PAXgene Blood RNA MDx Kit Handbook
Contents
1. Al ny DNase digestion Wash 3x Pure RNA PAXgene Blood RNA MDx Kit Handbook 04 2010 Equipment and Reagents to Be Supplied by User When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier For all protocols e PAXgene Blood RNA Tubes cat no 762165 e step dispenser such as the Multipette plus from Eppendorf recommended for optimal processing e Square well blocks optional cat no 19585 e Elution Microtubes CL racked optional cat no 1030483 e Caps for Elution Microtubes optional cat 1030481 e Disposable Troughs 30 ml optional cat 9232764 e Disposable gloves e 96 100 ethanol p a e Centrifuge capable of attaining 3000 5000 x g equipped with a swing out rotor and buckets to hold PAXgene Blood RNA Tubes PAXgene 96 Incubator Block cat 9238279 e Incubator capable of 80 C e Scalpel or spatula to remove the lower plate from the elution microtube rack e Heavy plate to prevent caps from opening during incubation at 80 C Ice For the BioRobot MDx protocol e BioRobot MDx workstation cat no 900600 e multitube vortexer such as the VX2500 from VWR recommended for optimal processing For the BioRobot Universal System protocol e BioRobot Universal System cat no 9001092. A processing error occurred during sample preparation Information about the status of the samples being processed when the protocol was interrupted may have been lost The tip disposal bag in the tip disposal container was not emptied leading to a tip jam After the protocol has stopped carefully shake the container in the position beneath the tip disposal station and try to pull it out Empty the tip disposal bag remove the jammed tips replace the tip disposal container and continue the protocol PAXgene Blood RNA MDx Kit Handbook 04 2010 e Low white blood cell count Low ratio a RNA purity measured in water b Spectrophotometer not properly zeroed General handling a Clogging of the PAXgene 96 RNA plate b Variable elution volumes c Result of single samples marked as invalid in the report file d Some positions in the Sample result column in the report file are empty e Zmovement blocked during tip disposal Comments and suggestions The tip disposal bag was not inserted thoroughly into the tip disposal container leading to a tip jam The bag must fit tightly to the walls of the container so that ejected tips fall down freely Carefully shake the container in the position beneath the tip disposal station and try pull it out Empty the tip disposal bag remove the jammed tips and make sure that the bag fits tightly to the container Replace the tip disposal container
3. Second Edition Blood RNA MDx Kit Handbook For automated purification of cellular RNA from whole blood using the BioRobot MDx or the BioRobot Universal System Important To be used only in conjunction with PAXgene Blood RNA Tubes For Research Use Only Not for use in diagnostics procedures April 20 1 0 A QIAGEN BD Company Trademarks PAXgene PreAnalytiX PreAnalytiX GmbH QIAGEN BioRobot QIAGEN Group BD Hemogard BD Vacutainer Hemogard Safety Lok Becton Dickinson and Company Franklin Lakes NJ USA Eppendorf Multipette Eppendorf Netheler Hinz GmbH NASBA Organon Teknika Speed Vac Savant Instruments Inc The PAXgene Blood RNA MDx System is for Research Use Only Not for use in diagnostics procedures No claim or representation is intended to provide information for the diagnosis prevention or treatment of a disease The PCR process is covered by the foreign counterparts of U S Patents Nos 4 683 202 and 4 683 195 owned by F Hoffmann La Roche Ltd 2005 PreAnalytiX GmbH all rights reserved PreAnalytiX Company PreAnalytiX GmbH Feldbachstrasse CH 8634 Hombrechtikon Switzerland www preanalytix com PreAnalytiX Distributors PreAnalytiX products are manufactured for PreAnalytiX by QIAGEN or BD and are distributed for PreAnalytiX by QIAGEN or BD Products cannot be ordered at PreAnalytiX GmbH Please see the last page for contact information for your local PreAnaly
4. Secondary Hemogard Closures QCard PAXgene RNA MDx Handbook Contains a guanidine salt See page 6 for safety information Buffer BRA is supplied as a concentrate Before using for the first time add 4 volumes of ethanol 96 100 purity grade p a as indicated on the bottle to obtain a working solution PAXgene Blood RNA MDx Kit Handbook 04 2010 Also available separately See page 25 for ordering information Shipping and Storage Except for the RNase Free DNase Set the remaining components of the PAXgene Blood RNA MDx Kit can be stored at room temperature 15 25 C The PAXgene Blood RNA MDx Kit contains ready to use proteinase K solution that can be stored at room temperature 15 25 C To store for extended periods of time we suggest keeping the proteinase K at 2 8 C The QIAGEN RNase Free DNase Set is shipped at room temperature All components should be stored immediately upon receipt at 2 8 C When stored at 2 8 C and handled correctly the buffers and the lyophilized enzyme can be kept for at least 9 months without showing any reduction in performance Product Use Limitations For Research Use Only Not for use in diagnostics procedures No claim or representation is intended to provide information for the diagnosis prevention or treatment of a disease The performance characteristics of this product have not been fully established Product Warranty and Satisfaction Guarantee PreAnalytiX
