Protocol - Norgen Biotek Corp.
Contents
1. 10 mL using Nuclease free water and proceed as outlined below 1 Place 10 mL of cell free urine in a 50 mL tube provided by the user Add 3 mL Binding Solution K and mix well by vortexing for 10 seconds 2 Transfer 4 5 mL of the mixture from Step 1 into a Midi Spin column assembled with one of the provided collection tubes Centrifuge for 3 minutes at 1 000 x g 2 200 RPM Discard the flowthrough and reassemble the spin column with its collection tube 3 Repeat Step 2 two more times to transfer the remaining mixture into the Midi Spin column 4 Apply 400 uL of Lysis Buffer A to the column and centrifuge for 3 minutes at 500 x g 1 600 RPM Do Not Discard the flowthrough 5 Apply an additional 400 uL of Lysis Buffer A to the column and centrifuge for 3 minutes at 500 x g 1 600 RPM Do Not Discard the flowthrough 6 Reload the 800 uL flowthrough from Step 5 back to the column and let stand at room temperature for 20 minutes Centrifuge for 3 minutes at 1 000 x g 2 200 RPM Do Not Discard the flowthrough 7 To the flowthrough from Step 6 add 400 uL 100 Isopropanol and mix well by pipetting 11 12 13 14 Transfer 600 uL of the mixture from Step 7 to a Mini Spin column assembled with one of the provided collection tubes Centrifuge for 2 minutes at 3 300 x g 7 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube Repeat Step 8 one more time to transfer the remaining mixture i2. and mix well by vortexing for 10 seconds Transfer 13 mL of the mixture from Step 1 into a Maxi Spin column assembled with one of the provided collection tubes Centrifuge for 3 minutes at 1 000 x g 2 200 RPM Discard the flowthrough and reassemble the spin column with its collection tube Repeat Step 2 two more times to transfer the remaining mixture into the Maxi Spin column Apply 600 uL of Lysis Buffer A to the column and centrifuge for 3 minutes at 500 x g 1 600 RPM Do Not Discard the flowthrough Apply an additional 600 uL of Lysis Buffer A to the column and centrifuge for 3 minutes at 500 x g 1 600 RPM Do Not Discard the flowthrough Reload the 1 2 mL flowthrough from Step 5 back to the column and let stand at room temperature for 30 minutes Centrifuge for 3 minutes at 1 000 x g 2 200 RPM Do Not Discard the flowthrough To the flowthrough from Step 6 add 600 uL 100 Isopropanol and mix well by pipetting Transfer 600 uL of the mixture from Step 7 to a Mini Spin column assembled with one of the provided collection tubes Centrifuge for 2 minutes at 3 300 x g 7 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube Repeat Step 8 two more time to transfer the remaining mixture into the Mini Spin column Apply 600 uL of Wash Solution A to the column and centrifuge for 1 minute at 3 300 x g 7 000 RPM Discard the flowthrough and reassemble the spin column with its collection tub
3. back to the Mini Spin column assembled with one of the provided collection tubes Centrifuge for 2 minutes at 3 300 x g 7 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube 8 Apply 600 uL of Wash Solution A to the column and centrifuge for 1 minute at 3 300 x g 7 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube 9 Repeat Step 8 one more time for a total of two washes 10 Spin the column empty for 2 minutes at 14 000 x g 14 000 RPM Discard the collection tube 11 Transfer the spin column to a fresh 1 7 mL Elution tube Apply 50 uL of Elution Solution A to the column and let stand at room temperature for 2 minutes Centrifuge for 1 minute at 200 x g 2 000 RPM followed by 2 minutes at 5 200 x g 8 000 RPM 12 For maximum recovery transfer the eluted buffer back to the column and let stand at room temperature for 2 minutes Centrifuge for 1 minute at 400 x g 2 000 RPM followed by 2 minutes at 5 800 x g 8 000 RPM gt Urine cfc RNA is ready for the downstream application of your choice For an explanation of expected yields and recommendations for quantification of the RNA please refer to Appendix A Section 2 Urine Cell Free Circulating RNA Purification Midi Kit Cat 57000 Note The procedure outlined below is for 10 mL inputs of urine If processing a sample volume lower than 10 mL urine simply bring the volume of your samples up to
4. 0 Purifications 25 30 minutes 40 45 minutes 45 50 minutes Size of RNA Purified All sizes including miRNA and small RNA lt 200 nt Average Yields Variable depending on specimen Please check page 6 for Average Urine Yields and Common RNA Quantification Methods Customer Supplied Reagents and Equipments e Benchtop microcentrifuge Swinging bucket centrifuges Vortexer Micropipettors 96 100 ethanol 100 Isopropanol B Mercaptoethanol Storage Conditions and Product Stability All buffers should be kept tightly sealed and stored at room temperature 15 25 C for up to 2 year without showing any reduction in performance It is recommended to warm Lysis Buffer A for 20 minutes at 60 C if any salt precipitation is observed Quality Control In accordance with Norgen s Quality Management System each lot of Norgen s Urine Cell Free Circulating RNA Purification Kits are tested against predetermined specifications to ensure consistent product quality Product Use Limitations Norgen s Urine Cell Free Circulating RNA Purification Kits are designed for research purposes only It is not intended for human or diagnostic use Product Warranty and Satisfaction Guarantee NORGEN BIOTEK CORPORATION guarantees the performance of all products in the manner described in our product manual The customer must determine the suitability of the product for its particular use Safety Information Ensure that a suitable lab coat d
