than intended The kit contents are unfit for consumption Authorized Representative EMERGO EUROPE Molenstraat 15 E C 2513 BH The Hague The Netherlands R Norgen Biotek Corp 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 2013 Norgen Biotek Corp PIDx29600 1
4. Urine Total RNA Purification Maxi Kit Dx Slurry Format does not provide a diagnostic result It is the sole responsibility of the user to use and validate the kit in conjunction with a downstream in vitro diagnostic assay Kit Components Component Product Dx29600 50 samples RNA Lysis Solution 3 x 90 mL RNA Wash Solution RNA Elution Solution Label Legend Q a boR Yy a m A Do not Use by Batch reuse Code Made in Canada Norgen Biotek Catalogue Number Consult instructions for use In Vitro Diagnostic Medical Device Manu facturer Contains sufficient for lt n gt tests Temper ature limitation Advantages e CE IVD marked in accordance with EU Directive 98 79 EC Fits into in vitro diagnostic workflows Sample collection is non invasive and painless Fast and easy processing using a rapid spin column format Isolate high quality total RNA Compatible with preserved urine samples collected using Norgen s Urine Preservative please see Related Products Table Storage Conditions and Product Stability All solutions should be kept tightly sealed and stored at room temperature All solutions and plastics can be used until the expiration date specified on their labels Precautions 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 a
5. ditional two minutes to ensure that the liquid is able to flow completely through the column 13 Why my RNA is degraded e RNase contamination RNases may be introduced during the use of the kit Ensure proper procedures are followed when working with RNA Please refer to Working with RNA at the beginning of this user guide e Procedure not performed quickly enough In order to maintain the integrity of the RNA it is important that the procedure be performed quickly e The cells are old Older samples contain prematurely lysed cells which release RNase and can degrade RNA Fresh urine samples are recommended Related Products Product 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 5 cc 50 tubes 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 through email at techsupport norgenbiotek com Product Use Restriction Urine Total RNA Purification Maxi Kit Dx Slurry Format provides a fast reliable and simple procedure for isolating total RNA from urine for subsequent in vitro diagnostic use Purification is based on the use of Norgen s proprietary resin as the separation matrix and the kit purifies all s
6. faces with commercially available RNase decontamination solutions e When working with purified RNA samples ensure that they remain on ice during downstream applications Notes prior to use 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 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 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 RNA Wash Solution by adding 66 mL of 96 100 ethanol provided by the user to the supplied bottle containing the concentrated RNA Wash Solution This will give a
7. g x Urine Total RNA Purification Maxi Kit Dx Slurry Format CE Dx29600 Intended Use NORGEN BIOTEK wie CORPORATION 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 905 227 8848 Fax 905 227 1061 Email techsupport norgenbiotek com Product Insert Piox29600 1 Norgen s Urine Total RNA Purification Maxi Kit Dx Slurry Format provides a fast reliable and simple procedure for isolating total RNA from urine for subsequent in vitro diagnostic use Purification is based on the use of Norgen s proprietary resin as the separation matrix and the kit purifies all sizes of RNA from large mRNA and ribosomal RNA down to small RNAs This kit is designed to be used with any downstream application employing enzymatic amplification or other enzymatic modifications of RNA followed by signal detection or amplification Any diagnostic results generated using the RNA isolated with Urine Total RNA Purification Maxi Kit Dx Slurry Format in conjunction with an in vitro diagnostic assay should be interpreted with regard to other clinical or laboratory findings To minimize irregularities in diagnostic results suitable controls for downstream applications should be used Norgen s Urine Total RNA Purification Maxi Kit Dx Slurry Format is intended for use by professional users such as technicians physicians and biologists experienced and trained in molecular biological techniques including RNA isolation Norgen s
