PAXgene 96 Blood RNA Kit Handbook
Contents
1. Russell D W 2001 Molecular Cloning A Laboratory Manual 3rd ed Cold Spring Harbor NY Cold Spring Harbor Laboratory Press 24 PAXgene 96 Blood RNA Handbook 02 2014 Appendix D Handling PAXgene Blood RNA Tubes The following recommendations from BD may be helpful when handling PAXgene Blood RNA Tubes See the PAXgene Blood RNA Tube Product Circular for more information about PAXgene Blood RNA Tubes Instructions for removal of BD Hemogard Closure D1 Grasp the PAXgene Blood RNA Tube with one hand placing the thumb under the BD Hemogard Closure For added stability place arm on solid surface With the other hand twist the BD Hemogard Closure while simultaneously pushing up with the thumb of the other hand ONLY UNTIL THE TUBE STOPPER IS LOOSENED D2 Move thumb away before lifting closure DO NOT use thumb to push closure off tube Caution If the tube contains blood an exposure hazard exists To help prevent exposure during closure removal it is important that the thumb used to push upward on the closure be removed from contact with the tube as soon as the BD Hemogard Closure is loosened D3 Lift closure off tube In the unlikely event of the plastic shield separating from the rubber stopper DO NOT REASSEMBLE CLOSURE Carefully remove rubber stopper from tube Instructions for insertion of Secondary BD Hemogard Closure D1 Replace closure over tube D2 Twist and push down firmly until stopper is fully reseated Comple2. pH 7 5 1 15 dilution Measure absorbance of diluted sample in a cuvette RNase free A260 0 2 Concentration of RNA sample 44 x Azo x dilution factor 44x0 2x 15 132 ug ml Total yield concentration x volume of sample in milliliters 132 ug ml x 0 12 ml 15 8 ug RNA When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate safety data sheets SDSs available from the product supplier 22 PAXgene 96 Blood RNA Handbook 02 2014 Purity of RNA The ratio of the readings at 260 nm and 280 nm 0 provides an estimate of the purity of RNA with respect to contaminants that absorb UV light such as protein However the A260 A2g0 ratio is influenced considerably by pH Lower pH results in a lower Az 0 Azg 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 an 0 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 spe
3. RNA from 2 5 ml human whole blood collected into PAXgene Blood RNA Tubes Principle and procedure The simple isolation procedure begins with a centrifugation step to pellet the contents of each PAXgene Blood RNA Tube Each pellet is washed resuspended and incubated in optimized buffers together with proteinase K to digest proteins Lysates are applied to a PAXgene 96 Filter Plate and centrifuged to remove residual cell debris see flowchart next page Ethanol is added to the flow through to adjust binding conditions and the lysates are applied to a PAXgene 96 RNA Plate RNA is selectively bound to the PAXgene 96 RNA membrane as contaminants pass through 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 A final heat treatment of the eluate enhances performance in downstream applications Typical yields of RNA isolated from 2 5 ml human whole blood are between 4 and 20 ug However the yield is donor dependent and in some cases higher or lower yields may be achieved The purified RNA is ready for immediate use Downstream applications that use RNA include RT PCR cDNA synthesis and quantitative RT PCR including TaqMan technology PAXgene 96 Blood RNA Handbook 02 2014 7 The PAXgene 96 Blood RNA Procedure Blood Cantrifuge to obtain pallet Transfer pellet to plate Proteinase K digestion 65 C L
4. necessary for the protocol The vacuum must be switched off and the manifold ventilated between pipetting steps to maintain uniform conditions for each sample All steps of the PAXgene 96 Blood RNA protocol for isolation of total RNA should be performed at room temperature Avoid interruptions during the procedure All centrifugation steps in the protocol are performed in a Centrifuge 4 168 or Centrifuge 4 16KS see page 11 Things to do before starting Buffer BR2 may form a precipitate upon storage If necessary warm to 37 C to redissolve Buffer BRA is supplied as a concentrate Before using for the first time add 4 volumes of ethanol 96 100 to obtain a working solution Heat an incubator to 65 C for the proteinase K digestion step If one incubator is used for the entire procedure set the temperature to 80 C after the proteinase K digestion to heat up in time for the final heat denaturation step Place the PAXgene 96 Incubator Block into the incubator Prepare DNase for the on plate DNase digest in step 16 according to the protocol in the appendix page 21 Procedure 1 14 Place the waste tray inside the QlAvac 96 base and place the top plate squarely over the base PAXgene 96 Blood RNA Handbook 02 2014 2 Place the PAXgene 96 RNA Plate in the QlAvac 96 top plate making sure that the plate is seated tightly 3 Attach the QlAvac 96 manifold to the vacuum source Note Keep the vacuum switched off