5. 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 Email techsupport norgenbiotek com BIOTEK wie CORPORATION S2 NORGEN P Urine Cell Free Circulating RNA Purification Kits Product Insert Product 56900 57000 57100 Introduction Recent evidence indicates that cell free circulating RNA cfc RNA including exosomal RNA in urine contains valuable information for the discovery of biomarkers that can help with early detection of certain cancer types and for monitoring the disease status The advantage for using urine as a source for cancer biomarkers is that it can be acquired in large quantities without using invasive procedures In addition repeated sampling from the same individual is applicable which facilitates longitudinal studies There are many advantages favouring the use of urinary nucleic acids for cancer biomarker discovery over blood tissue samples or other bodily fluids including 1 urine is non infectious for HIV and less infectious for many other pathogens 2 the profile of urinary nucleic acids is similar to that in plasma or serum 3 nucleic acid purification from urine is technically much easier because of its low protein concentration 1000 fold lower than blood Norgen s Urine Cell Free Circulating RNA Purification Kits provide a fast reliable reproducible and simple procedure for isolating circulating RNA and exosomal RNA from various urine inputs ran
6. RNAkit 25 500ng uL 25 250 ng 50 2000 pg uL 50 5000 5000 5 500 ng uL 5 250 ng uL pg uL pg uL 50 2000 pg uL Quantitation 6 P 3 2 NanoDrop 2000 gt Detection Limit 2 ng ul dsDNA 3 Quant iT RiboGreen RNA Assay Kit gt Quantitation Range 1 200 ng 4 qPCR Standard Curve generated by Norgen Standard Curve a a ae or ne a oe a Serer E Log Starting Quantity O Standard X Unknown SYBR E 296 1 R 2 0 890 Sjope 1 673 y int 32 707 Frequently Asked Questions 1 What If a variable speed cenirifuge is not available e A fixed speed centrifuge can be used however reduced yields may be observed 2 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 3 What if added more or less of the specified reagents volume e Adding more or less than the specified volumes may reduce both the quality and the quantity of the purified RNA Eluting your RNA in high volumes will increase the yield but will lower the concentration Eluting in small volumes will increase the concentration but will lower the overall yield 4 What if I forgot to do a dry spin before my final elution step e Your purified RNA will be contaminated with the Wash Solution A This may reduce the quality of your purified RNA and will interfere with your downstream applications 5 Can I
7. d on any urine preservative It has been noticed that urine samples stored at 70 20 or at 4 will develop some precipitation due to the aggregation of some of the highly abundant proteins in urine Eliminating these precipitates using centrifugation or filtration may cause the loss of some of the cfc protein bound RNA Furthermore these precipitates may affect the quality of the purified nucleic acid We recommend the use of Norgen s Urine Preservative when collecting urine samples Norgen s Urine Preservative is designed for the preservation of nucleic acids and proteins in fresh urine samples at ambient temperatures therefore no protein precipitation will occur and the purified nucleic acids will be of a higher quality 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 as 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 Kits Specifications Mini Kit Midi Kit Maxi Kit Cat 56900 Cat 57000 Cat 57100 Sample Type Fresh or frozen urine Fresh or frozen urine Fresh or frozen urine Sample volume Range 250 uL to 2 mL 2 to 10 mL 10 to 30 mL Minimum Elution Volume 50 uL 50 uL 50 uL Maximum Elution Volume 100 uL 100 uL 100 uL Time to Complete 1
8. e Repeat Step 10 one more time for a total of two washes Spin the column empty for 2 minutes at 14 000 x g 14 000 RPM Discard the collection tube Transfer the spin column to a fresh 1 7 mL Elution tube Apply 50 uL of Elution Solution A to the column and let stand at room temperature for 2 minutes Centrifuge for 1 minute at 200 x g 2 000 RPM followed by 2 minutes at 5 200 x g 8 000 RPM For maximum recovery transfer the eluted buffer back to the column and let stand at room temperature for 2 minutes Centrifuge for 1 minute at 400 x g 2 000 RPM followed by 2 minutes at 5 800 x g 8 000 RPM gt Urine cfc RNA is ready for the downstream application of your choice For an explanation of expected yields and recommendations for quantification of the RNA please refer to Appendix A Appendix A Cell Free Circulating RNA Yield Urine cfc RNA like RNA in other cell free bodily fluids is normally found in very low amounts 1 100 pg uL therefore measuring cell free RNA concentration using common quantification methods is very difficult and challenging Typical yields of urine cfc RNA vary significantly from sample to sample Variability is also observed between samples collected from the same donor at different times during the day and therefore there is no absolute yield for RNA purified from bodily fluids including urine Cell free circulating RNA yield varies depending on a number of factors including age sex diet exerc