5. PAXgene Blood RNA MDx Kit Handbook 04 2010 21 Appendix Quantification and Determination of Quality of Total RNA Quantification of RNA The concentration of RNA should be determined by measuring the absorbance at 260 nm in a spectrophotometer To ensure significance readings should be in the linear range of the spectrophotometer An absorbance of 1 unit at 260 nm corresponds to 44 pg of RNA per ml 1 44 pg ml This relation is valid only for measurements in 10 mM Tris Cl pH 7 5 Therefore if it is necessary to dilute the RNA sample this should be done in 10 mM Tris Cl As discussed below see Purity of RNA the ratio between the absorbance values at 260 and 280 nm gives an estimate of RNA purity When measuring RNA samples be certain that cuvettes are RNase free Use the buffer in which the RNA is diluted to zero the spectrophotometer and make sure to add the same volume of Buffer BR5 as the volume of eluted RNA to be diluted Buffer BR5 has high absorbance at 220 nm which can lead to high background absorbance levels if the spectrophotometer is not properly zeroed An example of the calculation involved in RNA quantification is shown below Volume of RNA sample 120 pl Dilution 10 pl of RNA sample 140 pl 10 mM Tris Cl pH 7 5 1 15 dilution Measure absorbance of diluted sample in a cuvette RNase free 0 2 Concentration RNA sample 44 x A x dilution factor 44x0 2x15 132 pg
6. For 48 samples two 2 ml tubes with DNase reaction mixture are required for 96 samples four tubes are required Kunitz units are the commonly used units for measuring DNase defined as the amount of DNase that causes an increase in of 0 001 per minute per milliliter at 25 C pH 5 0 with highly polymerized DNA as the substrate Kunitz M 1950 J Gen Physiol 33 349 and 363 14 PAXgene Blood RNA MDx Kit Handbook 04 2010 Procedure 1 Centrifuge the PAXgene Blood RNA Tubes for 10 min at 3000 5000 x using a swing out rotor Note Use only round bottomed tube adapters Tubes may break during centrifugation if centrifuge adaptors with conical bottoms are used To save time the BioRobot Universal System can be set up during this centrifugation step Start the QIAsoft 5 Operating System and run the PAXgene 96 Blood RNA Protocol Set up the plasticware tips and buffers according to the protocol instructions on the BioRobot Universal System 2 After centrifugation remove the supernatant by decanting Discard the supernatant and save the pellet Carefully dry the rim of the tube with a clean paper towel 3 Add 290 pl Buffer BR1 and 35 pl proteinase K Transfer the tube to the Shaker Adapter 24 tube PAXgene To save time mix the appropriate volumes of Buffer BR and proteinase for 48 samples 14 5 ml Buffer BR1 and 1750 pl proteinase K for 96 samples 29 0 ml Buffer BR1 and 3500 pl proteinase K and d
7. 70 C For accurate quantification of RNA by absorbance at 260 nm we recommend diluting the sample in 10 mM Tris Cl pH 7 5 Dilution of the sample in RNase free water may lead to inaccurately low values Use the buffer in which the RNA is diluted to zero the spectrophotometer and make sure to add the same volume of Buffer BR5 as the volume of eluted RNA to be diluted Buffer BR5 has high absorbance at 220 nm which can lead to high background absorbance levels if the spectrophotometer is not properly zeroed zo 5 O x Note For quantification in Tris buffer use the relationship Ago 1 44 pg ml 9 Check the report file generated at the end of each run to ensure that the automated procedure performed correctly 10 After a 48 sample run store the PAXgene 96 RNA plate and the PAXgene 96 Filter plate in its original packaging with a clean paper towel at the bottom PAXgene Blood RNA MDx Kit Handbook 04 2010 13 Protocol Purification of RNA on the BioRobot Universal System from Whole Blood Collected in PAXgene Blood RNA Tubes Important points before starting e Blood must be collected in PAXgene Blood RNA Tubes cat no 762165 e Use of a step dispenser such as the Multipette plus from Eppendorf is recommended e All steps of the PAXgene Blood RNA BioRobot Universal System protocol for purification of total RNA should be performed at 18 25 C o n 2 E 5 2 9 c X en
8. and continue the protocol The tip disposal container was not pushed back completely leading to a tip jam Remove the tip disposal container and the jammed tips empty the container and insert it again Push it back until a metallic click is heard Continue the protocol Sufficient vacuum was not reached After the protocol has paused open the worktable hood and check if the PAXgene 96 RNA plate fits well to the elution microtubes If necessary correct the position of the PAXgene 96 RNA plate close the hood and continue the protocol There is no contact between the sensor and the system liquid container Ensure that the container is positioned correctly in the container holder and that the outsides of the container and sensor are dry Drops adjacent to the sensor inside or outside the waste container cause sensing of a full container Make sure that the container is positioned correctly in the container holder and that the outside of the container is dry Drops adjacent to the sensor inside or outside the vacuum trap cause sensing of a full bottle Make sure that the bottle is positioned correctly in the container holder and that the outside of the container is dry Vacuum error during elution QIAsoft software prompts operator to refill the system liquid container although the container is filled QIAsoft software prompts operator to empty the waste container although the container is empty QIAsoft software prom
