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1. 9 Reload the eluted RNA from Step 8 back to the Midi Spin column and let stand at room temperature for 2 minutes Centrifuge for 2 minutes at 500 x g 1 600 RPM 10 To the elution from Step 9 add 300 uL of Lysis Buffer A and mix well by vortexing for 10 seconds Oo NO3 11 Add 400 uL of 96 100 Ethanol provided by the user Mix well by vortexing for 10 seconds 12 Transfer 700 uL of the mixture from Step 11 into a Mini Spin column assembled with one of the provided collection tubes Centrifuge for 2 minutes at 3 300 x g 6 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube 13 Repeat Step 12 one more time to transfer the remaining mixture into the Mini Spin column Optional Step An optional On Column DNA Removal Protocol is provided in Appendix A for maximum removal of residual DNA that may affect sensitive downstream applications This step should be performed at this point in the protocol 14 Apply 400uL of Wash Solution A to the column and centrifuge for 1 minute at 3 300 x g 6 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube 15 Repeat Step 14 two more times for a total of three washes 16 Spin the column empty for 2 minutes at 13 000 x g 14 000 RPM Discard the collection tube 17 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 Cent2. 2 has been transferred to the Maxi Spin column 5 Apply 5 mL of Wash Solution A to the column and centrifuge for 3 minutes at 1 000 x g 2 200 RPM Discard the flowthrough and reassemble the spin column with its collection tube Note Make sure that lid of the tubes is not tightly closed during centrifugation Repeat step 5 one more time for a total of two washes Spin the column empty for 3 minutes at 2 000 x g 3 000 RPM Discard the collection tube Transfer the Maxi Spin column to a fresh 50 mL tube not provided Apply 800 uL of Elution Buffer F to the column and let stand at room temperature for 2 minutes Centrifuge for 2 minutes at 500 x g 1 600 RPM 9 Reload the eluted RNA from Step 8 back to the Maxi Spin column and let stand at room temperature for 2 minutes Centrifuge for 2 minutes at 500 x g 1 600 RPM 10 To the elution from Step 9 add 600 uL of Lysis Buffer A and mix well by vortexing for 10 seconds 11 Add 800 uL of 96 100 Ethanol provided by the user Mix well by vortexing for 10 seconds 12 Transfer 750 uL of the mixture from Step 11 into a Mini Spin column assembled with one of the provided collection tubes Centrifuge for 2 minutes at 3 300 x g 6 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube 13 Repeat Step 12 two more times to transfer the remaining mixture into the Mini Spin column Optional Step An optional On Column DNA Removal Protocol is provided
3. are protected from degradation by RNAses they can be efficiently recovered from biological fluids such as plasma or serum Norgen s Plasma Serum RNA Purification Kits provide a fast reliable reproducible and simple procedure for isolating circulating RNA and exosomal RNA from small plasma serum inputs ranging from 50 uL and up to 5 mL with various kit formats addressing different plasma serum 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 Plasma Serum RNA Purification Kits provide a clear advantage over other available kits in that they do not require phenol chloroform or any protease treatments RNA can be isolated from either fresh or frozen samples using these kits Moreover the kits allow the user to elute into a flexible elution volume ranging from 10 uL to 100 uL Typical yields of free circulating and exosomal RNA vary depending on the input sample as the amount of RNA present in plasma and serum will depend upon the health status of the individual Normally the RNA yield from plasma or serum RNA is highly variable ranging from 1 to 100 ng mL Variability is also observed between samples collected from the same donor at different times during the day This kit is suitable for the isolation of RNA from fresh or frozen serum or plasma p
4. in Appendix A for maximum removal of residual DNA that may affect sensitive downstream applications This step should be performed at this point in the protocol 14 Apply 400 uL of Wash Solution A to the column and centrifuge for 1 minute at 3 300 x g 6 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube 15 Repeat Step 14 two more times for a total of two washes 16 Spin the column empty for 2 minutes at 13 000 x g 14 000 RPM Discard the collection tube co NO 17 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 400 x g 2 000 RPM followed by 2 minutes at 5 800 x g 8 000 RPM 18 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 Plasma Serum 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 B Appendix A Protocol for Optional On Column DNA Removal Norgen s Plasma Serum RNA Purification Kits isolate 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 dow
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 wi CORPORATION S2 NORGEN P Plasma Serum RNA Purification Kits Product Insert Product 55000 56100 56200 Cell free circulating RNA including exosomal RNA in plasma or serum has the potential to provide biomarkers for certain cancers and disease states and includes tumor specific extracellular RNA in the blood Exosomes are 40 100 nm membrane 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 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 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 As the RNA molecules encapsulated within exosomes
