Norgen`s Blood RNA Preservation and
Contents
1. RNA Size of RNA Purified lt 200 nt Time to Complete 10 Purifications 45 minutes Average Yield 6 25 ug per 3 mL preserved human blood Kit Components Component ae ae RNA Extraction Buffer A 75 mL 2x 75mL RNA Extraction Buffer B 75 mL 2x 75mL Resuspension Buffer 30 mL 2 x 30 mL Wash Solution 22 mL 2x22 mL Elution Solution 5 mL 2x5mL Mini Spin Columns 48 96 Collection Tubes 48 96 Elution tubes 1 7 mL 48 96 Product Insert 1 1 Customer Supplied Reagents and Equipment Norgen s Blood RNA Preservative Tubes please see Related Products table Centrifuge Temperature controlled with a swinging bucket rotor capable of 6 000 RPM Benchtop microcentrifuge Vortexer Micropipettes with an accuracy range between 10 100 uL and 100 1000 uL Disposable powder free gloves Sterile nuclease free aerosol barrier micropipettor tips 96 100 ethanol B mercaptoethanol 1x PBS Ca2 Mg2 free 50 mL Conical Tubes Storage Conditions and Product Stability The Blood RNA Extraction Buffer A and RNA Extraction Buffer B should be kept tightly sealed and stored upon arrival at 4 C for up to 1 year without showing any reduction in performance All other kit components should be kept tightly sealed and stored at room temperature 18 25 C for up to 1 year without showing any reduction in performance General Precautions e Follow universal precautions Use gloves eye protection and other per2. blood can be disinfected using 1 volume of commercial bleach solution 5 sodium hypochlorite per 9 volumes of the Blood RNA Preservative solution and blood mixture Sample preparation waste such as supernatants from centrifugation steps in the RNA purification procedure is to be considered potentially infectious Before disposal the waste must be autoclaved or incinerated to destroy any infectious material 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 defense 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 samples ensure that they remain on ice during downstream applications Procedures All centrifuga
3. ORGEN 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 2013 Norgen Biotek Corp PI52600 1
4. aa 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 gt Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 BIOTEK si CORPORATION Email techsupport norgenbiotek com Preserved Blood RNA Purification Kit Product Insert for use with Norgen Blood RNA Preservative Tubes Product 52600 52700 Intended Use Norgen s Preserved Blood RNA Purification Kit for use with Norgen Blood RNA Preservative Tubes is intended for the isolation and purification of total RNA from blood that has been preserved using Norgen s Blood RNA Preservative Tubes Please see Norgen s Blood RNA Preservative Tubes Product Insert for detailed information about the use of Norgen s Blood RNA Preservative Tubes Summary Norgen has developed a system that enables the collection stabilization storage and transportation of whole blood specimens together with a rapid and efficient protocol for purification of intracellular RNA The system requires the use of Norgen s Blood RNA Preservative Tubes for blood collection and RNA stabilization followed by RNA purification using Norgen s Preserved Blood RNA Purification Kit for use with Norgen Blood RNA Preservative Tubes Collection of whole blood is the first step in many molecular assays used to study cellular RNA and gene expression However a major problem in such assays is the instability of the cellular RNA profile in vitro Studies have shown that the copy numbers of individual MRNA species in whole
5. blood can change more than 1000 fold during storage or transport at room temperature due to rapid RNA degradation and by induced expression of certain genes after the blood is drawn Such changes in the RNA expression profile interfere with reliable studies of gene expression A method that preserves the RNA expression profile is therefore essential for accurate analysis of gene expression in human whole blood Performance Characteristics of Norgen s Blood RNA Preservative Tubes After blood has been collected into Norgen s Blood RNA Preservative Tubes the intracellular RNA profile remains stable for 12 days at room temperature 18 C to 25 C 21 days at 4 C and for extended periods of time at 20 C and below Isolation of RNA from Preserved Blood Samples Norgen s Preserved Blood RNA Purification Kit purifies all sizes of RNA from large mRNA and ribosomal RNA down to microRNA miRNA and small interfering RNA siRNA The RNA is preferentially purified from other cellular components such as proteins without the use of phenol or chloroform 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 Specifications Kit Specifications Maximum Column Binding Capacity 50 ug Maximum Column Loading Volume 650 uL ee All sizes including small