9. applications see last page for contact Comments and suggestions Check for RNase contamination of buffers Although all buffers have been tested and are guaranteed RNase free RNases can be introduced during use Be careful not to introduce any RNases during the procedure or later handling RNA degraded RNase contamination RNA does not perform well in downstream applications Ensure that Buffer BR4 is at room temperature 15 25 Ensure that incubation of the eluate at 80 C is performed in the PAXgene 96 Incubator Block Do not concentrate the eluate by vacuum centrifugation e g in a Speed Vac or similar instrument This can introduce RNases and concentrate salts in the eluate which can interfere with downstream applications Ensure that 2 5 ml blood is collected in the PAXgene Blood RNA Tube see Product Circular for the PAXgene Blood RNA Tube RNA concentration must be measured in 10 mM Tris Cl pH 7 5 for accurate quantification After removing the supernatant by decanting it is sufficient to dab the rim of the tube 5 10 times with a clean paper towel Excess drying such as placing the tubes upside down in a rack or wiping the inner walls of the tube is not recommended Incubate blood in the PAXgene Blood RNA Tube for at least 2 h after collection Incubation of the PAXgene Blood RNA Tube overnight may increase yields slightly in some cases a Salt carryover during elution b No incubation of
10. 2103 29 22000 Technical 02103 29 12400 Hong Kong Orders 800 933 965 Fax 800 930 439 Technical 800 930 425 Ireland Orders 1800 555 049 Fax 1800 555 048 Technical 1800 555 061 Italy Orders 800 789 544 Fax 02 334304 826 Technical 800 787980 Japan Telephone 03 6890 7300 Fax 03 5547 0818 Technical 03 6890 7300 Korea South Orders 080 000 7146 Fax 02 2626 5703 Technical 080 000 7145 Luxembourg Orders 8002 2076 Fax 8002 2073 Technical 8002 2067 Mexico Orders 01 800 7742 639 Fax 01 800 1122 330 Technical 01 800 7742 436 The Netherlands Orders 0800 0229592 Fax 0800 0229593 Technical 0800 0229602 Norway Orders 800 18859 Fax 800 18817 Technical 800 18712 Singapore Orders 1800 742 4362 Fax 65 6854 8184 Technical 1800 742 4368 Spain Orders 91 630 7050 Fax 91 630 5145 Technical 91 630 7050 Sweden Orders 020 790282 Fax 020 790582 Technical 020 798328 Switzerland Orders 055 254 22 11 Fax 055 254 22 13 Technical 055 254 22 12 UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 www giagen com www PreAnalytiX com 30 PAXgene Blood RNA MDx Kit Handbook 04 2010 www PreAnalytiX com 31 Argentina Uruguay and Paraguay Orders 011 4551 7100 Australia Orders 1 800 656 100 e Fax 1 800 656 110 Austria Orders 43 1 7063660 e Fax 43 1 7063660 11 Belgium Orders 32 53
11. 4 e Shaker Adapter 24 tube PAXgene cat no 9016753 This is not a complete list of suppliers and does not include many important vendors of biological supplies Eight square well blocks 4 x 96 Elution Microtube CL racks 55 x 8 Caps for Elution Microtubes and 20 Disposable Troughs 30 ml are supplied with the kit If one or more runs with 48 samples are processed extra Elution Microtubes CL and Caps for Elution Microtubes are required and it may be convenient to have additional extra plasticware available see page 25 for ordering information 10 PAXgene Blood RNA MDx Kit Handbook 04 2010 Protocol Purification of RNA on the BioRobot MDx from Whole Blood Collected in PAXgene Blood RNA Tubes Important points before starting e Blood must be collected in PAXgene Blood RNA Tubes cat 762165 e Use of a step dispenser such as the Multipette plus from Eppendorf and a multitube vortexer such as the VX2500 from VWR is recommended e All steps of the PAXgene Blood RNA BioRobot MDx protocol for purification of total RNA should be performed at 18 25 C zo 5 O x Things to do before starting e After collection of the blood sample it is important to incubate the PAXgene Blood RNA Tube for at least 2 hours at room temperature 15 25 C before RNA purification Incubation of the PAXgene Blood RNA Tube overnight may increase RNA yields in some cases If the blood samples in the PAXgene
12. 720408 Fax 32 53720558 Brazil Orders 0800 055 5654 Canada Orders 800 268 5430 Fax 800 565 0897 Denmark Orders 45 43 43 45 66 Fax 45 6 East Europe Middle East 8 Africa EMA Orders 971 4 3379525 Fax 971 4 03379551 Finland Orders 358 9 88 70 780 Fax 358 9 88 70 7817 France Orders 33 476 683636 Fax 33 476 683693 Germany Orders 49 6221305553 Fax 49 6221305377 Italy Orders 390248204775 Fax 390248204817 Technical 390248240264 The Netherlands Orders 31 20 6545 716 Fax 31 20 5829 421 New Zealand Orders 0800 572 468 Fax 0800 572 469 Spain Orders 34 91 848 8115 Fax 34 91 848 8104 Sweden Orders 46 8 775 51 00 e Fax 46 8 775 51 5 Switzerland Orders 41 61 4852222 Fax 41 61 4852200 UK Orders 44 1 865 781 666 Fax 44 1 865 781 528 USA Orders 888 237 2762 Fax 800 847 2220 Technical 800 631 0174 www bd com PAXgene Blood RNA MDx Kit Handbook 04 2010 PreAnalytiX A QIAGEN BD Company 4 1063012 04 2010
