58000 - Protocol (50 prep)
Contents
1. a Serer A 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 centrifuge 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 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 Exosomes contain very little RNA This varies from individual to individual based on numerous varia2. filters Vortex 96 100 Ethanol 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 General Precautions All biological samples should be considered as potentially infectious Proper biosafety measures should therefore be carried out when using this kit Quality Control In accordance with Norgen s ISO 9001 and ISO 13485 certified Quality Management System each lot of Norgen s Exosomal RNA Isolation Kit are tested against predetermined specifications to ensure consistent product quality Product Use Limitations Norgen s Exosomal RNA Isolation Kit 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 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 Lysis Buffer A contains guan
3. through the Column Do NOT exceed the centrifugation speed as this may affect RNA yield Vv vy Vv Section 1 Exosomal RNA Isolation from Exosomes Purified using Norgen s Urine Exosome Purification Mini Kit or Norgen s Plasma Serum Exosome Purification Mini Kit 1 Add 300 uL of Lysis Buffer A and 37 5 uL of Lysis Additive B to the 200 uL ExoR Buffer containing the purified Exosomes 2 Mix well by vortexing for 10 seconds then incubate at room temperature for 10 minutes 3 After incubation add 500 uL of 96 100 Ethanol to the mixture from Step 2 and mix well by vortexing for 10 seconds 4 Transfer 500 uL of the mixture from Step 3 into a Mini Spin column Centrifuge for 1 minute at 3 300 x g 6 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube 5 Repeat Step 4 one more time to transfer the remaining mixture from Step 3 into the Mini Spin Column 6 Apply 600 uL of Wash Solution A to the column and centrifuge for 30 seconds at 3 300 x g 6 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube 7 Repeat Step 6 one more time for a total of two washes Spin the column empty for 1 minute at 13 000 x g 14 000 RPM Discard the collection tube 9 Transfer the spin column to a fresh 1 7 mL Elution tube Apply 50 uL of Elution Solution A to the column and centrifuge for 1 minute at 2 000 RPM followed by 2 minutes at 8 000 RPM 10 For maximum recovery t
4. Fax 905 227 1061 BIOTEK amp CORPORATION Email techsupport norgenbiotek com gt k 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 i Phone 866 667 4362 e 905 227 8848 Exosomal RNA Isolation Kit Product Insert for use with Norgen s Urine Exosome Purification Kits or Plasma Serum Exosome Purification Kits Product 58000 Exosomes are 40 150 nm membrane vesicles which are secreted by most cell types Exosomes can be found in urine saliva plasma serum amniotic fluid and malignant ascite 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 These exosomes may play a functional role in mediating adaptive immune responses to infectious agents and tumours tissue repair neural communication and transfer of pathogenic proteins Recent work has demonstrated the presence of distinct subsets of microRNAs within exosomes and other extracellular vesicles EVs which depend upon the tumour cell type from which they are secreted For this reason exosomal RNA may serve as biomarkers for various diseases including cancer Norgen s Exosomal RNA Isolation Kit constitutes an all in one system for the isolation of exosomal RNA from exosomes previously purif
5. bles In order to increase the yield the amount of urine plasma or serum input could be increased 7 Why do the A260 280 ratio of the purified RNA is lower than 2 0 e Most of the Exosomal RNA is short RNA fragments with a very low concentration where the A260 280 ratio tends to decrease with the decrease in the RNA concentration 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 Exosomal RNA Isolation Kit 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 ther
6. column empty for 1 minute at 13 000 x g 14 000 RPM Discard the collection tube 9 Transfer the spin column to a fresh 1 7 mL Elution tube Apply 50 uL of Elution Solution A to the column and centrifuge for 1 minute at 2 000 RPM followed by 2 minutes at 8 000 RPM 10 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 ad gt Exosomal RNA is now ready for downstream applications Appendix A Exosomal RNA Yield Exosomal RNA like RNA in other cell free bodily fluids is normally found in very low amounts 1 100 pg uL therefore measuring exosomal RNA concentration using common quantification methods is very difficult and challenging Typical yields of exosomal 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 Exosomal RNA yield varies depending on a number of factors including age sex diet exercise 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 exosomes unl
7. efore 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 PI58000 1