5. pressure Always place the PAXgene 96 RNA Plate into the vacuum manifold with the beveled edges pointing to the right hand side For safety reasons do not use plates that have been damaged in any way Always place the QlAvac 96 vacuum manifold on a secure bench top or work area If dropped the manifold may crack Always store the QlAvac 96 vacuum manifold clean and dry To clean simply rinse all components with water and dry with paper towels Do not air dry as the screws may rust and need to be replaced Do not use abrasives Finally wipe manifold components with paper towels wetted with 7096 ethanol and dry with fresh paper towels PAXgene 96 Blood RNA Handbook 02 2014 The QlAvac 96 vacuum manifold and components are not resistant to ethanol methanol or other organic solvents when exposed for long periods If solvents are spilled on the unit rinse thoroughly with distilled water after the completion of the procedure Ensure that no residual buffers remain in the vacuum manifold To ensure consistent performance do not apply silicone or vacuum grease to any part of the QlAvac 96 vacuum manifold The spring lock on the top plate and the self sealing gasket provide an airtight seal when the vacuum is applied to the assembled unit To maximize gasket life rinse the gasket free of salts and buffers after each use and dry with paper towels before storage Table 1 Pressure conversions To convert from millibars
6. Add 350 ul 100 ethanol to each collection microtube and mix by pipetting up and down 3 times Pipet complete samples 1040 ul into the wells of the PAXgene 96 RNA Plate Apply vacuum until transfer is complete 15 60 s Switch off vacuum and ventilate the QlAvac 96 manifold Mark the PAXgene 96 RNA Plate for later identification Set the multichannel pipet to 1200 ul to transfer the entire sample to the PAXgene 96 RNA Plate Make sure the QlAvac 96 vacuum manifold is assembled correctly before loading The flow through is collected in the waste tray Note Cover unused wells with adhesive tape Do not use the AirPore Tape Sheets supplied with the PAXgene 96 Blood RNA Kit Use either adhesive tape or Tape Pads QIAGEN cat no 19570 Note The vacuum must be switched off and the manifold ventilated between pipetting steps to maintain uniform conditions for each sample Add 500 ul Buffer BR3 to each well of the PAXgene 96 RNA Plate Switch on the vacuum source and apply vacuum until transfer is complete 15 60 s Switch off the vacuum and ventilate the QlAvac 96 manifold Collect wash fractions in the same waste tray used in step 14 PAXgene 96 Blood RNA Handbook 02 2014 16 17 18 19 20 21 22 Pipet 80 ul of the DNase I incubation mix see Things to do before starting page 14 directly onto the PAXgene membrane in each well of the PAXgene 96 RNA Plate Seal the plate with an AirPore Tape Sheet Incubat
7. M Tris Cl pH 7 5 for accurate quantification c Supernatant not Ensure the entire supernatant is removed If the completely removed in supernatant is decanted remove drops from step 5 the rim of the tube by dabbing onto a paper towel PAXgene 96 Blood RNA Handbook 02 2014 19 Comments and suggestions d Blood incubated for 2 h after collection Low A260 A280 a RNA purity measured in b Spectrophotometer not 20 water properly zeroed 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 Use 10 mM Tris Cl pH 7 5 to dilute RNA before measuring purity 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 PAXgene 96 Blood RNA Handbook 02 2014 Appendix A On Plate DNase Digestion with the RNase Free DNase Set The RNase Free DNase Set from QIAGEN provides on plate digestion of DNA during RNA purification The DNase is removed in subsequent wash steps Note Standard DNase buffers are not compatible with on membrane DNase digestion Use of other buffers may affect the binding of the RNA to the PAXgene membrane reducing the yield and inte
