PAXgene® - PreAnalytiX
Contents
1. 407 203 uyy Aniqionpoadey 5 Blood RNA Kit Handbook 04 2008 22 Reproducibility of RT PCR between Users 2 1 d o 2 3 T T A B User AA Figure 7 RNA purified in the experiment described in Figure 6 was used for realtime RT PCR Relative tran script levels of EY FOS and IL1B were determined by real time duplex RT PCR using 18S rRNA as an internal standard The values for all samples are plotted relative to the values for user 1 10 donor pools x 3 kit lots x 4 replicates 120 data sets for each with means red lines and standard deviations black bars for all samples shown The dashed lines indicate the 3x total precision of the assays FOS 2 34 ILIB 1 93 Cj PAXgene Blood RNA Kit Handbook 04 2008 23 Reproducibility of RT PCR between Kit Lots Lot Lot Figure 8 RNA purified in the experiment described in Figure 6 was used for realtime RT PCR Relative transcript levels of EY FOS and ILTB were determined by realtime duplex RT PCR using 185 rRNA as an internal standard The values for all samples are plotted relative to the values for kit lot 1 10 donor pools x 3 users x 4 replicates 120 data sets for each gene with means red lines and standard deviations black bars for all samples shown The dashed lines indicate the 3x total precision of the assays FOS 2 34 ILIB 1 93 24 PAXgene Blood RNA Kit H2. Blood Kit Version 2 IVD Handbook The PAXgene Blood RNA System consists of a blood collection tube PAXgene Blood RNA Tube and nucleic acid purification kit PAXgene Blood RNA Kit It is intended for the collection storage and transport of blood and stabilization of intracellular RNA in a closed tube and subse quent isolation and purification of intracellular RNA from whole blood for RT PCR used in molecular diagnostic testing Performance characteristics for the PAXgene Blood RNA System have only been established with FOS and IL1B gene transcripts The user is responsible for establishing appropriate PAXgene Blood RNA System performance characteristics for other target transcripts For in vitro diagnostic use 762174 1051083 PreAnalytiX GmbH Feldbachstrasse CH 8634 Hombrechtikon Produced by QIAGEN GmbH for PreAnalytiX RI 1051083 e 8 April 2008 lad Pre Analyti Trademarks PAXgene PreAnalytiX PreAnalytiX GmbH QIAGEN GlAcube QIAGEN Group BD Vacutainer BD Hemogard Safety Lok Becton Dickinson and Company Franklin Lakes NJ USA Eppendorf Eppendorf Netheler Hinz GmbH PAXgene Blood RNA Kits are not available in all countries please inquire Limited License Agreement Use of this product signifies the agreement of any purchaser or user of the PAXgene Blood RNA Kit to the following terms The PAXgene Blood RNA Kit ma
3. g 4 6 4 9 4 12 4 13 T T T 1 2 3 Storage days Storage days Figure 1 Blood was drawn from 10 donors with duplicate samples and stored at 18 25 for the indicated number of days followed by total RNA purification Blood was collected and stored in PAXgene Blood RNA Tubes BRT and total RNA was purified using the PAXgene Blood RNA Kit Bl Blood was collected and stored in standard blood collection tubes with EDTA as an anticoagulant and total RNA was purified using a standard organic extraction method with silica membrane based RNA cleanup Relative transcript levels of FOS were determined by real time duplex RT PCR using 185 rRNA as an internal standard The values for all samples are plotted with means and standard deviations of all samples shown The dashed lines indicate the 3 total precision of the assay 2 34 12 PAXgene Blood RNA Kit Handbook 04 2008 RNA Stability in Blood Samples at 18 25 IL1B 4 a ee E E E go i i 1 i 1 i ee ale a 4 T 0 1 2 3 Storage days Storage days Figure 2 Blood was drawn and total RNA purified after storage at 18 25 C as described in Figure 1 Relative transcript levels of IL1B were determined by real time duplex RT PCR using 185 rRNA as an internal standard The values
4. e Variablespeed microcentrifuge capable of attaining 1000 8000 x g and equipped with a rotor for 2 ml microcentrifuge tubes e Shaker incubator capable of incubating at 55 C and 65 C and shaking at 2400 rpm not exceeding 1400 rpm e g Eppendorf Thermomixer Compact or equivalent For the automated protocol e QlAcube QIAGEN cat no 9001292 110 V cat no 9001293 230 e QlAcube consumables FilterTips 1000 pl 1024 QIAGEN cat no 990352 e Reagent Bottles 30 ml 6 QIAGEN cat no 990393 e Rotor Adapters 10 x 24 QIAGEN cat no 990394 QlAcube accessories e Reagent Bottle Rack QIAGEN cat no 990390 e Rotor Adapter Holder QIAGEN cat no 990392 e Scissors Ensure that instruments have been checked maintained and calibrated regularly according to the manufacturer s recommendations t Ensure that you are familiar with the guidelines on handling RNA Appendix A page 52 For the addition of ethanol to Buffer BRA concentrate 5 Also included in the Starter Pack GlAcube QIAGEN cat 990395 30 PAXgene Blood RNA Kit Handbook 04 2008 Important Notes Using the QlAcube Ensure that you are familiar with operating the QlAcube Please read the GlAcube User Manual and any additional information supplied with the QlAcube paying careful attention to the safety information before beginning the automated PAXgene Blood RNA protocols Starting the QlAcube Close the QlAcube d
5. PAXgene Blood RNA Tubes 100 100 Blood Collection Tubes 762165 To 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 Use Holder Case only for 13 mm and 364815 16 mm diameter 1000 case BD Vacutainer Plus Serum Tubes 13 x 75 mm 4 0 ml draw with 368975 Red BD Hemogard closure and paper label 100 box 1000 case PAXgene Blood RNA Kit Handbook 04 2008 57 This page intentionally left blank 58 PAXgene Blood RNA Kit Handbook 04 2008 PreAnalytiX Worldwide PreAnalytiX products are distributed by QIAGEN and BD companies Australia Orders 03 9840 9800 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 Canada Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 0086 21 3865 3865 Fax 0086 21 3865 3965 Technical 800 988 0325 800 988 0327 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 02103 29 22000 Technical 02103 29 12400 Hong Kong
6. RNA Purity Aj Values Automated Processing 2 3 2 1 RNA purity comme osoo om B Figure 10 RNA was purified by 3 different operators using 3 different lots 1 2 3 of the PAXgene Blood RNA Kit in the experiment described in Figure 9 values of all individual samples are shown for every operator lot combination RNA Purity Genomic DNA Contamination Automated Processing Genomic DNA w w Figure 11 RNA was purified by 3 different operators A B C using 3 different lots 1 2 3 of the PAXgene Blood RNA Kit in the experiment described in Figure 9 Genomic DNA amounts w w in all individual samples are shown for every operator lot combination 28 PAXgene Blood RNA Kit Handbook 04 2008 The automated protocol of RNA purification using the PAXgene Blood RNA System provides highly reproducible and repeatable RT PCR results as shown in Figure 12 making it highly robust for clinical diagnostic tests Reproducibility of RT PCR between Automated and Manual Protocols 1 Figure 12 RNA was purified by 3 different operators using 3 different lots 1 2 3 of the PAXgene Blood RNA Kit using the automated protocol in the experiment described in Figure 10 In parallel RNA was purified from