6. d should be dispensed in a fume hood It is important to work quickly during this procedure e IMPORTANT o Ifthe preserved blood sample was less than 9 mL make up the difference by adding enough 1x PBS Ca Mg free to bring the total volume to 9 mL o If the preserved blood sample was more than 9 mL discard the difference by pipeting enough to bring the total volume to 9 mL o Add RNA Extraction Buffer A and RNA Extraction Buffer B in the same sequence outlined in Step 1 below Transfer the preserved blood to a 50 mL conical tube by decanting Add 1 5 mL RNA Extraction Buffer A followed by the addition of 1 5 mL RNA Extraction Buffer B to the preserved blood to bring the volume to 12 mL Replace the cap on the tube then vortex the tube vigorously at maximum vortex speed for at least 30 seconds to ensure proper mixing IMPORTANT Vortexing for less than 30 seconds may cause clogging of the spin column IMPORTANT The tube MUST be held vertically during vortexing NOT TILTED to ensure that the lysate travels to the top of the tube Tilting the preserved blood mixture during vortexing will cause inadequate mixing that will lead to poor RNA isolation Note Frothing of the sample after vortexing is normal 4 Incubate immediately at 20 C for 10 minutes 5 Centrifuge the tube at 4 C at 3000 5000 x g minimum 4500 rpm on a Beckman JB 6 or equivalent swing bucket centrifuge for 30 minutes 6 Carefully pour off the
7. ed in Appendix A for maximum removal of residual DNA that may affect sensitive downstream applications It is recommended that Norgen s RNase Free DNase Kit Product 25710 be used for this step This step should be performed at this point in the protocol 18 Repeat Step 16 and Step 17 to wash the Mini Spin Columns a second time 19 Wash Mini Spin Column a third time by adding another 400 uL of Wash Solution and centrifuging for 1 minute 20 Discard the flowthrough and reassemble the Mini Spin Column with its collection tube 21 Spin the column for 2 minutes in order to thoroughly dry the resin Discard the collection tube 22 Place the column into a fresh 1 7 mL Elution tube provided with the kit 23 Add 100 uL of Elution Solution to the column Centrifuge for 2 minutes at 200 x g 2 000 RPM followed by 1 minute at 14 000 x g 14 000 RPM Note the volume eluted from the column If the entire 100 uL has not been eluted spin the column at 14 000 x g 14 000 RPM for 1 additional minute Storage of RNA gt The purified RNA sample may be stored at 20 C for a few days It is recommended that samples be placed at 70 C for long term storage Rapid Flow Chart Procedure 6 Transfer the resuspended pellet into a Mini Spin Column r l l l v Centrifuge at 14 000 x g 14 000 RPM 1 min 1 Transfer Stabilized Blood to 50 mL Conical Tube then dilute with 1 5 mL RNA Extraction Buffer A and 1 5 mL RNA Extrac
8. ing 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 Blood RNA Purification Procedure up Step 7 and reassemble the Mini Spin Column with a new collection tube 3 Apply 100 uL of the RNase free DNase solution prepared in Step 1 to the column and centrifuge at 14 000 x g 14 000 RPM for 1 minute Note Ensure that the entire DNase solution passes through the column If needed spin at 14 000 x g 14 000 RPM for an additional minute 4 After the centrifugation in Step 3 pipette the flowthrough that is present in the collection tube back onto the top of the column Note Ensure Step 4 is performed in order to ensure maximum DNase activity and to obtain maximum yields of RNA in particular for small RNA species Incubate the column assembly at 25 30 C for 15 minutes Without any further centrifugation proceed directly to the Step 8 from the Preserved Blood RNA Purification Procedure 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 Total Blood RNA Isolation Kit or N
9. rom human whole blood 4 8 x 10 1 1 x 10 leukocytes ml for gene expression applications It is not for the purification of genomic DNA or viral nucleic acids from human whole blood 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 To avoid the risk of infection e g from HIV or hepatitis B viruses or injury when working with biological and chemical materials always wear a suitable lab coat disposable gloves and protective goggles The Resuspension Buffer contains guanidine hydrochloride and should be handled with care Guanidine hydrochloride 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 buffers 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 The preserved