13. Blood RNA Tubes were frozen they must be thawed at room temperature for at least 2 hours before RNA purification e Fillthe ethanol bottle with 500 ml ethanol 96 100 p a and add 500 pl Buffer BR1 This allows the BioRobot MDx to detect the fill level in the ethanol bottle by conductivity measurements e Buffer BR2 may form a precipitate upon storage If necessary warm to 37 C to redissolve e Buffer BRA is supplied as a concentrate Before using for the first time add 4 volumes of ethanol 96 100 p a to obtain a working solution e Heatan incubator to 80 C for the final heat denaturation step Place the PAXgene 96 Incubator Block into the incubator e Prepare DNase stock solution Dissolve the solid DNase 1500 Kunitz units per vial in 550 pl of RNase free water provided in the set Take care that no DNase is lost when opening the vial Do not vortex the reconstituted DNase I DNase is especially sensitive to physical denaturation Mixing should only be carried out by gently inverting the tube 10 times Fill 2 ml tubes with 260 pl of this DNase stock solution each and add 1800 pl Buffer RDD For 48 samples two 2 ml tubes with DNase reaction mixture are required for 96 samples four tubes are required Kunitz units are the commonly used units for measuring DNase defined as the amount of DNase that causes an increase in Az of 0 001 per minute per milliliter at 25 C pH 5 0 with highly polymerized DNA as
14. Things to do before starting e Afer collection of the blood sample it is important to incubate the PAXgene Blood RNA Tube for at least 2 hours at room temperature before RNA purification Incubation of the PAXgene Blood RNA Tube overnight may increase RNA yields in some cases If the blood samples in the PAXgene Blood RNA Tubes were frozen they must be thawed at room temperature for at least 2 hours before RNA purification e the ethanol bottle with 500 ml ethanol 96 100 p a and add 500 yl Buffer This allows the BioRobot Universal System to detect the fill level in the ethanol bottle by conductivity measurements e Buffer BR2 may form a precipitate upon storage If necessary warm to 37 C to redissolve e Buffer BRA is supplied as a concentrate Before using for the first time add 4 volumes of ethanol 96 1006 p a to obtain a working solution e Heatan incubator to 80 C for the final heat denaturation step Place the PAXgene 96 Incubator Block into the incubator e Prepare DNase stock solution Dissolve the solid DNase 1500 Kunitz units per vial in 550 pl of RNase free water provided in the set Take care that no DNase is lost when opening the vial Do not vortex the reconstituted DNase DNase is especially sensitive to physical denaturation Mixing should only be carried out by gently inverting the tube 10 times Fill 2 ml tubes with 260 pl of this DNase stock solution each and add 1800 pl Buffer RDD
15. ation PAXgene Blood RNA MDx Kit Handbook 04 2010 15 After incubation chill the elution microtubes immediately on ice Put the bottom plate back onto the rack for storage Store the purified RNA at 20 C or 70 C For accurate quantification of RNA by absorbance at 260 nm we recommend diluting the sample in 10 mM Tris Cl pH 7 5 Dilution of the sample in RNase free water may lead to inaccurately low values Use the buffer in which the RNA is diluted to zero the spectrophotometer and make sure to add the same volume of Buffer BR5 as the volume of eluted RNA to be diluted Buffer BR5 has high absorbance at 220 nm which can lead to high background absorbance levels if the spectrophotometer is not properly zeroed Note For quantification in Tris buffer use the relationship Aj 1 44 pg ml Check the report file generated at the end of each run to ensure that the automated procedure performed correctly After a 48 sample run store the PAXgene 96 RNA plate and the PAXgene 96 Filter plate in its original packaging with a clean paper towel at the bottom PAXgene Blood RNA MDx Kit Handbook 04 2010 N 2 a 2 9 x X ea Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or molecular biology
16. d run the PAXgene 96 Blood RNA Protocol Set up the plasticware tips and buffers according to the protocol instructions on the BioRobot MDx To save time this step can be performed during the 10 min centrifugation in step 1 5 Place the tubes in the racks onto the BioRobot MDx sample input area and proceed with the PAXgene 96 Blood RNA Protocol 6 After the BioRobot MDx protocol finishes remove the elution microtubes and seal them with the appropriate caps 7 Remove the lower plate from the elution microtube rack using a scalpel or spatula Place the elution microtube rack onto the PAXgene 96 Incubator Block preheated in the 80 C incubator and incubate for 10 min in the incubator at 80 C Place a heavy plate over the caps fo prevent them from popping open Denaturation of the eluate is essential for maximum efficiency in downstream applications such as RT PCR other amplification reactions or CONA synthesis It is not necessary to denature samples more than once samples remain denatured after freezing and thawing Note The temperature of the eluate must reach at least 65 C but should not exceed 70 C Using a heat block or other methods to denature the RNA may result in higher temperatures leading to RNA degradation 12 PAXgene Blood RNA MDx Kit Handbook 04 2010 8 After incubation chill the elution microtubes immediately on ice Put the bottom plate back onto the rack for storage Store the purified RNA at 20 C or