8. Note Always place the PAXgene 96 RNA Plate into the vacuum manifold with the beveled edge pointing to the right hand side 4 Centrifuge the PAXgene Blood RNA Tube for 10 min at 3000 5000 x g using a swing out rotor Note The tube adaptors need to be round bottomed Tubes may break during centrifugation if centrifuge adaptors with conical bottoms are used 5 Remove the supernatant by decanting or pipetting Add 4 ml RNase free water to the pellet and close the tube using a fresh Secondary BD Hemogard Closure If the supernatant is decanted dry the rim of the tube with a clean paper towel 6 Thoroughly resuspend the pellet by vortexing and centrifuge for 10 min at 3000 5000 x g Remove and discard the entire supernatant Small amounts of debris remaining in the supernatant after vortexing will not affect the procedure If the supernatant is decanted dry the rim of the tube with a clean paper towel Note Incomplete removal of the supernatant will dilute the lysate and inhibit lysis which will affect the binding conditions 7 Thoroughly resuspend the pellet in 350 ul Buffer BR1 and transfer to a Round Well Block Transfer all samples to the Round Well Block before adding proteinase K or Buffer BR2 8 Add 40 ul Proteinase K and 300 ul Buffer BR2 to all samples in the Round Well Block Seal the wells of the Round Well Block with the appropriate caps and shake the Round Well Block vigorously for 10 s upside down Incubate the Rou
9. PAXgene 96 Blood RNA Handbook For isolation of cellular RNA from whole blood Important To be used only in conjunction with PAXgene Blood RNA tubes For research use only Not for use in diagnostic procedures February 2014 Trademarks PAXgene PreAnalytiX PreAnalytiX GmbH QIAGEN QIAGEN Group BD Vacutainer DB Hemogard Safety Lok Becton Dickinson and Company Matrix Matrix Technologies Corporation TaqMan Roche Group Limited License Agreement for the PAXgene 96 Blood RNA Kit Use of this product signifies the agreement of any purchaser or user of the product to the following terms 1 OP E CON The product may be used solely in accordance with the protocols provided with the product and this handbook and for use with components contained in the kit only PreAnalytiX grants no license under any of its intellectual property to use or incorporate the enclosed components of this kit with any components not included within this Kit except as described in the protocols provided with the product this handbook and additional protocols available at www preanalytix com Other than expressly stated licenses PreAnalytiX makes no warranty that this kit and or its use s do not infringe the rights of third parties This kit and its components are licensed for one time use and may not be reused refurbished or resold PreAnalytiX specifically disclaims any other licenses expressed or implied other than those
10. and Square Well Blocks or elution microtubes If unsupported the holders will collapse under high g force Remove the holders during test runs Standard 96 well microplates may be centrifuged in the holders if the g force of 500 x g is not exceeded PAXgene 96 Blood RNA Handbook 02 2014 Square Well Blocks Two Square Well Blocks are supplied per kit If several PAXgene 96 RNA Plates are processed per day it may be convenient to have extra Square Well Blocks available To reuse the Square Well Blocks rinse them thoroughly with tap water incubate for 2 hours or overnight in 0 1 N NaOH 1 mM EDTA rinse in distilled water and dry at 50 C Note Do not use bleach PAXgene 96 Blood RNA Handbook 02 2014 13 Protocol Isolation of RNA from Whole Blood Collected into PAXgene Blood RNA Tubes Important points before starting Blood must be collected in PAXgene Blood RNA Tubes cat no 762165 After collection of the blood sample it is important to incubate the PAXgene Blood RNA Tube for at least 2 hours at room temperature 18 25 C before RNA purification Incubation of the PAXgene Blood RNA Tube overnight may increase yields in some cases Use of a multichannel pipet is recommended Pour buffers into reagent reservoirs for multichannel pipets Use reservoirs from a freshly opened package or clean them as described for Square Well Blocks see page 13 A vacuum source capable of generating a vacuum pressure of 800 to 900 mbar is