9. ging from 250 uL and up to 30 mL with various kit formats addressing different urine input volumes Purification is based on spin column chromatography that uses Norgen s proprietary resin separation matrix The kit is designed to isolate all sizes of circulating RNA including microRNA as well as all sizes of exosomal RNA Norgen s Urine Cell Free Circulating RNA Purification Kits provides a clear advantage over other available kits in that they do not require phenol chloroform or any protease treatments Moreover the kit allows the user to elute into a flexible elution volume ranging from 50 uL to 100 uL 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 Kit Descriptions and Components Mini Kit Midi Kit Maxi Kit Cat 56900 Cat 57000 Cat 57100 Number of Preps 50 preps 20 preps 10 preps Binding Solution K 25 mL 75 mL l a i Lysis Buffer A 30 mL 20 mL 20 mL Wash Solution A 18 mL 18 mL 18 mL Elution Solution A 6 mL 6 mL 6 mL Mini Spin Columns 50 20 10 Midi Spin Column 20 Maxi Spin Column 10 Collection Tubes 50 20 10 Elution tubes 1 7 mL 50 20 10 Product Insert 1 1 1 These kits are suitable for the isolation of cfc RNA from fresh urine samples frozen urine samples or urine samples collecte
10. ise and most importantly the health status of the donor Below is a list of the most common RNA quantification methods as well as the limit of detection for each of these methods Unfortunately none of these methods can be used reliably for measuring the concentration of RNA purified from urine unless large urine volumes have been processed This would only be applicable if urine contains the maximum amount of RNA that can fit within the specification range of these quantification tools It should be noted that the specifications outlined below are based on measuring a pure RNA which will not be the case for the RNA purified from urine Urine RNA is short fragmented RNA which is usually present in less than 1000 bp Purified urine RNA usually contains traces of proteins which will interfere with most quantification methods leading to the overestimation of the purified RNA concentration Therefore purified RNA contaminated with more proteins will be presented at a higher concentration as compared to RNA purified with less protein contaminants which in this case will depend on the method used for urine RNA purification The only reliable method that can assess the quality and the relative quantity of the purified urine RNA is RT qPCR amplification of a standard RNA using a small RNA amplicon such as the 5S rRNA housekeeping gene Common RNA Quantification Methods 1 Bioanalyzer RNA Quantification kits Po RNA 6000 Nano Kit RNA 6000 Pico Kit Small
11. isposable 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 Binding Solution K and Lysis Buffer A contain guanidium salt and should be handled with care Guanidium salt forms highly reactive compounds when combined with bleach thus care must be taken to properly dispose of any of these solutions If liquid containing these solutions is spilt clean with suitable laboratory detergent and water If the spilt liquid contains potentially infectious agents clean the affected area first with laboratory detergent and water and then with 1 v v sodium hypochlorite Important Notes 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 o
12. n Kits or NORGEN products in general please do not hesitate to contact us NORGEN customers are a valuable source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as well as to the researchers at NORGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information please 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 or call one of the NORGEN local distributors www norgenbiotek com or through email at techsupport norgenbiotek com 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 P156900 3
13. nly 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 ensure that they remain on ice during downstream applications Notes Prior to Use All centrifugation steps are performed at room temperature Ensure that centrifuge tubes used are capable of withstanding the centrifugal forces required The provided spin columns are optimized to be used with a benchtop centrifuges and not to be used on a vacuum apparatus Most standard benchtop microcentrifuges will accommodate Norgen s Mini Spin Columns Most standard swinging bucket centrifuges will accommodate Norgen s Midi and Maxi Spin Columns Do not use a fixed angle rotor Norgen s Midi and Maxi Spin Columns are centrifuged in 15 mL and 50 mL centrifuge tubes respectively Centrifuging Norgen s Spin columns at a speed higher than recommended may affect RNA yield Centrifuging Norgen s Spin columns at a speed lower than recommended will not affect RNA yield However centrifugation at a lower speed may require longer time for the solutions to pass through the spin column When placing Norgen s Midi and Maxi Spin Columns into the swinging bucket centrifuge make sure that lids of the tubes are not tightly closed Tightly closed lids may cause back pressure which may cause column clogging or disintegra