17. d show the container or label 6 PAXgene Blood RNA MDx Kit Handbook 04 2010 DNase in the RNase Free DNase Set Contains deoxyribonuclease sensitizer Risk and safety phrases RA2 43 522 24 26 36 37 24 hour emergency information Emergency medical information in English French and German can be obtained 24 hours a day from Poison Information Center Mainz Germany Tel 49 6131 19240 Quality Control In accordance with QIAGEN s ISO certified Total Quality Management System each lot of PAXgene Blood RNA Kit is tested against predetermined specifications to ensure consistent product quality Technical Assistance Technical assistance with PreAnalytiX products is provided by QIAGEN the distributor for PreAnalytiX The Technical Service Departments at QIAGEN are staffed by experienced scientists with extensive practical and theoretical expertise in molecular biology If you have any questions or experience any difficulties regarding the PAXgene Blood RNA MDx Kit please contact one of the Technical Service Departments listed the last page PreAnalytiX customers are a major 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 PreAnalytiX We therefore encourage you to contact us through QIAGEN s Technical Service Departments if you have any suggestions about product performance or new applications and technique
18. g information and product specific disclaimers see the respective PreAnalytiX or QIAGEN kit handbook or user manual PreAnalytiX kit handbooks are available at www preanalytix com or can be requested from QIAGEN Technical Services or your local distributor QIAGEN kit handbooks and user manuals are available at www giagen com or can be requested from QIAGEN Technical Services or your local distributor 26 PAXgene Blood RNA MDx Kit Handbook 04 2010 27 Notes PAXgene Blood RNA MDx Kit Handbook 04 2010 Notes 28 PAXgene Blood RNA MDx Kit Handbook 04 2010 29 Notes PAXgene Blood RNA MDx Kit Handbook 04 2010 PreAnalytiX Worldwide PreAnalytiX products are distributed by QIAGEN and BD companies Australia Orders 1 800 243 800 Fax 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800 28 10 10 Fax 0800 28 10 19 Technical 0800 28 10 11 Belgium Orders 0800 79612 Fax 0800 79611 Technical 0800 79556 Brazil Orders 0800 557779 Fax 55 1 1 5079 4001 Technical 0800 557779 Canada Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 86 21 3865 3865 Fax 86 21 3865 3965 Technical 800 988 0325 Denmark Orders 80 885945 Fax 80 885944 Technical 80 885942 Finland Orders 0800 914416 Fax 0800 914415 Technical 0800 914413 France Orders 01 60 920 926 Fax 01 60 920 925 Technical 01 60 920 930 Offers 01 60 920 928 Germany Orders 02103 29 12000 Fax 0
19. guarantees the performance of all products in the manner described in our literature PreAnalytiX products are manufactured for PreAnalytiX by QIAGEN or BD and are distributed for PreAnalytiX by QIAGEN or BD Should any product fail to perform satisfactorily due to any reason other than misuse QIAGEN the distributor of PreAnalytiX products will replace it free of charge or refund the purchase price PreAnalytiX reserves the right to change alter or modify any product to enhance its performance and design If a PreAnalytiX product does not meet your expectations simply call your local QIAGEN Technical Service Department or other PreAnalytiX distributor We will credit your account or exchange the product as you wish PAXgene Blood RNA MDx Kit Handbook 04 2010 5 Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles 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 For more information please consult the appropriate material safety data sheets MSDSs These are available online in convenient and compact PDF format at www preanalytix com rna_msds asp where you can find view and print the MSDS for each PreAnalytiX kit and kit component CAUTION DO NOT add bleach or acidic solutions directly to the sa
20. istribute 325 pl of the mixture into each tube using a step dispenser 4 Start the QIAsoft 5 Operating System and run the PAXgene 96 Blood RNA Protocol Set up the plasticware tips and buffers according to the protocol instructions on the BioRobot Universal System Da o e 6 EL 5 a To save time this step can be performed during the 10 min centrifugation in step 1 5 Place the tubes in the Shaker Adapter onto the shaker unit of the BioRobot Universal System and proceed with the PAXgene 96 Blood RNA Protocol 6 After the BioRobot Universal System protocol finishes remove the elution microtubes and seal them with the appropriate caps 7 Remove the lower plate from the elution microtube rack using a scalpel or spatula Place the elution microtube rack onto the PAXgene 96 Incubator Block preheated in the 80 C incubator and incubate for 10 min in the incubator at 80 C Place a heavy plate over the caps to prevent them from popping open Denaturation of the eluate is essential for maximum efficiency in downstream applications such as RT PCR other amplification reactions or cDNA synthesis It is not necessary to denature samples more than once samples remain denatured after freezing and thawing Note The temperature of the eluate must reach at least 65 C but should not exceed 70 C Using a heat block or other methods to denature the RNA may result in higher temperatures leading to RNA degrad