11. ction list in the display field by turning the knob Press the knob and turn it again to select the rotor bucket combination 09100 09158 for the Plate Rotor 2 x 96 Confirm entry by pressing the knob Entering the rotor number automatically sets the time and speed limits for centrifugation for that particular rotor eliminating the danger of the centrifuge running too fast Select Speed by turning the knob Press the knob and turn it again to set the speed to 6000 Confirm entry by pressing the knob The corresponding relative centrifugal force RCF is calculated from the rotor number and speed and appears automatically in the RCF field It is also possible to enter the RCF value 5788 x g manually in the RCF field after selecting RCF in the same way Select Time by turning the knob Press once and turn the knob again to set the time as required Confirm entry by pressing the knob Open the lid place the 96 well plates with the metal carriers in the buckets and close the lid The start and lid keys light up Push Start to start the centrifuge When the centrifuge is running the lid key will not be lit Each run can be interrupted by pushing Stop At the end of the run the lid key will light up Open the centrifuge lid by pressing the lid key Remove the plates All preset parameters remain after a run has finished Warning Do not centrifuge the Plate Rotor 2 x 96 metal holders without the PAXgene 96 RNA Plates
12. ctrophotometric assessment of nucleic acid purity BioTechniques 22 474 PAXgene 96 Blood RNA Handbook 02 2014 23 Appendix C General Remarks on Handling 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 to 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 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 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
13. e at room temperature for 15 min Note Be sure to pipet the DNase incubation mix directly onto the center of the PAXgene membrane DNase digestion will be incomplete if some of the mix sticks to the walls or the O rings of the PAXgene 96 RNA Plate Remove the AirPore Tape and add 500 ul Buffer BR3 to each well of the PAXgene 96 RNA Plate Apply vacuum until transfer is complete 15 60 s Switch off the vacuum and ventilate the QlAvac 96 manifold Collect wash fractions in the same waste tray used in step 14 Lift the top plate carrying the PAXgene 96 RNA Plate from the base and empty the waste tray Reassemble the QlAvac 96 vacuum manifold Add 1 ml of Buffer BRA to each well of the PAXgene 96 RNA Plate Apply vacuum until transfer is complete 10 30 s Switch off the vacuum and ventilate the QlAvac 96 manifold Note Ensure that ethanol is added to Buffer BRA see Things to do before starting on page 14 Place the PAXgene 96 RNA Plate on top of a Square Well Block Add a further 1 ml Buffer BRA to each well of the PAXgene 96 RNA Plate Load the Square Well Block and PAXgene 96 RNA Plate into the holder of the plate rotor and place the whole assembly in the rotor bucket Centrifuge at 6000 rpm 5600 x g for 10 min at room temperature to allow Buffer BRA to pass through and to dry the membranes Important Ensure that the centrifuge is properly balanced Note It is important to dry the PAXgene membranes since residual
14. e back onto the rack for storage Store the purified RNA at 20 C or 70 C Denaturation of the eluate is essential for maximum efficiency in some downstream applications such as RT PCR other amplification reactions or cDNA synthesis It is not necessary to denature samples more than once and samples remain denatured after freezing and thawing For accurate quantification of RNA by absorbance at 260 nm we recommend dilution of the sample in 10 mM Tris Cl pH 7 5 Dilution of the sample in RNase free water may lead to inaccurately low values 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 elution buffer Buffer BR5 as the volume of eluted RNA to be diluted Elution buffer 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 A260 1 gt 44 ug ml When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate safety data sheets SDSs available from the product supplier 18 PAXgene 96 Blood RNA Handbook 02 2014 Troubleshootin
15. ers 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 02103 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 UKe 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 Argentina Urugua