8. ess large volumes have been processed This would only be applicable if exosomes contain 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 sample which will not be the case for exosomal RNA Exosomal RNA is short fragmented RNA which is usually present in less than 1000 bp Purified exosomal 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 The only reliable method that can assess the quality and the relative quantity of the purified plasma serum 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 RNAkit 25 500 ng L 25 250 nga 50 2000 pg uL 50 5000 a r 5000 5 500 ng L 5 250 ng uL pg uL pg uL 50 2000 pg uL Quantitation 6 P 5 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
9. idine thiocyanate and should be handled with care Guanidine thiocyanate forms highly reactive compounds when combined with bleach thus care must be taken to properly dispose of any of this solution If liquid containing these buffers is spilled clean with suitable laboratory detergent and water If the spilled 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 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 ensure that they remain o
10. ied using Norgen s Urine Exosome Purification Kits or Norgen s Plasma Serum Exosome Purification Kits This kit also allows for the isolation of RNA from intact extracellular vesicles EVs purified from different urine or plasma serum sample volumes The purification is based on spin column chromatography that employs Norgen s proprietary resin The kit is designed to isolate all sizes of extracellular vesicle RNA including microRNA The kit provides a clear advantage over other available kits in that it does not require any special instrumentation protein precipitation reagents extension tubes phenol chloroform or 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 Component Product 58000 50 preps Lysis Buffer A 20 mL Lysis Additive B 2 mL Wash Solution A 18 mL Elution Solution A 6 mL Mini Spin Columns 50 Collection Tubes 50 Elution tubes 1 7 mL 50 Product Insert 1 Please check page 5 for Average Yields and Common RNA Quantification Methods Customer Supplied Reagents and Equipment e Disposable powder free gloves Benchtop microcentrifuge Micropipettors Sterile pipette tips with
11. ly 50 uL of Elution Solution A to the column and centrifuge for 1 minute at 2 000 RPM followed by 2 minutes at 8 000 RPM 10 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 Exosomal RNA is now ready for downstream applications Section 3 Exosomal RNA Isolation from Exosomes Purified using Norgen s Urine Exosome Purification Maxi Kit or Norgen s Plasma Serum Exosome Purification Maxi Kit 1 Add 900 uL of Lysis Buffer A and 125 uL of Lysis Additive B to the 600 uL ExoR Buffer containing the purified Exosomes 2 Mix well by vortexing for 10 seconds then incubate at room temperature for 20 minutes 3 After incubation add 1 5 mL of 96 100 Ethanol to the mixture from Step 2 and mix well by vortexing for 10 seconds 4 Transfer 750 uL of the mixture from Step 3 into a Mini Spin column Centrifuge for 1 minute at 3 300 x g 6 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube 5 Repeat Step 4 two more times to transfer the remaining mixture from Step 3 into the Mini Spin Column 6 Apply 600 uL of Wash Solution A to the column and centrifuge for 30 seconds at 3 300 x g 6 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube 7 Repeat Step 6 one more time for a total of two washes Spin the
12. n ice during downstream applications Notes Prior to Use gt All centrifugation steps are performed at room temperature gt Ensure that centrifuge tubes used are capable of withstanding the centrifugal forces required gt 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 Micro Spin Columns 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 Ensure that all solutions are at room temperature prior to use 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 18 mL 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 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
13. ransfer 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 Exosomal RNA is now ready for downstream applications Section 2 Exosomal RNA Isolation from Exosomes Purified using Norgen s Urine Exosome Purification Midi Kit or Norgen s Plasma Serum Exosome Purification Midi Kit 1 Add 600 uL of Lysis Buffer A and 75 uL of Lysis Additive B to the 400 uL ExoR Buffer containing the purified Exosomes 2 Mix well by vortexing for 10 seconds then incubate at room temperature for 15 minutes 3 After incubation add 1 mL of 96 100 Ethanol to the mixture from Step 2 and mix well by vortexing for 10 seconds 4 Transfer 750 uL of the mixture from Step 3 into a Mini Spin column Centrifuge for 1 minute at 3 300 x g 6 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube 5 Repeat Step 4 two more times to transfer the remaining mixture from Step 3 into the Mini Spin Column 6 Apply 600 uL of Wash Solution A to the column and centrifuge for 30 seconds at 3 300 x g 6 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube 7 Repeat Step 6 one more time for a total of two washes Spin the column empty for 1 minute at 13 000 x g 14 000 RPM Discard the collection tube 9 Transfer the spin column to a fresh 1 7 mL Elution tube App
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