7. T Batch code a 8 8 lt lt Material number COMP Components Number STERILE Method of sterilization using irradiation Kunitz units Temperature limitation Upper limit of temperature Manufacturer Important note Write down the current date after adding ethanol to the bottle Upon arrival Adding gt PAXgene Blood RNA Kit Handbook 04 2008 Contains RCNS Reconstituted Deoxyribonuclease Ethanol GITC Guanidine isothiocyanate RNase Free DNase Set RNlase Free DNase Set Leads to PAXgene Blood RNA Kit Handbook 04 2008 5 Kit Contents PAXgene Blood RNA Kit 50 Catalog no 762174 Number of preps 50 BRI Resuspension Buffer 20 ml BR2 Binding Buffer 18 ml BR3 Wash Buffer 1 WASH BUF 1 A5 ml BR4 Wash Buffer 2 11 ml concentrate WASH BUF 2 CONC BR5 Elution Buffer 6 ml RNFW RNase Free Water 2 x bottle WASH 125 ml PK Proteinase K 2x green lid 1 4 ml PRC PAXgene Spin 5x 10 Columns red PAXgene COL PT Processing Tubes 6 x 50 due Hemogard Secondary BD 50 Hemogard Closures Microcentrifuge Tubes 3 x 50 1 5 ml MIC TUBE 1x10 RNFD DNase RNase Free 1500 lyophilized Kunitz units RDD DNA Digestion Buffer 2x2 white lid DIG BUF DRB DNase Resuspension 2 ml Buffer tube lilac lid DNase RES BUF PSC PAXgene Shredder 5x10 Spin C
8. 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 02 33430411 Fax 02 33430426 Technical 800 787980 Japan Telephone 03 5547 0811 Fax 03 5547 0818 Technical 03 5547 0811 Korea South Orders 1544 7145 Fax 1544 7146 Technical 1544 7145 Luxembourg Orders 8002 2076 Fax 8002 2073 Technical 8002 2067 The Netherlands Orders 0800 0229592 Fax 0800 0229593 Technical 0800 0229602 Norway Orders 800 18859 Fax 800 18817 Technical 800 18712 Singapore Orders 65 67775366 Fax 65 67785177 Technical 65 67775366 Spain Orders 9 1 630 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 Australia BD North Ryde Orders 612 8875 7000 Fax 612 8875 7000 Canada BD Oakville Orders 800 268 5430 Fax 800 565 0897 France BD Le Pont de Claix Orders 33 4 76 68 36 36 Fax 33 4 76 68 34 95 Poland BD Warsaw Orders 48 22 65 1 75 88 Fax 48 22 65 1 75 89 USA BD Diagnostics Preanalytical Systems 1 Becton Drive Franklin Lakes NJ 07417 Orders 888 237 2762 Fax 800 847 2220 Technical support
9. closure supplied with the kit If the supernatant is decanted take care not to disturb the pellet and dry the rim of the tube with a clean paper towel 3 Vortex until the pellet is visibly dissolved and centrifuge for 10 minutes at 3000 5000 x g using a swing out rotor Remove and discard the entire supernatant Small debris remaining in the supernatant after vortexing but before centrifugation will not affect the procedure 1 Incomplete removal of the supernatant will inhibit lysis and dilute the lysate and therefore affect the conditions for binding RNA to the PAXgene membrane 4 Add 350 pl resuspension buffer BR1 and vortex until the pellet is visibly dissolved PAXgene Blood RNA Kit Handbook 04 2008 41 o 2 i a 2 42 Pipet the sample into a 1 5 ml microcentrifuge tube MCT Add 300 pl binding buffer BR2 and 40 pl proteinase K PK Mix by vortexing for 5 seconds and incubate for 10 minutes at 55 C using a shaker incubator at 400 1400 rpm After incubation set the temperature of the shaker incubator to 65 C for step 20 ID Do not mix binding buffer BR2 and proteinase PK together before adding them to the sample Pipet the lysate directly into a PAXgene Shredder spin column PSC lilac placed in a 2 ml processing tube PT and centrifuge for 3 minutes at maximum speed but not to exceed 20 000 x D Carefully pipet the lysate into the spin column PSC and vis
10. page 16 is transferred from the PAXgene Blood RNA Tube into processing tubes PT which are placed into the thermoshaker unit on the GlAcube worktable The operator selects and starts the PAXgene Blood RNA Part A protocol from the menu The QlAcube performs the steps of the protocol through to elution of RNA in elution buffer BR5 The operator transfers the microcentrifuge tubes containing the purified RNA into the thermoshaker unit of the QlAcube The operator selects and starts the PAXgene Blood RNA Part B protocol from the menu and heat denaturation is performed by the GlAcube Average sample preparation time based on dota from 12 sample preps is 125 minutes with only approximately 20 minutes of hands on time RNA yields from 2 5 ml healthy human whole blood are 23 pg for 29576 of the samples processed Figure 9 indicates the RNA yields from a total of 288 samples prepared using the automated protocol with 3 kit lots by 3 operators As pooled blood samples instead of individual PAXgene Blood RNA Tubes BRT were used for these studies the results do not reflect the RNA yield expected from single samples of individual blood draws Since yields are highly donor dependent individual yields may vary Figure 9 26 PAXgene Blood RNA Kit Handbook 04 2008 RNA Yield Automated Processing 16 Li RNA yield 9 2 5 ml blood 1 1 2 3 1 2 3 1 2 3 A B c Figure 9 Bl
11. 18 D gr processing fewer than 12 samples make sure to load the centrifuge rotor balanced radially see Figure 19 All centrifuge buckets must be mounted before starting a protocol run even if fewer than 12 samples are to be processed A single sample or 11 samples cannot be processed Loading the Centrifuge Figure 18 Load the assembled rotor adapters into the centrifuge buckets 36 PAXgene Blood RNA Kit Handbook 04 2008 Loading the Centrifuge and Shaker 1 1 17e o1 7 1706 7 OO 2 samples 3 samples el 9 5 se suo suo LLL WY ILLNM amm RE 189 Je ell 2 el 8 4 samples 5 samples H OO 9 5 Isie OI suo 5 N gu 2 ge en e 280 O 6 samples 4 7 samples 3 v 5 M OO OO Offhe 7 6 Oi m e o 8 samples 9 samples v 2 ee e o 10 samples Figure 19 Centrifuge and shaker positions are shown for processing from two 2 samples to ten 10 samples samples One or 11 samples cannot be processed PAXgene Blood RNA Kit Handbook 04 2008 37 Processing tubes Remove any processing tubes PT left in the microcentrifuge tube slots from previous runs see Figure 15 page 34 Fill 3 processing tubes PT with the amount of reagents given in Table 5 Label the tubes PT clearly wi
12. 20 Incubate the eluate for 5 minutes at 65 C in the shaker incubator from step 5 without shaking After incubation chill immediately on ice 1 This incubation at 65 C denatures the RNA for downstream applications Do not exceed the incubation time or temperature o 2 O a Pl e 21 If the RNA samples will not be used immediately store at 20 C or 70 C Since the RNA remains denatured after repeated freezing and thawing it is not necessary to repeat the incubation at 65 C If using the RNA samples in a diagnostic assay follow the instructions supplied by the manufacturer 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 BR5 as the volume of eluted RNA to be diluted Elution 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 1 44 pg ml See Appendix B page 53 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 fro
13. 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 page 54 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 elution buffer BR5 as the volume of eluted RNA to be diluted Elution 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 80 yl 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 3 Concentration of RNA sample 44 x Az x dilution factor 44x0 3x15 198 pg ml Total yield concentration x volume of sample in milliliters 198 pg ml x 0 08 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 PAXgene Blood RNA Kit Handbook 04 2008 53 Purity of RNA The ratio of the
14. E LL 80 974 soj Jes 69 6 60 6 Bd Bl x Bd 6 pak AD 45 AD as uw AD QS AD dS w 2 01 01 OL u sio OL X 8 6 ood 5 OL X 9 9 jood u s o2 01 L S 5101 V pup 4207 91 480 880 S 990 8240 8l rel 6501 OL 680 848 asn g 10 ve ZIZ 888 vi 880 179 el A 5 9 101 Ol 70 S87 6 0 818 8 890 968 L 060 702 V asn 10 O iei iwc are 2 o A0 7 D asn Z 40 8 9 004 OL 140 024 6 282 86 ZL VOL 609 9 asn Z 40 o eel An l 681 826 EL 860 ZEZ Hsn Z 40 EL 6 1 ZVOL vl WL LOL 9 S90 1 01 9 484 101 6 9 0 O68 l ZZL Z88 IZ y l 8E6 G A Z g sn 101 9 670 967 9 170 ZSZ OL 660 556 08 asn 40 69 6 pak 80 6 Bd 60 x Bd 68 pak jo AD as AD dS AD QS ww AD dS w 2 OL 01 01 14 19 01 8 6 ood OL X 9 9 jood 5 92 01 X L S jood 1951 pup 407 203 Appiqionpoadeg v 21 Blood RNA Kit Handbook 04 2008 jood Jouop SIOUOP B