16. ethanol may interfere with subsequent reactions The 10 min centrifugation ensures that residual traces of salt are removed and that no ethanol is carried over during elution Place the PAXgene 96 RNA Plate on top of a clean elution microtubes rack To elute the RNA add 45 60 ul Buffer BR5 to each well and seal the PAXgene 96 RNA Plate with a new sheet of AirPore Tape Incubate for 1 min at room temperature Then centrifuge at 6000 rpm 5600 x g for 4 min Note Be sure to pipet the Buffer BR5 directly onto the center of the PAXgene membrane Elution will be incomplete if some of the buffer sticks to the walls or the O rings of the PAXgene 96 RNA Plate PAXgene 96 Blood RNA Handbook 02 2014 17 23 24 25 Remove the AirPore Tape Repeat step 22 once with a second 45 60 pl Buffer BR5 Note Repeating the elution step is required for complete recovery of RNA The elution volume will be approximately 15 ul less membrane dead volume than the volume of Buffer BR5 added to the membrane Remove the PAXgene 96 RNA Plate and seal the elution microtubes with the appropriate caps Remove the bottom 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 at 80 C Place a heavy plate over the caps to prevent them from popping open Following incubation chill immediately on ice Put the bottom plat
17. expressly stated The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above PreAnalytiX may enforce the prohibitions of this Limited License Agreement in any Court and shall recover all its investigative and Court costs including attorney fees in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the Kit and or its components For updated license terms see www preanalytix com O 2014 PreAnalytiX GmbH all rights reserved PreAnalytiX 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 PreAnalytiX distributor Contents Kit Contents Shipping and Storage Intended Use Safety Information Quality Control Introduction Principle and procedure Equipment and Reagents to Be Supplied by User Important Notes QlAvac 96 vacuum manifold handling guidelines Centrifugation Protocol a Isolation of RNA from Whole Blood Collected into PAXgene Blood RNA Tubes Troubleshooting Guide Appendix A on Plate DNase Digestion with the RNase Free DNase Set Appendix B Quantification and Determination
18. g Guide This troubleshooting guide may be helpful in solving any problems that may arise For more information see also the Frequently Asked Questions page at our Technical Support Center www giagen com FAQ FAQList aspx The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about the information and protocol in this handbook or sample and assay technologies for contact information see the last page or visit www giagen com Comments and suggestions RNA degraded RNase contamination 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 does not perform well in downstream experiments a Salt carryover during Ensure that Buffer BR4 is at room temperature elution 18 25 C b Ethanol carryover Ensure that during the second wash with Buffer BRA plates are centrifuged at 6000 rpm for 10 min to dry the membranes c No incubation of the Ensure that incubation of the eluate at 80 C is RNA eluate at 80 C performed in the alloy block Low RNA yield a Less than 2 5 ml blood Ensure that 2 5 ml blood is collected in the collected in the PAXgene PAXgene Blood RNA Tube see Product Circular Blood RNA Tube for the PAXgene Blood RNA Tube b RNA concentration RNA concentration must be measured in measured in water m
19. grity of the RNA After washing with Buffer BR3 the RNA is treated with DNase while bound to the silica gel membrane The DNase is removed by a second wash with Buffer BR3 Washing with Buffer BRA and elution are then performed according to the protocol One RNase Free DNase Set contains RNase free reagents and buffers for 50 RNA preps Prepare DNase stock solution before using the RNase Free DNase Set for the first time Dissolve the solid DNase 1500 Kunitz units in 550 ul RNase free water provided e Take care that no DNase is lost when opening the vial Note DNase l is especially sensitive to physical denaturation Mixing should only be carried out by gently flicking or inverting the tube Do not vortex For each sample to be processed add 10 ul DNase stock solution to 70 ul Buffer RDD Processing of multiple samples For 48 samples dissolve the solid DNase 1500 Kunitz units in 550 ul RNase free water to make a stock solution Add 3850 ul Buffer RDD Mix by gently flicking the tube and centrifuge briefly to collect residual liquid from the sides of the tube For 96 samples dissolve two sets of DNase in 550 ul RNase free water each Pool the stock solutions and add 7700 ul Buffer RDD Mix by gently flicking the tube and centrifuge briefly to collect residual liquid from the sides of the tube Buffer RDD is supplied with the RNase Free DNase Set For long term storage of DNase l divide the