21. ml Total yield concentration x volume of sample in milliliters 132 pg ml x 0 12 ml 15 8 pg RNA When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier 22 PAXgene Blood RNA MDx Kit Handbook 04 2010 Purity of RNA The ratio of the readings at 260 nm and 280 nm A260 A2g0 provides an estimate of the purity of RNA with respect to contaminants that absorb UV light such as protein However the A o Asso ratio is influenced considerably by pH Lower pH results in a lower A260 A2g0 ratio and reduced sensitivity to protein contamination For accurate values we recommend measuring absorbance in 10 mM Tris Cl pH 7 5 Pure RNA has A260 A2g0 ratio of 1 8 2 2 in 10 mM Tris Cl pH 7 5 Use the buffer in which the RNA is diluted to zero the spectrophotometer and make sure to add the same volume of Buffer BR5 as the volume of eluted RNA to be diluted Buffer BR5 has high absorbance at 220 nm which can lead to high background absorbance levels if the spectrophotometer is not properly zeroed Wilfinger W W Mackey M and Chomezynski P 1997 Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity BioTechniques 22 474 PAXgene Blood RNA MDx Kit Handbook 04 2010 23 Ordering Information Product Contents Cat no Produc
22. mple preparation waste Buffer BR2 contains guanidine thiocyanate and Buffer BR3 contains a small amount of guanidine thiocyanate which can form highly reactive compounds when combined with bleach If liquid containing this buffer 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 following risk and safety phrases apply to components of the PAXgene Blood RNA MDx Kit Buffer BR2 Contains guanidine thiocyanate harmful Risk and safety phrases R20 21 22 32 S13 26 36 46 Buffer BR3 Contains ethanol flammable Risk and safety phrases 0 Proteinase K Contains proteinase K sensitizer irritant Risk and safety phrases R36 37 38 42 43 7 R10 Flammable R20 21 22 Harmful by inhalation contact with skin and if swallowed R32 Contact with acids liberates very toxic gas R36 37 38 Irritating to eyes respiratory system and skin R42 43 May cause sensitization by inhalation and skin contact 513 Keep away from food drink and animal feedingstuffs S23 Do not breathe vapor 24 Avoid contact with skin S26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice 36 Wear suitable protective clothing 536 37 Wear suitable protective clothing and gloves 546 If swallowed seek medical advice immediately an
23. ndling RNA Handling RNA Ribonucleases RNases are very stable and active enzymes that generally do not require cofactors to function Since RNases are difficult to inactivate and even minute amounts are sufficient destroy RNA do not use any plasticware or glassware without first eliminating possible RNase contamination Great care should be taken to avoid inadvertently introducing RNases into the RNA sample during or after the purification procedure In order to create and maintain an RNase free environment precautions must be taken during pretreatment and use of disposable and non disposable vessels and solutions while working with RNA General handling Proper microbiological aseptic technique should always be used when working with RNA Hands and dust particles carry bacteria and molds and these are the most common sources of RNase contamination Always wear latex or vinyl gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin or from dusty laboratory equipment Change gloves frequently and keep tubes closed whenever possible Keep purified RNA on ice when aliquots are pipetted for downstream applications Protocols for removing RNase contamination from glassware and solutions can be found in general molecular biology guides such as Sambrook J and Russell D W 2001 Molecular Cloning A Laboratory Manual 3rd ed Cold Spring Harbor NY Cold Spring Harbor Laboratory Press
24. pplied to a PAXgene 96 RNA Plate RNA is selectively bound to the PAXgene 96 RNA membrane and contaminants pass through The bound RNA is washed with Buffer BR3 and ethanol Residual DNA is removed through a DNase digestion on the PAXgene 96 membrane Remaining contaminants are removed in three efficient wash steps and RNA is eluted in Buffer BR5 Following purification a final heat treatment of the eluate enhances performance in downstream applications Typical yields of RNA isolated from 2 5 ml healthy human whole blood 4 8 x 10 1 1 x 107 leukocytes ml are lt 3 pg for gt 95 of the samples processed Since yields are highly donor dependent individual yields may vary The purified RNA is ready for immediate use Downstream applications that use RNA include RT PCR and NASBA cDNA synthesis quantitative RT PCR RNase and S1 nuclease protection expression array and expression chip analysis poly A RNA selection northern dot and slot blot analysis and primer extension 8 PAXgene Blood RNA MDx Kit Handbook 04 2010 The PAXgene Blood RNA MDx Procedure Blood Centrifuge to obtain pellet Add proteinase K Resuspend pellet BioRobot MDx protocol uoioJodeud jdwps jonuow Transfer tubes to BioRobot workstation Resuspend pellet BioRobot Universal System protocol Proteinase K digestion 65 C Transfer to PAXgene 96 Filter Plate Lysate clearing Bind RNA Wash 2x