14. nto the Mini Spin column Apply 600 uL of Wash Solution A to the column and centrifuge for 1 minute at 3 300 x g 7 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube Repeat Step 10 one more time for a total of two washes Spin the column empty for 2 minutes at 14 000 x g 14 000 RPM Discard the collection tube Transfer the spin column to a fresh 1 7 mL Elution tube Apply 50 uL of Elution Solution A to the column and let stand at room temperature for 2 minutes Centrifuge for 1 minute at 200 x g 2 000 RPM followed by 2 minutes at 5 200 x g 8 000 RPM For maximum recovery transfer the eluted buffer back to the column and let stand at room temperature for 2 minutes Centrifuge for 1 minute at 400 x g 2 000 RPM followed by 2 minutes at 5 800 x g 8 000 RPM gt Urine cfc RNA is ready for the downstream application of your choice For an explanation of expected yields and recommendations for quantification of the RNA please refer to Appendix A Section 3 Urine Cell Free Circulating RNA Purification Maxi Kit Cat 57100 Note The procedure outlined below is for 30 mL inputs of urine If processing a sample volume lower than 30 mL urine simply bring the volume of your samples up to 30 mL using Nuclease free water and proceed as outlined below 1 2 11 12 13 14 Place 30 mL of cell free urine in a 50 mL tube provided by the user Add 9 mL Binding Solution K
15. perform a second elution e Yes but it is recommended that the 2 elution be in a smaller volume 50 of 1 Elution It is also recommended to perform the 2 elution into a separate elution tube to avoid diluting the 1 elution 6 Why do my samples show low RNA yield e 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 7 Why do the A260 280 ratio of the purified RNA is lower than 2 0 e Most of the Free Circulating Urine RNA is short RNA fragments The A260 280 ratio is normally between 1 1 6 This low A260 280 ratio will not affect any downstream application 8 Why does my isolated RNA not perform well in downstream applications e lf 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 Technical Assistance NORGEN s Technical Service Department is staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of NORGEN products If you have any questions or experience any difficulties regarding Norgen s Urine Cell Free Circulating RNA Purificatio
16. rine into a fresh 15 50 mL conical tube Cell Free Urine is now ready for the purification of cell free RNA Please check the product and proceed to the appropriate section for your Urine cfc RNA Purification Section 1 Urine Cell Free Circulating RNA Purification Mini Kit Cat 56900 Note The procedure outlined below is for 2 mL inputs of urine If processing a sample volume lower than 2 mL urine simply bring the volume of your samples up to 2 mL using Nuclease free water and proceed as outlined below 1 Place 2 mL of cell free urine in a 15 mL tube provided by the user Add 400 uL Binding Solution K and mix well by vortexing for 10 seconds 2 Transfer 800 uL of the mixture from Step 1 into a Mini Spin column assembled with one of the provided collection tubes Centrifuge for 2 minutes at 3 300 x g 7 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube 3 Repeat Step 2 two more times to transfer the remaining mixture into the Mini Spin column 4 Apply 500 uL of Lysis Buffer A to the column and centrifuge for 1 minute at 3 300 x g 7 000 RPM Do Not Discard the flowthrough 5 Reload the flowthrough from Step 4 back to the column and let stand at room temperature for 10 minutes Centrifuge for 1 minute at 3 300 x g 7 000 RPM Do Not Discard the flowthrough 6 To the flowthrough from Step 5 add 250 uL 100 Isopropanol and mix well by pipetting 7 Transfer the entire mixture from Step 6
17. tion gt Ensure that all solutions are at room temperature prior to use gt It is highly recommended to warm up Lysis Buffer A at 60 C for 20 minutes and mix well until the solutions become clear again if precipitates are present gt 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 18mL of 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 gt 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 Lysis Buffer A gt If any of the solutions do not go through the Spin Columns within the specified centrifugation time spin for an additional 1 2 minutes until the solution completely passes through the Column Do NOT exceed the centrifugation speed as this may affect RNA yield VV V VV VV WV v Preparation of Cell free Urine Sample 1 Collect and transfer 15 50 mL of the urine into a conical tube and centrifuge at 200 x g 1 000 RPM for 10 minutes to remove urine exfoliated cells and debris Decant cell free urine into new 15 50 mL conical tube 2 Centrifuge the cell free urine at 1 800 x g 3 000 RPM for 10 minutes to remove any residual debris or bacterial cells 3 Transfer cell free u
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