6. RT PCR Frozen plasma or serum samples should be centrifuged for 2 minutes at 400 x g 2 000 RPM before processing Only clear supernatant should be processed as column clogging may be encountered if frozen samples are directly processed 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 Please check the product and proceed to the appropriate section for your l Plasma Serum RNA Purification Section 1 Plasma Serum RNA Purification Mini Kit Product 55000 Note The procedure outlined below is for 200 uL inputs of Plasma Serum If processing a sample volume lower than 200 uL Plasma Serum simply bring the volume of your samples up to 200 uL using Nuclease free water and proceed as outlined below 1 Place 200 uL of plasma serum sample in a 2 mL tube provided by the user and add 600 uL of Lysis Buffer A Mix well by vortexing for 10 seconds 2 Add 800 uL of 96 100 ethanol provided by the user Mix well by vortexing for 10 seconds 3 Transfer 650 uL of the mixture from Step 2 into a Micro Spin column Centrifuge for 2 minutes at 3 300 X g 6 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube 4 Repeat Step 3 two more times until all the mixture from Step 2 has been transferred to the Micr
7. ct 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 guanidinium salts and should be handled with care Guanidinium salts form highly reactive compounds when combined with bleach thus care must be taken to properly dispose of any of these solutions Plasma or serum 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 plasma or serum 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 The RNA area should be located away from microbiological work stations 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 There should be designated solutions tips tubes lab coats p
8. e the case for the RNA purified from plasma or serum Plasma Serum RNA is short fragmented RNA which is usually present in less than 1000 bp Purified plasma serum 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 plasma serum RNA purification 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 Quantitative range 25 500 ng pL 25 250ngpl 50 2000 pg L Quantitation R z 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 DNA Standard Curve generated by Norgen Standard Curve I te Log Starting Quantity R 2 0 890 Sjope 1 673 y int 32 707 O Standard X Unknown SYBR E 296 1 Frequently Asked Questions 1 What If a variable speed cenirifuge is not available e A fixed speed c
9. entrifuge can be used however reduced yields may be observed 2 At what temperature should I 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 DNA will be contaminated with the Wash Solution A This may reduce the quality of your purified DNA 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 Plasma Serum samples contain very little RNA This varies from individual to individual based on numerous variables In order to increase the yield the amount of Plasma 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 Free Circulating Plasma Serum RNA is short RNA fragme
10. ipettes etc for RNA only All RNA solutions should be prepared using at least 0 05 DEPC treated autoclaved water or molecular biology grade nuclease free water Clean all surfaces with commercially available RNase decontamination solutions When working with purified RNA ensure that they remain on ice during downstream applications Notes Prior to Use VV V V V VV WV v 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 Micro and 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
11. may cause column clogging or disintegration 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 Prepare a working concentration of the Wash Solution A gt Add 42 mL of 96 100 ethanol provided by the user to the supplied bottle containing 18 mL of the concentrated Wash Solution A This will give a final volume of 60 mL gt Add 90 mL of 96 100 ethanol provided by the user to the supplied bottle containing 38 mL from the concentrated Wash Solution A This will give a final volume of 128 mL gt The labels on the bottles have a box that may be checked to indicate that the ethanol has been added The use of 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 Ensure that samples have not undergone more than one freeze thaw cycle as this may lead to RNA degradation It is recommended to not work with samples that were hemolyzed as this will affect the RNA profile outcome This kit is suitable for the isolation of RNA from serum or plasma prepared from blood collected on either EDTA or citrate Plasma samples prepared from blood collected on heparin should not be used as heparin can significantly interfere with many downstream applications such as
12. nstream 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 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 4 Mini Format or up to Step 13 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 solution 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 min
13. nts 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 9 Do I need to do a DNase treatment for my RNA Elution e You may need to do a DNase treatment to your isolated Plasma Serum 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 on 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 Plasma Serum RNA 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 con
14. o Spin column Optional Step An optional On Column DNA Removal Protocol is provided in Appendix A for maximum removal of residual DNA that may affect sensitive downstream applications This step should be performed at this point in the protocol 5 Apply 400 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 6 Repeat step 5 two more times for a total of three washes 7 Spin the column empty for 2 minutes at 13 000 x g 14 000 RPM Discard the collection tube 8 Transfer the spin column to a fresh 1 7 mL Elution tube Apply from 10 uL up to 25 uL of Elution Solution A 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 9 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 Plasma Serum 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 B Section 2 Plasma Serum RNA Purification Midi Kit Product 56100 Note The procedure outlined below is for processing 250 uL to 1 5 mL inputs of Plasma Serum If the sample volume is l
