Protocol - Norgen Biotek Corp.
Contents
1. Cell Free Urine is now ready for the purification of cell free DNA er i ea es a ee Please check the product and proceed to the appropriate section for I your Urine cfc RNA Purification I Section 1 Urine Cell Free Circulating and Viral Nucleic Acid Purification Mini Kit Cat 59900 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 from2. Step 6 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 Optional Step If only RNA is the primary interest an optional On Column DNA Removal Protocol is provided in Appendix A for the maximum removal of DNA that may affect sensitive downstream applications This step should be performed at this point in the protocol 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 Buffer F 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 nucleic acids are ready for the downstream application of your choice For an explanation of expected yields and recommendations for quantification of the nu
3. gt k 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 i Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 BIOTEK ci CORPORATION Email techsupport norgenbiotek com Urine Cell Free Circulating and Viral Nucleic Acid Purification Kits Product Insert Product 59900 60000 60100 Introduction Recent evidence indicates that cell free circulating cfc nucleic acids such as DNA and RNA including exosomal RNA in urine contains valuable information for the discovery of biomarkers that can help with early detection of certain cancer types for monitoring the disease status as well as for the detection fetal DNA Moreover urine has been widely used for the detection of viral infections including HIV HCV and HBV The advantage for using urine as a source for cancer biomarkers is that it can be acquired in large quantities without using invasive procedures and 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 Exosomes are 40 100 nm memb
4. 0 uL 100 Isopropanol and mix well by pipetting 8 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 9 Repeat Step 8 two more time to transfer the remaining mixture into the Mini Spin column Optional Step If only RNA is the primary interest an optional On Column DNA Removal Protocol is provided in Appendix A for the maximum removal of DNA that may affect sensitive downstream applications This step should be performed at this point in the protocol 10 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 11 Repeat Step 10 one more time for a total of two washes 12 Spin the column empty for 2 minutes at 14 000 x g 14 000 RPM Discard the collection tube 13 Transfer the spin column to a fresh 1 7 mL Elution tube Apply 50 uL of Elution Buffer F 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 14 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 0
5. 00 RPM gt Urine nucleic acid is ready for the downstream application of your choice For an explanation of expected yields and recommendations for quantification of the nucleic acid please refer to Appendix B Appendix A Protocol for Optional On Column DNA Removal Norgen s Urine Cell Free Circulating and Viral Nucleic Acid Purification Kits isolate total nucleic acid RNA and DNA in one elution However an optional protocol is provided below for the maximum removal of DNA that may affect sensitive downstream applications if your primary interest is cell free circulating RNA 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 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 procedure up to Step 7 Mini Format or up to Step 9 Midi and Maxi Format 3 Apply 400 uL of Wash Solution A to the column and centrifuge for 30 seconds at 6 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube 4 Apply 50 uL of the RNase free DNase I solution
6. 62 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 PI59900 1
7. 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 9 Repeat Step 8 one more time to transfer the remaining mixture into the Mini Spin column Optional Step If only RNA is the primary interest an optional On Column DNA Removal Protocol is provided in Appendix A for the maximum removal of DNA that may affect sensitive downstream applications This step should be performed at this point in the protocol 10 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 11 Repeat Step 10 one more time for a total of two washes 12 Spin the column empty for 2 minutes at 14 000 x g 14 000 RPM Discard the collection tube 13 Transfer the spin column to a fresh 1 7 mL Elution tube Apply 50 uL of Elution Buffer F 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 14 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 nucleic acid is ready for the downstream application of your choice F
8. cleic acids please refer to Appendix B Section 2 Urine Cell Free Circulating and Viral Nucleic Acid Purification Midi Kit Cat 60000 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 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 8 Transfer 600 uL of the mixture from