25. pts operator to empty the vacuum trap although the bottle is empty f g h PAXgene Blood RNA MDx Kit Handbook 04 2010 19 Comments and suggestions Sample tubes were not positioned correctly in the tube holders of the sample tracking system Turn the tubes so that the bar codes face the bar code reader on the left of the BioRobot MDx Scan the sample tubes again and continue with the run once all samples have been correctly identified Bar code labels should be stuck to the sample tubes such that the bar code lines are horizontal If some bar code labels were incorrectly oriented remove the unidentified tubes from the sample tracking system tube holder and enter their identification codes into the table either manually or using the hand held bar code reader Put the sample tubes back into the sample tracking system tube holder and continue with the protocol Check that the type of bar code used can be read by the QlAsoft MDx Operating System refer to the BioRobot MDx User Manual for a list of bar code systems that the software can interpret Remove the unidentified tubes from the sample tracking system tube holder and manually enter their identification codes into the table Replace the sample tubes in the sample tracking system tube holder and continue with the protocol PAXgene Blood RNA MDx Kit Handbook 04 2010 BioRobot MDx Some bar codes not identified i 20 Appendix A General Remarks on Ha
26. ries that can be ordered from QIAGEN PAXgene 96 Incubator Block Block for denaturation of eluates in 9238279 PAXgene 96 procedures Shaker Adapter 24 tube Adapter for PAXgene Blood RNA 9016753 PAXgene Tubes on the shaker unit of the BioRobot Universal System Square Well Blocks 24 96 well blocks with 2 2 ml wells for 19585 PAXgene 96 procedures 24 per case Elution Microtubes CL 1 x 96 Nonsterile polypropylene tubes 1030483 0 85 ml maximum capacity less than 0 7 ml storage capacity 0 4 ml elution capacity 96 in a rack for PAXgene 96 procedures Caps for Elution Microtubes Nonsterile polypropylene caps for 1030481 55 x 8 Elution Microtubes CL 440 in strips of 8 Disposable Troughs Nonsterile plastic disposable 9232764 30 ml 10 troughs for PAXgene 96 procedures on the BioRobot MDx workstation 10 troughs These blood collection accessories represent typical products that can be used with PAXgene Blood RNA Tubes To find out more about these accessories including how to order visit www bd com vacutainer products venous US only CE marked for European use PAXgene Blood RNA MDx Kit Handbook 04 2010 25 Ordering Information Product Contents Cat no Disposable Filter Tips Conducting disposable filter tips 9012598 1100 pl 960 pack of 960 Tape Pads 5 Adhesive tape sheets for sealing 19570 multiwell plates and blocks 25 sheets per pad 5 pads per pack For up to date licensin
27. s R42 43 May cause sensitization by inhalation and skin contact S22 Do not breathe dust S24 Avoid contact with skin S26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice 36 37 Wear suitable protective clothing and gloves PAXgene Blood RNA MDx Kit Handbook 04 2010 7 Introduction The PAXgene Blood RNA MDx Kit allows the automated high throughput purification on the BioRobot MDx or BioRobot Universal System of total RNA from 2 5 ml human whole blood collected into PAXgene Blood RNA Tubes The PAXgene Blood RNA MDx Kit is for laboratory use Principle and procedure The simple procedure begins with a centrifugation step to pellet the contents of each PAXgene Blood RNA Tube Proteinase K and an optimized incubation buffer are added to digest proteins With the BioRobot MDx the pellets are resuspended manually and the tubes are then transferred to the workstation On the BioRobot Universal System the tubes are transferred to the workstation and the pellets are automatically resuspended by the integrated shaker on the workstation After setup of buffers and plasticware guided by the QIAsoft Operating System the BioRobot MDx or BioRobot Universal System carries out the fully automated RNA purification procedure Lysates are applied to a PAXgene 96 Filter Plate see flowchart next page Ethanol is added to the flow throughs to adjust binding conditions and the resulting samples are a