15. ower than 1 5 mL Plasma Serum simply bring the volume of your sample up to 1 5 mL using Nuclease free water and proceed as outlined below 1 Place 1 5 mL of plasma serum sample in a 15 mL tube provided by the user and add 4 5 mL of Lysis Buffer A Mix well by vortexing for 10 seconds 2 Add 3 mL of 100 Ilsopropanol provided by the user Mix well by vortexing for 10 seconds 3 Transfer 4 5 mL of the mixture from Step 2 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 Note Make sure that lid of the tubes is not tightly closed during centrifugation 4 Repeat Step 3 one more time until all the mixture from Step 2 has been transferred to the Midi Spin column 5 Apply 3 mL of Wash Solution A to the column and centrifuge for 3 minutes at 1 000 x g 2 200 RPM Discard the flowthrough and reassemble the spin column with its collection tube Note Make sure that lid of the tubes is not tightly closed during centrifugation Repeat step 5 one more time for a total of two washes Spin the column empty for 3 minutes at 2 000 x g 3 000 RPM Discard the collection tube Transfer the Midi Spin column to a fresh 15 mL tube not provided Apply 400 uL of Elution Buffer F to the column and let stand at room temperature for 2 minutes Centrifuge for 2 minutes at 500 x g 1 600 RPM
16. repared from blood collected on either EDTA or Citrate Plasma samples prepared from blood collected on heparin should not be used as heparin can significantly interfere with many downstream applications such as RT PCR Kit Descriptions and Components Component Mini Kit Midi Kit Maxi Kit p Cat 55000 Cat 56100 Cat 56200 Number of Preps 50 preps 20 preps 10 preps 1x 130 mL Lysis Buffer A 30 mL 100 mL 1x30 mL i 1 x 38 mL Wash Solution A 18 mL 1x18 mL 38 mL Elution Solution A 6 mL 6 mL 6 mL Elution Buffer F 15 mL 15 mL Micro Spin Columns 50 Mini Spin Columns 20 10 Midi Spin Columns 20 Maxi Spin Columns 10 Collection Tubes 50 20 10 Elution tubes 1 7 mL 50 20 10 Product Insert 1 1 1 For the preparation of working solutions please see Important Notes Notes prior to use Kits Specifications Mini Kit Midi Kit Maxi Kit Cat 55000 Cat 56100 Cat 56200 Sample Type Plasma Serum Plasma Serum Plasma Serum Anti coagulant for Plasma t EDTA or Citrate EDTA or Citrate EDTA or Citrate Sample Volume Range 50 to 200 uL 250 uL to 1 5 mL 2to5mL Minimum Elution Volume 10 uL 50 uL 50 uL Maximum Elution Volume 25 uL 100 uL 100 uL Time to Complete 10 Purifications 15 20 minutes 35 40 minutes 35 40 minutes Size of RNA Purified All sizes including miRNA and small RNA lt 200 nt Average Yields Variable depending on specimen t This kit i
17. rifuge for 1 minute at 400 x g 2 000 RPM followed by 2 minutes at 5 800 x g 8 000 RPM 18 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 Plasma Serum 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 B Section 3 Plasma Serum RNA Purification Maxi Kit Product 56200 Note The procedure outlined below is for processing 2 mL to 5 mL inputs of Plasma Serum If the sample volume is lower than 5 mL Plasma Serum simply bring the volume of your sample up to 5 mL using Nuclease free water and proceed as outlined below 1 Place 5 mL of plasma serum sample in a 50 mL tube provided by the user and add 15 mL of Lysis Buffer A Mix well by vortexing for 10 seconds 2 Add 10 mL of 100 Isopropanol provided by the user Mix well by vortexing for 10 seconds 3 Transfer 15 mL of the mixture from Step 2 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 Note Make sure that lid of the tubes is not tightly closed during centrifugation 4 Repeat Step 3 one more time until all the mixture from Step
18. s suitable for the isolation of RNA from fresh or frozen serum or plasma prepared from blood collected on either EDTA or Citrate Plasma samples prepared from blood collected on heparin should not be used as heparin can significantly interfere with many downstream applications such as RT PCR Please check page 7 for Average Plasma Serum Yields and Common RNA Quantification Methods Customer Supplied Reagents and Equipments e Benchtop microcentrifuge Swinging bucket centrifuges Vortexer Micropipettors 96 100 ethanol 100 Isopropanol Optional 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 years 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 Plasma Serum RNA Purification Kits is tested against predetermined specifications to ensure consistent product quality Product Use Limitations Norgen s Plasma Serum 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 produ
19. tact 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 P155500 4 M14
20. utes 7 Without any further centrifugation proceed directly to the second wash step in Step 6 Mini Format or to the second wash step in Step 15 Midi and Maxi Format Appendix B Cell Free Circulating RNA Yield Plasma Serum 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 plasma serum 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 plasma or serum Cell free circulating 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 plasma or serum unless large plasma serum volumes have been processed This would only be applicable if plasma serum 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 b
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