15. Yield for Different Operators and PAXgene Blood RNA Kit Lots Using Pooled Blood Samples RNA yield 2 5 ml blood ON tk CV of RNA yield Donor pool Figure 6 Blood samples from 30 different donors were collected in PAXgene Blood RNA Tubes BRT 12 tubes per donor 360 tubes in total The contents of the tubes from 3 donors were pooled and subsequently re aliquoted into 36 samples These 36 samples per 3 donor pool were manually processed by 3 different operators Each operator used 3 different PAXgene Blood RNA Kit lots for the extraction and processed quadruplicate samples from each of the 10 donor pools EY RNA yield and standard deviation for every operator lot combination Quadruplicate blood samples from 10 donor pools were processed by 3 different operators A B C with each of 3 kit lots 1 2 3 The mean yields columns and standard deviations error bars per quadruplicate sample from the same donor pool for different operator and different kit lot are presented El CV of RNA yield per donor pool for all operator lot combinations A B C 1 2 3 as calculated from the mean yield and standard deviation of the yield shown in Figure 6A 20 PAXgene Blood RNA Kit Handbook 04 2008 OL Oll 6801 YEL ZOOL 11 SLL 9900 860 784 S10 O 61 61 978 OZ 051 84 607 868 l lel 2 sioj g LL 780 187 OL ZZO 794 EL 261
16. for all samples are plotted with means and standard deviations of all samples shown The dashed lines indicate the 3x total precision of the assay 1 93 PAXgene Blood RNA Kit Handbook 04 2008 13 RNA Stability in Blood Samples at 2 8 C FOS 12 4 13 T T T T T 12 4 13 T T T T T Storage days Figure Blood was drawn from 10 donors with duplicate samples and stored at 2 8 C for the indicated number of days followed by total RNA purification EY Blood was collected and stored PAXgene Blood RNA Tubes BRT and total RNA was purified using the PAXgene Blood RNA Kit Bl Blood was collected and stored in standard blood collection tubes with EDTA as an anticoagulant and total RNA was purified using a standard organic extraction method with silica membrane based RNA cleanup Relative transcript levels of FOS were determined by real time duplex RT PCR using 185 rRNA as an internal standard The values for all samples are plotted with means and standard deviations of all samples shown The dashed lines indicate the 3x total precision of the assay 2 34 14 PAXgene Blood RNA Kit Handbook 04 2008 RNA Stability in Blood Samples at 2 8 C IL1B AAC Storage days Storage days Figure 4 Blood was drawn and total RNA purified after storage at 2 8 C as described in Figure 3 Relative transc
17. readings at 260 nm and 280 nm provides an estimate of the purity of RNA with respect to contaminants that absorb in the UV such as protein However the ratio is influenced considerably by pH Lower pH results in a lower As o Asso 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 Asso 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 elution buffer BR5 as the volume of eluted RNA to be diluted Elution 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 Chomczynski P 1997 Effect of pH and ionic strength on the spectro photometric assessment of nucleic acid purity BioTechniques 22 474 54 PAXgene Blood RNA Kit Handbook 04 2008 Appendix Handling PAXgene Blood RNA Tubes 1 The following recommendations from BD may be helpful when handling PAXgene Blood RNA Tubes BRT See the PAXgene Blood RNA Tube Product Circular for more information about PAXgene Blood RNA Tubes BRT Instructions for removal of BD Hemogard Closure 1 Grasp PAXgene Blood RNA Tube BRT with one hand placing the thumb under the BD Hemogard closure For added stability place arm on so
18. scientists with extensive practical and theoretical expertise in molecular biology and the use of PreAnalytiX products If you have any questions regarding the PAXgene Blood RNA Kit please do not hesitate to contact us For technical assistance and more information please call QIAGEN Technical Services see page 59 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 coot 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 MSDSs for this kit 8 PAXgene Blood RNA Kit Handbook 04 2008 Binding buffer BR2 and wash buffer 1 BR3 contain guanidine thiocyanate which can form highly reactive compounds when combined with bleach If Binding buffer BR2 or wash buffer 1 BR3 are spilt clean with suitable laboratory detergent and water If liquid containing potentially infectious agents is spilt clean the affected area first with laboratory detergent and water and then with 1 v v sodium hypochlorite CAUTION DO NOT add bleach or acidic solutions directly to the sample preparation wa
19. step in many molecular assays used to study cellular RNA However a major problem in such experiments is the instability of the cellular RNA profile in vitro Studies at PreAnalytiX have shown that the copy numbers of individual mRNA species in whole blood can change more than 10004old during storage or transport at room temperature This is caused both by rapid RNA degradation and by induced expression of certain genes after the blood is drawn Such changes in the RNA expression profile make reliable studies of gene expression impossible A method that preserves the RNA expression profile during and after phlebotomy is therefore essential for accurate analysis of gene expression in human whole blood Principle and procedure PreAnalytiX has developed a new system that enables the collection stabilization storage and transportation of human whole blood specimens together with a rapid and efficient protocol for purification of intracellular RNA The system requires the use of PAXgene Blood RNA Tubes BRT US Patents 6 602 718 and 6 617 170 for blood collection and RNA stabilization followed by manual or automated RNA purification using the PAXgene Blood RNA Kit Both manual and automated protocols provide substantially equivalent performance with regards to RNA quality and yield Performance data for the manual protocol pages 18 25 and the automated protocol pages 26 29 are included in this handbook Sample collection and stabilization P
20. 2 by the instrument see Figure 17 page 36 12 Close the lids of all 1 5 ml microcentrifuge tubes MCT containing the purified RNA in the rotor adapters position 3 lid position L3 see Figure 17 page 36 Transfer the 1 5 ml microcentrifuge tubes MCT onto the GlAcube shaker adapter see Figure 15 page 34 13 Close the GlAcube instrument door see Figure 13 page 32 48 PAXgene Blood RNA Kit Handbook 04 2008 14 Select the PAXgene Blood RNA Part B protocol and start the protocol Follow the instructions given on the GlAcube touchscreen 1 This program incubates the samples at 65 and denatures the RNA for downstream applications Even if the downstream application includes a heat denaturation step do not omit this step Sufficient RNA denaturation is essential for maximum efficiency in downstream applications 15 After the PAXgene Blood RNA Part B program is finished open the QlAcube instrument door see Figure 13 page 32 Immediately place the microcentrifuge tubes MCT containing the purified RNA on ice WARNING Hot surface The shaker can reach temperatures of up to 70 C 158 F Avoid touching it when it is hot gt S 3 e ay 9 3 e D Do not let the purified RNA remain in the QlAcube Since the samples not cooled the purified RNA can be degraded Unattended overnight sample preparation runs are therefore not recommended 16 If the RN
21. 800 631 0174 www bd com www PreAnalytiX com PAXgene Blood RNA Kit Handbook 04 2008 59 1051083 127153017 a PreAnalytiX A QIAGEN BD Company