20. mbar to Multiply by Millimeters of mercury mm Hg 0 75 Kilopascals kPa 0 1 Inches of mercury inch Hg 0 0295 Torrs Torr 0 75 Atmospheres atmos 0 000987 Pounds per square inch psi 0 0145 Centrifugation The PAXgene 96 Blood RNA protocol utilizes a streamlined centrifugation procedure that allows preparation of RNA from up to 2 x 96 samples in parallel for direct use in downstream applications For optimal handling QIAGEN in cooperation with the centrifuge manufacturer Sigma Laborzentrifugen GmbH has developed a centrifugation system consisting of the Plate Rotor 2 x 96 and the table top Centrifuge 4 16S or Centrifuge 4 16KS A wide range of other rotors can be used with Centrifuge 4 165 or Centrifuge 4 16KS in addition to the Plate Rotor 2 x 96 Standard table top centrifuges and 96 well microplate rotors are not suitable for the PAXgene 96 Blood RNA procedure Usually 96 well microplate buckets are not deep enough to carry the complete PAXgene assembly without interfering with how the buckets swing out Furthermore high g forces 25500 x g are required for optimal performance of the PAXgene 96 Blood RNA Kit For further information about the centrifuges and Plate Rotor 2 x 96 please contact QIAGEN PAXgene 96 Blood RNA Handbook 02 2014 11 Abbreviated instructions for using the Centrifuge 4 16S and the Centrifuge 4 16KS 1 2 Switch on the centrifuge by pressing the main switch on the back Select the rotor sele
21. n that is stable for at least 1 year after delivery when stored at room temperature To store for more than 1 year 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 Intended Use For research use only Not for use in diagnostic procedures No claim or representation is intended to provide informationfor the diagnosis prevention or treatment of a disease All due care and attention should be exercised in the handling of the products We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to other applicable guidelines Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate safety data sheets SDSs These are available online in a convenient and compact PDF format at www qiagen com Safety where you can find view and print the SDS for each PreAnalytiX kit and kit component CAUTION DO NOT add bleach or acidic solutions directly to the sample preparation waste Buffer BR2 contains guanidine thiocyanate which can form highly reactive c
22. nd Well Block for 30 min at 65 C in an incubator Place a heavy plate over the caps to prevent them from popping open Shake once during the incubation Note lt is essential to mix the sample thoroughly to achieve optimal protein digestion Insufficient mixing can reduce RNA yield If one incubator is used for the entire procedure set the incubator to 80 C after the proteinase digestion for it to heat up in time for the final heat denaturation step Place the PAXgene 96 Incubator Block into the incubator Do not mix Buffer BR2 and proteinase K together before adding them to the sample PAXgene 96 Blood RNA Handbook 02 2014 15 9 10 11 12 13 14 15 Briefly centrifuge the block 10 s at 1000 x g Do not exceed the centrifugation time as longer centrifugation can lead to pellet formation Transfer the samples to a PAXgene 96 Filter Plate sitting on a rack of collection microtubes Mark the PAXgene 96 Filter Plate and collection microtube rack for later identification Note Set the multichannel pipet to 850 ul to transfer the entire sample to the PAXgene 96 Filter Plate Seal the PAXgene 96 Filter Plate with an AirPore Tape Sheet load the collection microtube rack and PAXgene 96 Filter Plate into the holder of the plate rotor and place the whole assembly into the rotor bucket Centrifuge at 6000 rpm 5600 x g for 10 min at room temperature 18 25 C Remove and discard the PAXgene 96 Filter Plate