9. d the quantity of the purified nucleic acids Eluting your nucleic acids 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 What If I forgot to do a dry spin before my final elution step e Your purified nucleic acids will be contaminated with the Wash Solution A This may reduce the quality of your purified nucleic acid and will interfere with your downstream applications Can I 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 Why do my samples show low nucleic acid yield e Urine samples contain very little nucleic acid This varies from individual to individual based on numerous variables In order to increase the yield the amount of urine input could be increased Why do the A260 280 ratio of the purified nucleic acid is lower than 2 0 e Most of the Free Circulating urine nucleic acid is short nucleic acid fragments The A260 280 ratio is normally between 1 1 6 This low A260 280 ratio will not affect any downstream application Why does my isolated RNA not perform well in downstream applications e If a different Elution Buffer was used other than the one provided in the kit the buffer should be checked for any components tha
10. d 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 disintegration gt Ensure that all solutions a
11. fication Methods 1 Bioanalyzer RNA Quantification Kits fC G RNA 6000 Nano Kit RNA 6000 Pico Kit Small RNAkit 25 500 ng L 25 250nguL 50 2000 pg uL 5 500ngpL 5 250ng L 5072900 E 5000 50 2000 pg uL Quantitation A o S 2 Bioanalyzer DNA Quantification Kits DNA1000Kit DNA 7500 Kit DNA 12000 Kit High Sensitivity DNA Kit 25 1000 bp 100 7500 bp 100 12000 bp 50 7000 bp 20 CV 20 CV 25 CV 20 CV 0 5 50 ng L 0 5 50 ng L 0 5 50 ng pL 5 500 pg pL 3 NanoDrop 2000 gt Detection Limit 2 ng ul dsDNA 4 Quant iT RiboGreen RNA Assay Kit gt Quantitation Range 1 200 ng 5 Quant iT Pico Green dsDNA Assay Kit gt Detection Limit 25 pg mL 6 qPCR Standard Curve generated using Norgen s Low Abundance DNA Quantification Kit Cat 57200 Standard Curve 1 0 1 2 3 4 Log Starting Quantity O Standard X Unknown SYBR E 296 1 R 2 0 890 Slope 1 673 y int 32 707 Frequently Asked Questions 1 2 What If a variable speed centrifuge is not available e A fixed speed centrifuge can be used however reduced yields may be observed 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 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 an
12. ins 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 59900 Cat 60000 Cat 60100 Sample Type Fresh or frozen urine Fresh or frozen urine Fresh or frozen urine Sample volume Range 250 uL to 2 mL 2to 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 10 Purifications 25 30 minutes 40 45 minutes 45 50 minutes Size of DNA Purified 2 50 bp Size of RNA Purified All sizes including miRNA and small RNA lt 200 nt Average Yields Variable de
13. ion is based on spin column chromatography that uses Norgen s proprietary resin separation matrix The kit is designed to isolate all sizes of cfc DNA and circulating RNA including microRNA as well as all sizes of exosomal RNA Norgen s Urine Cell Free Circulating and Viral Nucleic Acid 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 nucleic Acid is of the highest integrity and can be used in a any downstream applications Kit Descriptions and Components Mini Kit Midi Kit Maxi Kit Cat 59900 Cat 60000 Cat 60100 Number of Preps 50 preps 20 preps 10 preps Binding Solution K 25 mL 75 mL a a Lysis Buffer A 30 mL 20 mL 20 mL Wash Solution A 18 mL 18 mL 18 mL Elution Buffer F 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 total nucleic acid RNA and DNA from fresh urine samples frozen urine samples or urine samples collected on any urine preservative It has been found that urine samples stored at 70 20 or at 4 will develop some precipitation due to the aggregation of some of the highly abundant prote
14. or an explanation of expected yields and recommendations for quantification of the nucleic acid please refer to Appendix B Section 3 Urine Cell Free Circulating and Viral Nucleic Acid Purification Maxi Kit Cat 60100 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 Place 30 mL of cell free urine in a 50 mL tube provided by the user Add 9 mL Binding Solution K and mix well by vortexing for 10 seconds 2 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 3 Repeat Step 2 two more times to transfer the remaining mixture into the Maxi Spin column 4 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 5 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 6 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 7 To the flowthrough from Step 6 add 60