28. the RNA eluate at 80 C Eluate concentrated by vacuum centrifugation Low RNA yield a Less than 2 5 ml blood collected in the PAXgene Blood RNA Tube b concentration measured in water c Pellet overdried in step 2 d Blood incubated for lt 2 h after collection PAXgene Blood RNA MDx Kit Handbook 07 2005 17 Comments and suggestions RNA yields are highly donor dependent Blood samples with low leukocyte counts e g gt 4 8 x 10 leukocytes ml will give low yields RNA concentration must be measured in 10 mM Tris Cl pH 7 5 for accurate quantification To zero the spectrophotometer use a blank containing the same proportion of elution buffer Buffer BR5 and dilution buffers as in the samples to be measured Buffer BR5 has high absorbance at 220 nm which can lead to high background absorbance levels if the spectrophotometer is not properly zeroed Insufficient vacuum was applied If fewer than 96 samples are purified simultaneously ensure that unused wells in the PAXgene 96 RNA plate are sealed with a tape sheet cat no 19570 Blood samples with very high white blood cell counts were used This can lead to overloading and clogging Use less of the blood sample Processing of different blood samples may lead to variations in elution volume During sample aspiration from sample tubes no sample was detected After sample transfer not enough sample was detected in the square well block
29. the substrate Kunitz M 1950 J Gen Physiol 33 349 and 363 PAXgene Blood RNA MDx Kit Handbook 04 2010 11 Procedure 1 Centrifuge the PAXgene Blood RNA Tubes for 10 min at 3000 5000 x using a swing out rotor Note Use only round bottomed tube adapters Tubes may break during centrifugation if centrifuge adaptors with conical bottoms are used To save time the BioRobot MDx can be set up during this centrifugation step Start the QIAsoft MDx Operating System and run the PAXgene 96 Blood RNA Protocol Set up the plasticware tips and buffers according to the protocol instructions on the BioRobot MDx 2 After centrifugation remove the supernatant by decanting Discard the supernatant and save the pellet for resuspension in step 3 x Pu 2 e E ca Carefully dry the rim of the tube with a clean paper towel 3 Add 290 yl Buffer BR1 and 35 pl proteinase K Close the tubes with the Secondary Hemogard Closures provided and thoroughly resuspend the pellet by vortexing Remove the closures and transfer the tube to a sample tracking system tube rack To save time mix the appropriate volumes of Buffer BRI and proteinase for 48 samples 14 5 ml Buffer BRI and 1750 yl proteinase for 96 samples 29 0 ml Buffer BR1 and 3500 pl proteinase K and distribute 325 pl of the mixture into each tube using a step dispenser For resuspension use a multitube vortexer 4 Start the QlAsoft MDx Operating System an
30. tiX distributor 2 PAXgene Blood RNA MDx Kit Handbook 04 2010 co 04 JOG Ud 0 A Contents Kit Contents Shipping and Storage Product Use Limitations Product Warranty and Satisfaction Guarantee Safety Information Quality Control Technical Assistance Introduction Principle and procedure Equipment and Reagents to Be Supplied by User Protocols Purification of RNA on the BioRobot MDx from Whole Blood Collected in PAXgene Blood RNA Tubes EI Purification of RNA on the BioRobot Universal System from Whole Blood Collected in PAXgene Blood RNA Tubes Troubleshooting Guide Appendix A General Remarks on Handling RNA Appendix B Quantification and Determination of Quality of Total RNA Ordering Information PreAnalytiX Worldwide PAXgene Blood RNA MDx Kit Handbook 04 2010 4 762431 4x96 4 4 170 ml 2 x 220 ml 2 x 450 ml 2x 100 ml 8 10 ml 110 ml 2 x 10 ml For 8 x 50 reactions 1x16 8 4x96 55x 8 2 5 10 8 x 0 Kit Contents PAXgene Blood RNA MDx Kit Catalog no Number of preps PAXgene 96 RNA Plates PAXgene 96 Filter Plates Buffer BR1 Resuspension Buffer Buffer BR2 Binding Buffer Buffer BR3 Wash Buffer Buffer BRA Wash Buffer Buffer BR5 Elution Buffer Top Elute Fluid Proteinase K RNase Free DNase Set Bottle 500 ml Ethanol p a Sample Tube 2 ml Square Well Blocks Elution Microtubes CL racked Caps for Elution Microtubes Disposable Troughs 30
31. ts that can be ordered from QIAGEN PAXgene Blood RNA For 4 x 96 RNA preps on the 762431 MDx Kit 4 BioRobot MDx workstation 4 PAXgene 96 RNA Plates 4 PAXgene 96 Filter Plates Buffers Proteinase K RNase Free DNase Set Plasticware Collection Vessels To be used with PAXgene Blood RNA Tubes Products that can be ordered from BD PAXgene Blood RNA 100 Blood Collection Tubes To be 762165 Tubes 100 used with the PAXgene Blood RNA MDx Kit Robotic workstations that can be ordered from QIAGEN BioRobot MDx Robotic workstation computer 900600 controlled vacuum pump computer QIAsoft MDx Operating System laboratory cabinet accessory cabinet installation 1 year warranty on parts and labor BioRobot Universal System Robotic workstation computer 9001094 controlled vacuum pump computer QlAsoft 5 Operating System installation 1 year warranty on parts and labor Wash buffers are labeled with bar codes 24 PAXgene Blood RNA MDx Kit Handbook 04 2010 Ordering Information Product Contents Cat no Accessories that can be ordered from BD BD Vacutainer Safety Lok 21G 3 4 in needle 12 inch tubing 3672811 Blood Collection Set with luer adapter 50 box 367286 200 case BD Vacutainer One Use Case only for 13 mm and 16 mm 364815 Holder diameter 1000 case BD Vacutainer Plus Serum 13 x 75 mm 4 0 ml draw with Red 367812 Tubes BD Hemogard closure and paper 368975 label 100 box 1000 case Accesso
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