23. nvenient to have extra Square Well Blocks available PAXgene 96 Blood RNA Handbook 02 2014 Important Notes QlAvac 96 vacuum manifold handling guidelines Use of the convenient modular QlAvac 96 vacuum manifold facilitates RNA preps performed using the PAXgene 96 Blood RNA Kit The following recommendations should be followed when handling the QlAvac 96 vacuum manifold 10 QlAvac 96 operates with a vacuum pump We recommend using a vacuum pump with a capacity of 18 liter min Use of insufficient vacuum pressure may reduce RNA yield and purity A vacuum pressure of 800 to 900 mbar should develop when a PAXgene 96 RNA Plate sealed with tape is used on the QlAvac 96 Vacuum pressures exceeding 900 mbar should be avoided The vacuum pressure is the pressure differential between the inside of the manifold and the atmosphere standard atmospheric pressure 1013 mbar or 760 mm Hg and can be regulated and measured using a pressure gauge or vacuum regulator Vacuum recommendations are given in negative units to indicate the required reduction in pressure with respect to the atmosphere Table 1 page 12 provides pressure conversions to other units Between loading steps the vacuum must be switched off and the manifold ventilated to maintain uniform conditions for each sample This can be done with a vacuum regulator inserted between the vacuum source and the QlAvac 96 vacuum manifold Wear safety glasses when working near a manifold under
24. of Quality of Total RNA Quantification of RNA Purity of RNA Appendix C General Remarks on Handling RNA Handling RNA General handling Appendix D Handling PAXgene Blood RNA Tubes Ordering Information PreAnalytiX Worldwide PAXgene 96 Blood RNA Handbook 02 2014 NN OOO n 10 11 14 19 21 22 22 23 24 24 24 25 26 31 Kit Contents PAXgene 96 Blood RNA Kit 4 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 RNase free water Proteinase K RNase Free DNase Set Round Well Blocks Square Well Blocks Rack of Collection Microtubes Elution Microtube Racks Caps for blocks and tubes Caps for Elution Microtubes AirPore Tape Sheets Secondary BD Hemogard Closures Register Cards 96 well Handbook 762331 4x96 4 4 170 ml 220 ml 2 x 220 ml 4x55ml 60 ml 2 x 1000 ml 2x 10 ml 8 x 50 reactions 4 2 4 4 2 55 8 55x8 4x5 8x50 4 1 Contains guanidine thiocyanate which is not compatible with bleach For safety information see page 5 PAXgene 96 Blood RNA Handbook 02 2014 Shipping and Storage Except for the RNase Free DNase Set the remaining components of the PAXgene 96 Blood RNA Kit can be stored at room temperature 18 25 C The PAXgene 96 Blood RNA Kit contains ready to use proteinase K solutio
25. ompounds 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 196 v v sodium hypochlorite PAXgene 96 Blood RNA Handbook 02 2014 5 The RNA stabilization solution and blood mixture from the PAXgene Blood RNA Tube must be disinfected by using 1 volume of commercial bleach solution 596 sodium hypochlorite per 9 volumes of RNA stabilization 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 Disposal must be made according to official regulations 24 hour emergency information Chemical emergency or accident assistance is available 24 hours a day from CHEMTREC USA amp Canada Tel 1 800 424 9300 Outside USA amp Canada Tel 1 703 527 3887 collect calls accepted Quality Control In accordance with QIAGEN s ISO certified Total Quality Management System each lot of PAXgene 96 Blood RNA Kits is tested against predetermined specifications to ensure consistent product quality 6 PAXgene 96 Blood RNA Handbook 02 2014 Introduction The PAXgene 96 Blood RNA Kit allows the high throughput isolation of total
26. or 816101 81625 81620 Plate Rotor 2 x 96 Rotor for 2 QIAGEN 96 well 81031 plates for use with QIAGEN Centrifuges Vacuum Regulator For use with QlAvac manifolds 19530 Square Well Blocks 96 well blocks with 2 2 ml wells 19578 24 per case PAXgene 96 Block for incubating eluates in 9238279 Incubator Block PAXgene 96 procedures For up to date licensing information and product specific disclaimers see the respective PreAnalytiX or QIAGEN kit handbook or user manual PreAnalytiX and QIAGEN kit handbooks and user manuals are available at www giagen com or can be requested from QIAGEN Technical Services or your local distributor Japan North America UK Rest of World PAXgene 96 Blood RNA Handbook 02 2014 27 Notes 28 PAXgene 96 Blood RNA Handbook 02 2014 Notes PAXgene 96 Blood RNA Handbook 02 2014 29 Notes 30 PAXgene 96 Blood RNA Handbook 02 2014 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 Brazile Orders 0800 557779 Fax 55 11 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 Ord