15. pending on specimen Please check page 7 for Average Urine Yields and Common RNA Quantification Methods Customer Supplied Reagents and Equipments 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 and Viral Nucleic Acid Purification Kits are tested against predetermined specifications to ensure consistent product quality Product Use Limitations Norgen s Urine Cell Free Circulating and Viral Nucleic Acid Purification Kits are designed for research purposes only They are not intended for 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 disposable gloves and protective goggles are worn when working with chemicals For more information please consult the appropriate Mate
16. prepared in Step 1 to the column and centrifuge at 8 000 x g 10 000 RPM for 1 minute Note Ensure that the entire DNase solution passes through the column If needed spin at 13 000 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 in Step 9 Mini Format or to the second wash step in Step 11 Midi and Maxi Format Appendix B Cell Free Circulating Nucleic Acid Yield Urine total Nucleic Acid like RNA and DNA in other cell free bodily fluids is normally found in very low amounts 1 100 pg uL therefore measuring cell free RNA or DNA concentration using common quantification methods is very difficult and challenging Typical yields of urine Nucleic Acid 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 exercise and most importantly the heal
17. rane vesicles which are secreted by most cell types Exosomes can be found in saliva blood urine amniotic fluid and malignant ascitic fluids among other biological fluids Evidence has been accumulating recently that these vesicles act as cellular messengers conveying information to distant cells and tissues within the body The exosomes contain cell specific proteins lipids and RNAs which are transported to other cells where they can alter function and or physiology Recent work has demonstrated the presence of distinct subsets of microRNAs within exosomes which depend upon the tumour cell type from which they are secreted For this reason exosomal RNAs may serve as biomarkers for various diseases including cancer Cell free mitochondrial DNA cfmtDNA is also under investigation for its clinical significance In addition cell free fetal DNA has been widely used as a non invasive method for prenatal diagnosis including early identification of fetal sex genetic studies for families at high risk for inherited genetic disorders screening for Rhesus factor screening for aneuploidy and identification of preeclampsia Norgen s Urine Cell Free Circulating and Viral Nucleic Acid Purification Kits provide a fast reliable reproducible and simple procedure for isolating total circulating nucleic acids DNA and RNA from various urine inputs ranging from 250 uL and up to 30 mL with various kit formats addressing different urine input volumes Purificat
18. re 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 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 urine into a fresh 15 50 mL conical tube
19. rial 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 I CAUTION DO NOT add bleach or acidic solutions directly to the sample preparation waste 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 only e All RNA solutions should be prepare
20. t 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 Do I need to do a DNase treatment for my nucleic acid Elution e You may need to do a DNase treatment to your isolated urine miRNA It is recommended to use Norgen s RNase Free DNase Kit Cat 25710 Also please refer to the protocol for optional on column DNA removal outlined in Page 6 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 and Viral Nucleic Acid Purification 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 43
21. th status of the donor Below is a list of the most common RNA and DNA 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 the purified nucleic acid from urine unless large urine volumes have been processed This would only be applicable if urine contains the maximum amount of nucleic acid 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 DNA which will not be the case for the nucleic acid purified from urine Urine nucleic acid is short and fragmented and is usually present in less than 1000 bp Purified urine nucleic acid usually contains traces of proteins which will interfere with most quantification methods leading to the overestimation of the purified nucleic acid concentration Therefore purified nucleic acid contaminated with more proteins will be presented at a higher concentration as compared to nucleic acid purified with less protein contaminants which in this case will depend on the method used for urine nucleic acid purification The only reliable method that can assess the quality and the relative quantity of the purified urine nucleic acid is RT q gPCR RNA or qPCR DNA amplification of a standard RNA or DNA using a small amplicon such as the 5S rRNA housekeeping gene Common Nucleic Acid Quanti
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