10. sonal protective equipment to protect from blood splatter blood leakage and possible exposure to bloodborne pathogens e Handle all biological samples and blood collection sharps safely and according to the policies and procedures of your facility Obtain appropriate medical attention in the event of any exposure to biological samples since they may transmit HIV AIDS viral hepatitis or other infectious disease e Dispose of unused kit reagents and human specimens according to local provincial or federal regulations e Use proper pipetting techniques and maintain the same pipetting pattern throughout the procedure to ensure optimal and reproducible values e Do not substitute or mix reagents from different kit lots or from other manufacturers e Do not interchange reagent tube bottle caps as this may lead to contamination and compromise test results e Only use the protocol provided in this insert Alterations to the protocol and deviations from the times and temperatures specified may lead to erroneous results Quality Control In accordance with Norgen s ISO 9001 and ISO 13485 certified Quality Management System each lot of Norgen s Preserved Blood RNA Purification Kit is tested against predetermined specifications to ensure consistent product quality Product Use Limitations For research use only Not for use in diagnostic procedures The Preserved Blood RNA Purification Kit is intended for purification of intracellular RNA f
11. supernatant IMPORTANT Handle the tubes carefully so that you do not dislodge the RNA pellet from the bottom of the tube Note The RNA pellet is transparent and invisible 7 Leave the tube inverted on absorbent paper for 1 to 2 minutes 8 Blot the remaining drops of liquid off the rim of the tube with a clean absorbent paper 9 Pipette 570 uL of Resuspension Buffer into the tube then vortex briefly to resuspend the RNA pellet IMPORTANT To prevent washing any blood residue down the inside of the tube insert the pipette tip into the tube and add the resuspension solution to the bottom of the tube 10 The resuspended RNA can be kept on ice while preparing for the next steps 11 Assemble a spin column with one of the provided collection tubes 12 To the resuspended RNA add 330 uL 96 100 Ethanol then vortex briefly 13 Pipet 450 uL of the mixture from Step 12 into the Mini Spin column then centrifuge for 1 minute 14 Discard the flowthrough Reassemble the spin column with its collection tube 15 Repeat Step 13 and Step 14 to completely transfer the rest of the mixture from Step 12 16 Apply 400 uL of Wash Solution and centrifuge for 1 minute 17 Discard the flowthrough and reassemble the spin column with its collection tube Optional Step Norgen s Preserved Blood RNA Purification Kit isolates total blood RNA with minimal amounts of genomic DNA contamination However an optional On Column DNA Removal Protocol is provid
12. tion 7 Wash Three times with Buffer B 400 uL Wash Solution 2 Vortex vigorously for at least 30 sec ensuring that the lysate travels to the top of the tube Incubate immediately at 20 C for 10 minutes B B 8 Mini Spin Column Drying a Centrifuge at 14 000 x g 14 000 RPM 1 min Centrifuge at 14 000 x g 14 000 RPM 2 min pi a E E ee 3 Centrifuge at 4 C at 3000 5000 x g minimum 4500 rpm for 30 minutes and then carefully pour off supernatant 9 Elute Blood RNA with p 100 uL Elution Solution Centrifuge at 200 x g 2 000 RPM 2 min Centrifuge a 14 000 x g 14 000 RPM 1 min Purified Total Blood RNA Store at 20 C or 80 C for long term storage i 4 Resuspend RNA pellet in 570 uL Resuspension Buffer then vortex briefly 5 Add 330 uL 96 100 Ethanol then vortex briefly Appendix A Protocol for Optional On Column DNA Removal Norgen s Preserved Blood RNA Purification Kit isolates total blood 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 downstream 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 and 100 uL of Enzyme Incubation Buffer using Norgen s RNase Free DNase Kit Product 25710 Mix gently by invert
13. tion steps are carried out in a benchtop microcentrifuge Various speeds are required for different steps so please check your microcentrifuge specifications to ensure that it is capable of the proper speeds All centrifugation steps are performed at room temperature The correct rpm can be calculated using the formula RPM RCF 1 118 x 10 r where ACF required gravitational acceleration relative centrifugal force in units of g r radius of the rotor in cm and RPM the number of revolutions per minute required to achieve the necessary g force Notes prior to use All centrifugation steps are carried out in a benchtop microcentrifuge at 14 000 x g 14 000 RPM except where noted All centrifugation steps are performed at room temperature A variable speed centrifuge should be used for maximum kit performance If a variable speed centrifuge is not available a fixed speed centrifuge can be used however reduced yields may be observed Ensure that all solutions are at room temperature prior to use Prepare a working concentration of the Wash Solution by adding 50 mL of 96 100 ethanol provided by the user to the supplied bottle containing the concentrated Wash Solution This will give a final volume of 72 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added Add 10 uL of B mercaptoethanol provided by the user to each 1 mL of Resuspension Buffer B mercaptoethanol is toxic an
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