27. stock solution into single use aliquots and store at 20 C for up to 9 months Thawed aliquots can be stored at 2 8 C for up to 6 weeks Do not refreeze the aliquots after thawing PAXgene 96 Blood RNA Handbook 02 2014 21 Appendix B 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 ug of RNA per ml 1 gt 44 ug 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 mM Tris Cl As discussed see Purity of RNA below 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 ul Dilution 10 ul of RNA sample 140 ul 10 mM Tris Cl
28. te reinsertion of the stopper is necessary for the closure to remain securely on the tube during handling PAXgene 96 Blood RNA Handbook 02 2014 25 Ordering Information Product Contents Cat no PAXgene 96 Blood 4 PAXgene 96 RNA Plates 162331 RNA Kit 4 4 PAXgene 96 Filter Plates Buffers Proteinase K RNase free DNase Set AirPore Tape Sheets Collection Vessels To be used in conjunction with PAXgene Blood RNA Tubes Related products Products that can be ordered from BD and BD authorized distributors PAXgene Blood RNA 100 Blood Collection Tubes To 762165 Tubes 100 be used in conjunction with the PAXgene Blood RNA Kit 50 Blood Collection Set BD Vacutainer Safety Lok 367286 Blood Collection Set 21G 0 75 inch needle 12 inch tubing with luer adapter 50 per box 200 per case BD Vacutainer One Case only for 13 mm and 364815 Use Holder 16 mm diameter 1000 case BD Vacutainer Plus 4 0 ml draw with Red 368975 Serum Tubes 13 x BD Hemogard Closure and 75 mm paper label 100 box 1000 case Accessories QlAvac 96 Vacuum manifold for processing 19504 QIAGEN 96 well plates includes QlAvac 96 Top Plate Base Waste Tray Plate Holder Rack of Collection Microtubes 1 2 ml 26 PAXgene 96 Blood RNA Handbook 02 2014 Centrifuge 4 168 Universal laboratory centrifuge 81500 with brushless motor 815101 815255 81520 Centrifuge 4 16KS Refrigerated universal laboratory 81600 centrifuge with brushless mot
29. y and Paraguay Orders 011 4551 7100 Australia Orders 1 800 656 100 Fax 1 800 656 110 Austria Orders 43 1 7063660 Fax 43 1 7063660 11 Belgium Orders 32 53720408 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 43 43 41 66 East Europe Middle East amp 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 Fox 49 6221305377 Italy Orders 390248204775 Fax 390248204817 Technical 390248240264 The Netherlands Orders 31 20 6545 716 Fox 31 20 5829 421 New Zealand Orders 0800 572 468 Fax 0800 572 469 Spain Orders 34 91 848 8115 Fox 34 91 848 8104 Sweden Orders 46 8 775 51 00 Fax 46 8 775 51 95 Switzerland Orders 41 61 4852222 Fax 41 61 4852200 UK Orders 44 1 865 781 666 Fox 44 1 865 781 528 USA Orders 888 237 2762 Fax 800 847 2220 Technical 800 631 0174 www bd com www PreAnalytiX com PAXgene 96 Blood RNA Handbook 02 2014 31 PreAnalytiX A GIAGEN BD Company
30. ysate clearing Bind RNA DNase digestion Wash 3x PAXgene 96 Blood RNA Handbook 02 2014 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 safety data sheets SDSs available from the product supplier e PAXgene Blood RNA Tubes PreAnalytiX cat no 762165 e Multichannel pipet with tips For efficient liquid handling we recommend usingan electric multichannel pipet with a minimum capacity of 650 ul per pipet tip For example Matrix Technologies Corporation www matrixtechcorp com provides cordlesselectronic multichannel pipets with a unique expandable tip spacing system allowing transfer of liquid between different types of multiwell plate e Reagent reservoirs for multichannel pipets e Disposable gloves e Square Well Blocks cat no 19573 optional e 96 100 ethanol e QlAvac 96 vacuum manifold e Vacuum source capable of generating a vacuum pressure of 800 to 900 mbar e Centrifuge 4 16S or Centrifuge 4 16KS e Plate Rotor 2 x 96 e PAXgene 96 Incubator Block available from QIAGEN contact QIAGEN Technical Services This is not a complete list of suppliers and does not include many important vendors of biological supplies t Two Square Well Blocks are supplied with the kit They can be reused see page 13 If several plates are processed per day it may be co
Download Pdf Manuals
Related Search