8. hould store my samples e We recommend the use of Norgen s Urine Preservative for storage at ambient temperatures Please see Question 1 above 3 What if a variable speed centrifuge is not available e A fixed speed centrifuge can be used however reduced yields may be observed 4 What will happen if my centrifugation speed varied from the recommended speed e This may decrease the binding of the RNA to the column 5 At what temperature should centrifuge my samples e All centrifugation steps are performed at room temperature Centrifugation at 4 C will not adversely affect kit performance 6 My centrifuge speeds are defined in rpm and not in g force How can I convert g force to rpm e The correct rom 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 7 Can I process a different urine volume e Yes you can All the buffers included in this kit are in a linear relationship to the volume of urine sample processed Make sure that you do not deviate from the ratio specified in the product manual The minimum recommended urine input is 5 mL and the maximum recommended input is 10 mL 8 What if added more or less from the specified reagents volume e Adding less volume may reduce your RNA yields
9. izes of RNA from large mRNA and ribosomal RNA down to small RNAs This kit is designed to be used with any downstream application employing enzymatic amplification or other enzymatic modifications of RNA followed by signal detection or amplification Any diagnostic results generated using the RNA isolated with Norgen s Total RNA Purification Maxi Kit Dx Slurry Format in conjunction with an in vitro diagnostic assay should be interpreted with regard to other clinical or laboratory findings To minimize irregularities in diagnostic results suitable controls for downstream applications should be used Norgen s Total RNA Purification Maxi Kit Dx Slurry Format is intended for use by professional users such as technicians physicians and biologists experienced and trained in molecular biological techniques including RNA isolation Norgen s Total RNA Purification Maxi Kit Dx Slurry Format does not provide a diagnostic result lt is the sole responsibility of the user to use and validate the kit in conjunction with a downstream in vitro diagnostic assay The respective user is liable for any and all damages resulting from application of Norgen s Total RNA Purification Maxi Kit Dx Slurry Format for use deviating from the intended use as specified in the user manual All products sold by Norgen Biotek are subjected to extensive quality control procedures and are warranted to perform as described when used correctly Any problems should be
10. re every pipeting Vortex for 15 seconds Add 5 mL 95 100 Ethanol Vortex for 10 seconds SPIN Immediately 2 000 x g 5 min Gil RP Cll A Ga A Ciia Mm Di d eee A Add 500 uL RNA Wash Solution Supernatant Mix Transfer the whole content into a mini filter Column SPIN 14 000 xg 1 min W ash twice with 500 uL RNA Wash Solution ES tJ SPIN 3 14 000 x g 1 min l W ash with 500 uL 95 i 100 Ethanol SPIN 14 000 xg 1 min W 3 14 000 xg 3 min Incubate for 3 min at 60 C Dry SPIN as Elute RNA with RNA elution Solution 50 uL 150 uL Purified Total 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 recommended as your best defence 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 sur
11. reassemble the spin column with its collection tube 6 Repeat Step 5 7 Apply 500 uL of 95 100 ethanol provided by the user and centrifuge for 1 minute at 14 000 x g 14 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube 8 Spin the column empty for 3 minutes at 14 000 x g 14 000 RPM Discard the collection tube 9 Incubate the column with the lid open at 60 C for 3 minutes 10 Transfer the spin column to a fresh 1 7 mL Elution tube Apply 100 uL of RNA Elution Solution to the column and centrifuge for 2 minutes at 200 x g 2 000 RPM followed by 3 minute at 14 000 x g 14 000 RPM Frequently Asked Questions 1 If am not going to process my samples immediately is there any additional preservative for long term storage e We recommend the use of Norgen s Urine Preservative 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 2 If am not going to process my samples immediately how s
12. s convenient PDF files online at www norgenbiotek com The RNA Lysis Solution contains guanidine hydrochloride and should be handled with care Guanidinium salts forms highly reactive compounds when combined with bleach thus care must be taken to properly dispose of any of these solutions Urine of all human and animal subjects is considered potentially infectious All necessary precautions recommended by the appropriate authorities in the country of use should be taken when working with urine Customer Supplied Reagents and Equipment e Benchtop microcentrifuge e 15 mL conical tubes e 96 100 ethanol e B mercaptoethanol Procedure 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 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 Flow Chart Procedure for Purifying Urine Total RNA using Norgen s Urine Total RNA Purification Maxi Kit Dx Slurry Format In a 50mL tube add 5 mL Urine Sample Add 5 mL RNA Lysis Solution RNA Lysis Solution must be mixed well befo
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