22. A samples will not be used immediately store at 20 C or 70 C Since the RNA remains denatured after repeated freezing and thawing it is not necessary to repeat the heat incubation protocol PAXgene Blood RNA Part B D For quantification in Tris buffer use the relationship Azs 1 gt 44 pg ml See Appendix B page 53 17 Remove the reagent bottle rack from GlAcube worktable see Figure 15 page 34 and close all bottles with the appropriately labeled lids Buffer in bottles can be stored at room temperature 15 25 C for up to 3 months Remove and discard remaining reagents in the processing tubes PT in the QlAcube microcentrifuge tube slots see Figure 15 page 34 Remove and discard rotor adapters from the centrifuge see Figure 15 page 34 Empty the GlAcube waste drawer see Figure 13 page 32 Close the QlAcube instrument door and switch off the instrument with the power switch see Figure 13 page 32 PAXgene Blood RNA Kit Handbook 04 2008 49 Troubleshooting Guide This troubleshooting guide may be helpful in explaining any questions that may arise The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about the information and the manual and automated protocols in this handbook for contact information see page 59 or visit www qiagen com Comments and suggestions RNA degraded RNase contamination Low RNA yield a b d Less than 2 5 ml blo
23. AXgene Blood RNA Tubes BRT contain a proprietary reagent composition based on a patented RNA stabilization technology This reagent composition protects RNA molecules from degradation by RNases and minimizes ex vivo changes in gene expression PAXgene Blood RNA Tubes BRT are intended for the collection of human whole blood and stabilization of cellular RNA for up to 3 days at 18 25 Figures 1 and 2 pages 12 and 13 or up to 5 days at 2 8 C Figures 3 and 4 pages 14 and 15 Currently available data shows stabilization of cellular RNA for at least 24 months at 20 C or 70 C For more information from ongoing studies evaluating stability for longer time periods please contact QIAGEN Technical Services The actual duration of RNA stabilization may vary depending upon the species of cellular RNA and the downstream application used Due to the limited number of transcripts validated for stabilization specifications FOS and IL1B gene transcripts the performance characteristics have not been established for all transcripts Laboratory personnel should review the manufacturer s data and their own data to determine whether validation is necessary for other transcripts Rainen L et al 2002 Stabilization of mRNA expression in whole blood samples Clin Chem 48 1883 PAXgene Blood RNA Kit Handbook 04 2008 11 RNA Stability in Blood Samples at 18 25 FOS
24. Kit Handbook 04 2008 Blood Wash pellet Resuspend Transfer to processing tube PT and load into the QlAcube shaker l i E 1 i Ready to use RNA PAXgene Blood RNA Kit Handbook 04 2008 The Automated PAXgene Blood RNA Procedure Add proteinase K PK and binding buffer BR2 Incubate Transfer to PAXgene Shredder spin column PSC Add ethanol to flow through Load on PAXgene RNA Spin Column PRC Bind total RNA Wash Digest DNA Wash Transfer PAXgene RNA Spin Column PRC Elute Close lids of microcentrifuge tubes MCT and transfer to QlAcube shaker Heat to 65 C eqnoyqo eui uo ApnJ 17 Manual RNA purification In detail the resuspended pellet is incubated in optimized buffers together with proteinase K PK to bring about protein digestion An additional centrifugation through the PAXgene Shredder spin column PSC is carried out to homogenize the cell lysate and remove residual cell debris and the supernatant of the flow through fraction is transferred to a fresh microcentrifuge tube Ethanol is added to adjust binding conditions and the lysate is applied to a PAXgene RNA spin column PRC During a brief centrifugation RNA is selectively bound to the PAXgene silica membrane as contaminants pass through Remaini
25. V purification of DNA RNA or 9001293 proteins using QIAGEN spin column kits 1 year warranty on parts and labor Warranty PLUS 2 Full GlAcube 3 year warranty 48 hour 2 9240834 working days priority response all labor travel and repair parts Starter Pack GlAcube Pack includes reagent bottle 990395 racks 3 rack labeling strips 8 200 filter tips 1024 1000 pl filtertips 1024 1000 pl filtertips wide bore 1024 30 ml reagent bottles 18 rotor adapters 240 rotor adapter holder Filter Tips 1000 pl 1024 Sterile Disposable Filter Tips 990352 racked Reagent Bottles 30 ml 6 Reagent Bottles 30 ml with lids 990393 pack of 6 for use with the QlAcube reagent bottle rack US Canada and Japan Rest of world 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 56 PAXgene Blood RNA Kit Handbook 04 2008 Ordering Information Product Contents Cat no Rotor Adapters 10 x 24 For 240 preps 240 Disposable 990394 Rotor Adapters for use with the GlAcube Reagent Bottle Rack Rack for accommodating 990390 6 x 30 ml reagent bottles on the GlAcube worktable Rotor Adapter Holder Holder for 12 disposable rotor 990392 adapters for use with the GlAcube Products that can be ordered from BD and BD authorized distributors
26. Y sjunoo 39 poo q JO eui sjuese1dei junoo poo q ju seiooyne OL X 1 501 X 8p siunoo 2 Jo puuou eui jo sen p 19 0 y pup jeddn eui PUD junoo 2 eiu eui papajas sjood y sjood 1 jo sisA pup pe ioieq Oc 081 668 0 041 S 8 116591 99 GL 601 1e8n JD PUD stor 69 6 80 6 Bd 6 67 pak ds 9 as 9 as 9 as ju 9 OL X Z OL u 9 01 X t8 u 2 01 X 9 1 1 OL X L S OL 6 ood 9 jood s1es V s407 Aqiqionpouday aL eqD 0 081 026 ve 661 9 61 141 606 ZL 61 097 s1esn P 10 pz tae eL 951 G A 8 78 666 5S0 OZ 5 D Z 40 461 206 61 291 288 Yl YEL 886 6 690 964 5 40 69 6 pak 60 6 Bd 60 Bd 6d pak ads 9 as 9 as 9 as 2 OL X COL 2 OL X 78 5 92 01 X 9 u sio 01 X L S OL 6 ood 9 jood 5195 V
27. andbook 04 2008 Table 2 Summary of RT PCR Data from Figures 7 and 8 Test system FOS 18S rRNA assay IL1B 18S rRNA assay Comparison of data Mean SD Mean SD AAC AAC AAC Reproducibility within each user and between all lots All users lot 1 lot 1 0 00 0 00 0 00 0 00 All users lot 1 lot 2 0 03 0 48 20 07 0 66 All users lot 1 lot 3 0 21 0 52 0 11 0 71 Reproducibility within each lot and between all users All lots user A user A 0 00 0 00 0 00 0 00 All lots user A user B 0 46 0 44 0 06 0 69 All lots user A user C 0 31 0 60 0 15 0 71 User Technician performed the study Lot Number of kit lot used in this study SD Standard deviation Mean AAC values 120 and standard deviations are shown for the data presented in Figures 7 and 8 PAXgene Blood RNA Kit Handbook 04 2008 25 Automated RNA purification Sample preparation using the QlAcube follows the same steps as the manual procedure enabling you to continue using the PAXgene Blood RNA Kit for purification of high quality RNA See the QlAcube User Manual www giagen com MyQlAcube for more information about the GlAcube The automated RNA purification protocol consists of 2 parts or protocols PAXgene Blood RNA Part A and PAXgene Blood RNA Part B with a brief manual intervention between the 2 parts The centrifuged washed and resuspended nucleic acid pellet see RNA concentration and purification
28. ck that the sample is completely transferred to the spin column PRC Pipet 350 pl wash buffer 1 BR3 into the PAXgene RNA spin column PRC Centrifuge for 1 minute at 8000 20 000 x Place the spin column PRC in a new 2 ml processing tube PT and discard the old processing tube PT containing flow through PAXgene Blood RNA Kit Handbook 04 2008 12 Add 10 pl DNase I RNFD stock solution to 70 pl DNA digestion buffer RDD in a 1 5 ml microcentrifuge tube MCT Mix by gently flicking the tube and centrifuge briefly to collect residual liquid from the sides of the tube If processing for example 10 samples add 100 pl DNase RNFD stock solution to 700 pl DNA digestion buffer RDD Use the 1 5 ml microcentrifuge tubes MCT supplied with the kit D DNase is especially sensitive to physical denaturation Mixing should only be carried out by gently flicking the tube Do not vortex 0201014 13 Pipet DNase RNFD incubation mix 80 pl directly onto the PAXgene RNA spin column PRC membrane and place on the benchtop 20 30 C for 15 minutes D Ensure that the DNase RNFD incubation mix is placed directly onto the membrane DNase digestion will be incomplete if part of the mix is applied to and remains on the walls or the O ring of the spin column PRC 14 Pipet 350 pl wash buffer 1 BR3 into the PAXgene RNA spin column PRC and centrifuge for 1 minute at 8000 20 000 g Plac
29. d for less than 2 hours Low ratio a RNA diluted in water before 1 Use 10 mM Tris Cl pH 7 5 to dilute RNA purity is measured before measuring purity see Appendix B page 53 b Spectrophotometer not 1 To zero the spectrophotometer use a blank properly zeroed containing the same proportion of elution buffer BR5 and dilution buffers as in the samples to be measured Elution buffer BR5 has high absorbance at 220 nm which can lead to high background absorbance levels if the spectro photometer is not properly zeroed Instrument malfunction QlAcube not properly operated Read the QlAcube User Manual paying careful attention to the Troubleshooting section Make sure that the GlAcube is properly maintained as described in the QlAcube User Manual Wilfinger W W Mackey M and Chomczynski P 1997 Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity BioTechniques 22 474 PAXgene Blood RNA Kit Handbook 04 2008 51 Appendix A General Remarks on Handling RNA Handling RNA I 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
30. d in molecular diagnostic testing See the PAXgene Blood RNA Tube Product Circular for information about the use of PAXgene Blood RNA Tubes BRT Performance characteristics for the PAXgene Blood RNA System have only been established with FOS and ILIB gene transcripts The user is responsible for establishing appropriate PAXgene Blood RNA System performance characteristics for other target transcripts PAXgene Blood RNA Kit Handbook 04 2008 7 Product Use Limitations The PAXgene Blood RNA Kit is intended for purification of intracellular RNA from human whole blood 4 8 x 10 1 1 x 107 leukocytes ml for in vitro diagnostics applications It is not for the purification of genomic DNA or viral nucleic acids from human whole blood Due to the limited number of transcripts validated for stabilization specifications FOS and ILIB gene transcripts the performance characteristics have not been established for all transcripts Laboratory personnel should review the manufacturer s data and their own data to determine whether validation is necessary for other transcripts Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of PAXgene Blood RNA Kit is tested against predetermined specifications to ensure consistent product quality Technical Assistance At QIAGEN we pride ourselves on the quality and availability of our technical support Our Technical Service Departments are staffed by experienced
31. 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 D 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 down stream 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 52 PAXgene Blood RNA Kit Handbook 04 2008 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 yg of RNA per ml Ago 1
32. e the spin column PRC in new 2 ml processing tube PT and discard the old processing tube PT containing flow through 15 Pipet 500 pl wash buffer 2 BR4 into the PAXgene RNA spin column PRC and centrifuge for 1 minute at 8000 20 000 g Place the spin column PRC new 2 ml processing tube PT and discard the old processing tube PT containing flow through D Wash buffer 2 BRA is supplied as a concentrate Ensure that ethanol is added to wash buffer 2 BRA before use see Things to do before starting page 40 16 Add another 500 pl wash buffer 2 BR4 to the PAXgene RNA spin column PRC Centrifuge for 3 minutes at 8000 20 000 x g 17 Discard the processing tube PT containing the flow through and place the PAXgene RNA spin column PRC in a new 2 ml processing tube PT Centrifuge for 1 minute at 8000 20 000 x g 18 Discard the processing tube PT containing the flow through Place the PAXgene RNA spin column PRC in a 1 5 ml microcentrifuge tube MCT and pipet 40 pl elution buffer BR5 directly onto the PAXgene RNA spin column PRC membrane Centrifuge for 1 minute at 8000 20 000 x g to elute the RNA It is important to wet the entire membrane with elution buffer BR5 in order to achieve maximum elution efficiency PAXgene Blood RNA Kit Handbook 04 2008 43 19 Repeat the elution step step 18 as described using 40 pl elution buffer BR5 and the same microcentrifuge tube MCT
33. eAnalytiX products are manufactured for PreAnalytiX by GIAGEN 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 Explanation of Symbols 4 Kit Contents 6 Storage Conditions 7 Intended Use 7 Product Use Limitations 8 Quality Control 8 Technical Assistance 8 Safety Information 8 Introduction 11 Principle and procedure 11 Sample collection and stabilization 11 RNA concentration and purification 16 Manual RNA purification 18 Automated RNA purification 26 Equipment and Reagents to Be Supplied by User 30 Important Notes 31 Using the GlAcube 31 Starting the QlAcube 31 Installing protocols on the QlAcube 31 Loading the QlAcube 33 Protocols Manual Purification of Total RNA from Human Whole Blood Collected into PAXgene Blood RNA Tubes BRT 39 BE Automated Purification of Total RNA from Human Whole Blood Collected into PAXgene Blood RNA Tubes BRT 45 Troubleshooting Guide 50 Appendix A General Remarks on Handling RNA 52 Appendix B Quantification and Determination of Quality of Total RNA 53 Appendix C Handling PAXgene Blood RNA Tubes 55 Ordering Information 56 PAXgene Blood RNA Kit Handbook 04 2008 3 Explanation of Symbols A Z V Contains sufficient for lt N gt tests Consult instructions for use Use by Do not reuse In vitro diagnostic medical device Catalog number
34. ear suitable protective clothing 536 37 Wear suitable protective clothing and gloves 546 If swallowed seek medical advice immediately and show the container or label PAXgene Blood RNA Kit Handbook 04 2008 9 Proteinase x x Contains proteinase Tritirachium album sensitizer irritant Risk and safety phrases R36 37 38 42 43 523 24 26 36 37 DNase RNFD xx Contains deoxyribonuclease bovine sensitizer Risk and safety phrases R42 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 R10 Flammable R20 21 22 Harmful by inhalation in 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 13 Keep away from food drink and animal feedingstuffs S22 Do not breathe dust S23 Do not breathe spray 524 Avoid contact with skin 526 In case of contact with eyes rinse immediately with plenty of water and seek medical advice 536 Wear suitable protective clothing S36 37 Wear suitable protective clothing and gloves S46 If swallowed seek medical advice immediately and show the container or label 10 PAXgene Blood RNA Kit Handbook 04 2008 Introduction Collection of whole blood is the first
35. er see Figure 15 page 34 The sample positions are numbered for ease of loading Insert shaker rack plugs included with the QlAcube into the slots at the edge of the shaker rack next to each processing tube This enables detection of samples during the load check D Make sure that the correct shaker adapter Shaker Adapter 2 ml safe lock tubes marked with a 2 included with the QlAcube is installed D If processing fewer than 12 samples make sure to load the shaker rack as shown in Figure 19 page 37 One or 11 samples cannot be processed 5 o 2 un a im 2 amp EJ 9 Close the QlAcube instrument door see Figure 13 32 10 Select the PAXgene Blood RNA Part A protocol and start the protocol Follow the instructions given on the QlAcube touchscreen Make sure that both program parts part A and part are installed on the QlAcube instrument see Installing protocols on the GlAcube page 31 The GlAcube will perform load checks for samples tips rotor adapters and reagent bottles 11 After the PAXgene Blood RNA Part A protocol is finished open the QlAcube instrument door see Figure 13 page 32 Remove and discard the PAXgene RNA spin columns PRC from rotor adapters and the empty processing tubes PT from the shaker During the run spin columns are transferred from the rotor adapter position 1 lid position L1 to rotor adapter position 3 lid position L
36. fore 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 Kit Handbook 04 2008 33 Loading the Reagent Bottle Rack Position 1 Position 2 Binding buffer 96 100 BR2 ethanol Position 3 Position 4 Wash buffer Wash buffer 1 BR3 2 BR4 Labeling strip duis Position 5 Position 6 Empty Empty Figure 14 EY Schematic of positions and contents of bottles in the reagent bottle rack Bl Loading the rack onto the QlAcube Internal View of the GlAcube Figure 15 ll Centrifuge lid 8 Microcentrifuge tube slots Centrifuge Tip racks Shaker Disposal slots for tips and columns Bi Reagent bottle rack E Robotic arm Tip sensor 34 PAXgene Blood RNA Kit Handbook 04 2008 Spin columns PRC PSC microcentrifuge tubes MCT and GlAcube plasticware Place 2 tip racks filled with Filter Tips 1000 pl onto the QlAcube see Figure 15 Refill racks with tips when necessary Only use 1000 yl filter tips designed for use with the QlAcube Label rotor adapters and microcentrifuge tubes MCT for each sample using a permanent pen Open the PAXgene Shredder spin columns PSC to be used and cut the lids off completely using scissors see Figure 16 D For proper operation of the QlAcube robotic gripper completely remove cut off the lids and all plastic parts connecting the lid to the PAXgene S
37. hredder spin columns PSC see Figure 16 Otherwise the robotic gripper cannot grip the spin columns PSC PRC properly Load the PAXgene RNA spin column PRC PAXgene Shredder spin column PSC without lid and labeled microcentrifuge tube MCT into the appropriate positions in each labeled rotor adapter as shown in Table 4 and Figure 17 page 36 Make sure that the spin column PRC and microcentrifuge tube MCT lids are pushed all the way down to the bottom of the slots at the edge of the rotor adapter otherwise the lids will break off during centrifugation Loading a PAXgene Shredder Spin Column PSC Column PSC lid removed incorrectly part of lid is remaining Column PSC lid removed correctly Figure 16 The PAXgene Shredder spin column PSC is loaded into the middle position Cut off the lid before loading the column PSC PAXgene Blood RNA Kit Handbook 04 2008 35 Table 4 Labware in the rotor adapter Position Labware Lid position 1 PAXgene RNA spin column red PRC L1 2 PAXgene Shredder spin column lilac PSC cut off lid before placing in rotor adapter 3 Microcentrifuge tube MCT L3 Use the microcentrifuge tubes 1 5 ml MCT included in the PAXgene Blood RNA Kit L2 L1 L3 Figure 17 The rotor adapter has three tube positions 1 3 and three lid positions L1 L3 Loading the centrifuge Load the assembled rotor adapters into the centrifuge buckets as shown in Figure
38. ity of RNA yield and real time RT PCR performance As pooled blood samples instead of individual PAXgene Blood RNA Tubes BRT were used for these studies the results do not reflect the system repeatability including fluctuation between individual blood draws but only the repeatability of the sample preparation see Figure 6 18 PAXgene Blood RNA Kit Handbook 04 2008 Reproducible and Repeatable RNA Purification HA Hb 20 4 E 16 4 8 4 12 E 94 ES 44 0 T T T T T i 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Donor 1 244 204 i 164 5 gt 124 5 lt 87 5 3 44 gt 0 T T T T T 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Donor Figure 5 Quadruplicate blood samples from 14 donors were manually processed by each of 3 technicians A Three sets of equipment were used and all samples prepared by a single technician were processed using the same equipment EY Means and standard deviations of RNA yield per replicate samples from the same donors and different technicians are shown E Twelve replicate blood samples from each of 14 donors were processed by the 3 different technicians Means and standard deviations of RNA yield per samples from the same donors and all technicians are presented For all RNA samples 0 Asso ratios ranged from 1 8 to 2 2 PAXgene Blood RNA Kit Handbook 04 2008 19 Repeatability and Reproducibility of RNA
39. lid 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 2 Move thumb away before lifting closure DO NOT use thumb to push closure off tube BRT Caution If the tube BRT contains blood an exposure hazard exists To help prevent injury during closure removal it is important that the thumb used to push upward on the closure be removed from contact with the tube BRT as soon as the BD Hemogard closure is loosened 3 Lift closure off tube BRT In the unlikely event of the plastic shield separating from the rubber stopper DO NOT REASSEMBLE CLOSURE Carefully remove rubber stopper from tube BRT Instructions for insertion of Secondary BD Hemogard Closure 1 Replace closure over tube BRT 2 Twist and push down firmly until stopper is fully reseated Complete reinsertion of the stopper is necessary for the closure to remain securely on the tube during handling PAXgene Blood RNA Kit Handbook 04 2008 55 Ordering Information Product Contents Cat no PAXgene Blood RNA System Products that can be ordered from QIAGEN PAXgene Blood RNA Kit 50 50 PAXgene Spin Columns 762174 50 PAXgene Shredder Spin Columns Processing Tubes RNase Free DNase RNase Free Reagents and Buffers To be used in conjunction with the PAXgene Blood RNA Tubes QlAcube 110 V Robotic workstation for automated 9001292 QlAcube 230
40. m the prod uct supplier 44 PAXgene Blood RNA Kit Handbook 04 2008 Protocol Automated Purification of Total RNA from Human Whole Blood Collected into PAXgene Blood RNA Tubes BRT Important points before starting e sure that the kit box is intact and undamaged and that buffers have not leaked Do not use a kit that has been damaged e When using a pipet ensure that it is set to the correct volume and that liquid is carefully and completely aspirated and dispensed e To avoid transferring samples to the wrong tubes and plastic consumables ensure that all processing tubes PT microcentrifuge tubes MCT and rotor adapters are properly labeled using a permanent pen Label the lid and the body of each microcentrifuge tube MCT the body of each processing tube PT and the outer wall of each rotor adapter gt 3 V lt c e Spillages of samples and buffers during the procedure may reduce the yield and purity of RNA e Unless otherwise indicated all steps of this protocol including centrifugation steps should be carried out at room temperature 15 25 e Because of the sensitivity of nucleic acid amplification technologies the following precautions are necessary when handling samples to avoid cross contamination e Carefully pipet the sample into the processing tube PT on the bottom of the tube without moistening the rim of the tube e Always change pipet tip
41. mn PRC PSC membrane with the pipet tip e After vortexing or heating a microcentrifuge tube MCT briefly centrifuge it to remove drops from the inside of the lid Wear gloves throughout the entire procedure In case of contact between gloves and sample change gloves immediately e Close the spin column PRC PSC before placing it in the microcentrifuge Centrifuge as described in the procedure e Open only one spin column PRC PSC at a time and take care to avoid generating aerosols e For efficient parallel processing of multiple samples fill a rack with processing tubes PT to which the spin columns PRC PSC can be transferred after centrifugation Discard the used processing tubes PT containing flow through and place the new processing tubes PT containing spin columns PRC PSC directly in the microcentrifuge PAXgene Blood RNA Kit Handbook 04 2008 39 0201014 Things to do before starting e Blood must be collected in PAXgene Blood RNA Tubes according to the instructions in the PAXgene Blood RNA Tube Product Circular If necessary see Appendix C page 55 for recommendations on handling PAXgene Blood RNA Tubes BRT e Ensure that the PAXgene Blood RNA Tubes BRT are incubated for at least 2 hours at room temperature after blood collection to ensure complete lysis of blood cells Incubation of the PAXgene Blood RNA Tube BRT overnight may increase yields If the PAXgene Blood RNA T
42. ng contaminants are removed in several efficient wash steps Between the first and second wash steps the membrane is treated with DNase RNFD to remove trace amounts of bound DNA After the wash steps RNA is eluted in elution buffer BR5 and heatdenatured Total RNA purified using the PAXgene Blood RNA System is highly pure Using the manual protocol values are between 1 8 and 2 2 and lt 1 w w genomic DNA is present in 29576 of all samples as measured by quantitative real time PCR of a sequence of the beta actin gene At least 95 of samples show no inhibition in RT PCR when using up to 30 of the eluate Using the manual protocol average sample preparation time based on data from 12 sample preps is approximately 90 minutes with only 40 minutes of hands on time RNA yields from 2 5 ml healthy human whole blood are 23 pg for 29576 of the samples processed Since yields are highly donor dependent individual yields may vary For individual donors the PAXgene Blood RNA system provides highly reproducible and repeatable yields Figures 5 and 6 pages 19 and 20 and reproducible and repeatable RT PCR Figures 7 and 8 pages 23 and 24 making it highly robust for clinical diagnostic tests Figure 5 indicates the overall repeatability and reproducibility of the PAXgene Blood RNA System Additional studies were conducted to show the influence of different PAXgene Blood RNA kit lots and different operators on the reproducibil
43. od collected in the PAXgene Blood RNA BRT RNA concentration measured in water Cell debris transferred to the PAXgene RNA spin column PRC in steps 9 and 10 of the manual protocol Supernatant not completely removed in step 3 D Be careful not to introduce any RNases into the reagents during the procedure or later handling see Appendix A page 52 0 Ensure that 2 5 ml blood is collected in the PAXgene Blood RNA Tube BRT see PAXgene Blood RNA Tube Product Circular 1 RNA concentration must be measured in 10 mM Tris Cl pH 7 5 for accurate quantification see Appendix B page 53 D Avoid transferring large particles when pipetting the supernatant in step 7 of the manual protocol Transfer of small debris will not affect the procedure D Ensure the entire supernatant is removed If the supernatant is decanted remove drops from the rim of the tube by dabbing onto a paper towel Take appropriate precautions to prevent cross contamination 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 50 PAXgene Blood RNA Kit Handbook 04 2008 Comments and suggestions e After collection in the PAXgene Incubate blood in the PAXgene Blood RNA Blood RNA Tube BRT Tube BRT for at least 2 hours after collection blood is incubate
44. olumns lilac PAXgene SHRED COL Table continued overleaf Not compatible with disinfecting reagents containing bleach Contains a guanidine salt See page 8 for safety information t Wash Buffer 2 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 Kunitz units are the commonly used units for measuring DNase see page 40 or 46 for definition 6 PAXgene Blood RNA Kit Handbook 04 2008 PAXgene Blood RNA Kit 50 Catalog no 762174 Number of preps 50 PAXgene Blood RNA Kit FS 1 Handbook Version 2 Storage Conditions PAXgene RNA spin columns PAXgene Shredder spin columns PSC proteinase PK and buffers BR1 BR2 BR3 BRA and BR5 can be stored dry at the temperature indicated on the kit label The RNase Free DNase Set which contains DNase RNFD DNA digestion buffer RDD and DNase resuspension buffer DRB is shipped at ambient temperature Store all components of the RNase Free DNase Set immediately upon receipt at the temperature indicated on the label Intended Use The PAXgene Blood RNA Kit is for the purification of intracellular RNA from whole blood collected in the PAXgene Blood RNA Tube BRT When the kit is used in conjunction with the PAXgene Blood RNA Tube BRT the system provides purified intracellular RNA from whole blood for RT PCR use
45. ood samples from 48 different donors were collected in PAXgene Blood RNA Tubes BRT 6 tubes per donor 288 tubes in total The contents of the tubes from 6 donors were pooled and subsequently realiquoted into 36 samples These 36 samples per 6 donor pool were processed by 3 different operators B C Each operator used 3 different lots 1 2 3 of the PAXgene Blood RNA Kit for automated extraction and processed quadruplicate samples from each of the 8 donor pools RNA yields of all individual samples are shown for every operator lot combination At least 95 of samples show no inhibition in RT PCR when using up to 30 of the eluate Using the automated protocol cross contamination between samples is undetectable as measured by quantitative real time RT PCR of sequences of the beta globin and FOS transcripts in RNA negative samples water paired with RNA positive samples human whole blood in the same run RNA purified with the PAXgene Blood RNA System and the automated protocol is highly pure as shown by lack of RT PCR inhibition see above and values between 1 8 and 2 2 Genomic DNA is present at lt 1 w w in 295 of all samples as measured by quantitative realtime PCR of a sequence of the beta actin gene Figures 10 and 11 show the values and relative genomic DNA of a total of 288 samples prepared using the automated protocol with 3 kit lots by 3 operators PAXgene Blood RNA Kit Handbook 04 2008 27
46. oor and switch on the QlAcube with the power switch see Figure 13 A beeper sounds and the startup screen appears The instrument automatically performs initialization tests Installing protocols on the QlAcube An initial protocol installation is required before the first RNA preparation run on the QlAcube can be performed Install both PAXgene Blood RNA Part A and PAXgene Blood RNA Part B protocols Protocols are provided at www giagen com MyQlAcube and need to be downloaded to the USB stick supplied with the QlAcube and transferred to the QlAcube via the USB port The USB port located behind the protective panel see Figure 13 allows connection of the GlAcube to a USB stick supplied with the QlAcube Data files such as log files or report files can also be transferred via the USB port from the QlAcube to the USB stick The USB port is only for use with the USB stick provided by QIAGEN Do not connect other devices to this port 1 Do not remove the USB stick while downloading protocols or transferring data files or during a protocol run PAXgene Blood RNA Kit Handbook 04 2008 31 Front View of the GlAcube GACEN u n H Figure 13 H Touchscreen USB port behind protective panel B Door Power switch E RS232 serial port behind protective panel for use I Waste drawer by GIAGEN Instrument Service Specialists only 32 PAXgene Blood RNA Kit Handbook 04 2008 Loading the QlAcube To sa
47. processing tubes PT included in the PAXgene Blood RNA Kit 38 PAXgene Blood RNA Kit Handbook 04 2008 Protocol Manual Purification of Total RNA from Human Whole Blood Collected into PAXgene Blood RNA Tubes BRT Important points before starting Make sure that the kit box is intact and undamaged and that buffers have not leaked Do not use a kit that has been damaged When using a pipet ensure that it is set to the correct volume and that liquid is carefully and completely aspirated and dispensed To avoid transferring samples to the wrong tube or spin column ensure that all tubes and spin columns are properly labeled using a permanent pen Label the lid and the body of each tube PT MCT For spin columns label the body of its processing tube PT Close each tube or spin column after liquid is transferred to it Spillages of samples and buffers during the procedure may reduce the yield and purity of RNA Unless otherwise indicated all steps of this protocol including centrifugation steps should be carried out at room temperature 15 25 Because of the sensitivity of nucleic acid amplification technologies the following precautions are necessary when handling samples to avoid cross contamination e Carefully pipet the sample into the spin column PRC PSC without moistening the rim of the column e Always change pipet tips between liquid transfers Use aerosol barrier pipet tips e Avoid touching the spin colu
48. r measuring DNase I defined as the amount of DNase that causes an increase in Aygo 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 46 PAXgene Blood RNA Kit Handbook 04 2008 When reconstituting and aliquoting DNase RNFD ensure that you follow the guidelines for handling RNA Appendix A page 52 Install the correct shaker adapter included with the GlAcube use the adapter for 2 ml safe lock tubes marked with a 2 and place the shaker rack on top of the adapter Check the waste drawer and it if necessary Install the protocols if not already done for previous runs Install both PAXgene Blood RNA Part A and PAXgene Blood RNA Part B protocols See Installing protocols on the QlAcube page 31 Procedure 1 Close the QlAcube door and switch on the QlAcube with the power switch see Figure 13 page 32 A beeper sounds and the startup screen appears The instrument automatically performs initialization tests Open the QlAcube door and load the necessary reagents and plasticware into the QlAcube See Loading the GlAcube pages 33 38 To save time loading can be performed during one or both of the following 10 minute centrifugation steps steps 3 and 5 Centrifuge the PAXgene Blood RNA Tube BRT for 10 minutes at 3000 5000 x g using a swing out rotor I Ensure that the blood sample ha
49. ript levels of 18 were determined by realtime duplex RT PCR using 18S rRNA as an internal standard The values for all samples are plotted with means and standard deviations of all samples shown The dashed lines indicate the 3x total precision of the assay 1 93 PAXgene Blood RNA Kit Handbook 04 2008 15 RNA concentration and purification The PAXgene Blood RNA Kit is for the purification of total RNA from 2 5 ml human whole blood collected in a PAXgene Blood RNA Tube BRT The procedure is simple and can be performed using manual or automated procedures see flowchart In both protocols purification begins with a centrifugation step to pellet nucleic acids in the PAXgene Blood RNA Tube BRT The pellet is washed and resuspended followed by manual or automated RNA purification In principle both protocols follow the same protocol steps with the same kit components The Manual PAXgene Blood RNA Procedure Add proteinase Blood and binding buffer BR2 Incubate Transfer to PAXgene Shredder Spin Column PSC x Transfer supernatant of flow through J to microcentrifuge tul Add ethanol Load on PAXgene RNA Spin Column PRC Wash pellet Bind total RNA Wash Digest DNA Resuspend Transfer to microcentrifuge tube MCT Wash 4 m emi ema lt Elute 3 aid 9 cat 9 9 moh exci um mtb Heat to 65 C Ready to use RNA 16 PAXgene Blood RNA
50. s been incubated in PAXgene Blood RNA Tube BRT for a minimum of 2 hours at room temperature 15 25 in order to achieve complete lysis of blood cells 1 The rotor must contain tube adapters for round bottom tubes If other types of tube adapter are used the tubes may break during centrifugation Remove the supernatant by decanting or pipetting Add 4 ml RNase free water RNFW to the pellet and close the tube using a fresh secondary BD Hemogard closure supplied with the kit If the supernatant is decanted take care not to disturb the pellet and dry the rim of the tube with a clean paper towel Vortex until the pellet is visibly dissolved and centrifuge for 10 minutes at 3000 5000 x g using a swing out rotor Remove and discard the entire supernatant Small debris remaining in the supernatant after vortexing but before centrifugation will not affect the procedure 1 Incomplete removal of the supernatant will inhibit lysis and dilute the lysate and therefore affect the conditions for binding RNA to the PAXgene membrane PAXgene Blood RNA Kit Handbook 04 2008 47 a fe 3 a 2 y 5 fo e 8 6 Add 350 pl resuspension buffer BR1 and vortex until the pellet is visibly dis solved 7 Pipet the sample into a 2 ml processing tube PT 1 Use the 2 ml processing tubes PT included in the PAXgene Blood RNA Kit 8 Load the open processing tubes PT containing sample into QlAcube shak
51. s between liquid transfers Use aerosol barrier pipet tips e Avoid touching the spin column PRC PSC membrane with the pipet tip e After vortexing or heating a microcentrifuge tube MCT briefly centrifuge it to remove drops from the inside of the lid Wear gloves throughout the entire procedure In case of contact between gloves and sample change gloves immediately PAXgene Blood RNA Kit Handbook 04 2008 45 Things to do before starting e Blood must be collected in PAXgene Blood RNA Tubes according to the instructions in the PAXgene Blood RNA Tube Product Circular If necessary see Appendix C page 55 for recommendations on handling PAXgene Blood RNA Tubes BRT e Ensure that the PAXgene Blood RNA Tubes BRT are incubated for at least 2 hours at room temperature after blood collection to ensure complete lysis of blood cells Incubation of the PAXgene Blood RNA Tube BRT overnight may increase yields If the PAXgene Blood RNA Tube BRT was stored at 2 8 C or 20 C or 70 C after blood collection first equilibrate it to room temperature and then store it at room temperature for 2 hours before starting the procedure e Read the safety information on page 8 5 o 2 un a El 2 E amp EJ e Read Important Notes pages 31 38 e Read the guidelines on handling RNA Appendix page 52 e Readthe GlAcube User Manual and any additional information supplied with the GlAc
52. ste The RNA stabilizing solution and blood mixture from the PAXgene Blood RNA Tube can be disinfected using 1 volume of commercial bleach solution 5 sodium hypochlorite per 9 volumes of the RNA stabilizing 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 Disposal must be made according to official regulations The following risk and safety phrases apply to components of the PAXgene Blood RNA Kit See the PAXgene Blood RNA Tube Product Circular for safety information about PAXgene Blood RNA Tubes BRT Binding buffer BR2 x Contains guanidine thiocyanate harmful Xn Risk and safety phrases R20 21 22 32 513 26 36 46 Wash buffer 1 BR3 Contains ethanol flammable Risk phrase R10 R10 Flammable R20 21 22 Harmful by inhalation in 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 13 Keep away from food drink and animal feedingstuffs S22 Do not breathe dust S23 Do not breathe spray S24 Avoid contact with skin 26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice 536 W
53. th reagent names and place them into the appropriate position in the microcentrifuge tube slots as indicated in Table 6 Pipet the indicated volume of DNA digestion buffer RDD into a processing tube PT and add the indicated volume of DNase RNFD stock solution Mix by gently pipetting the complete mixture up and down 3 times using a 1000 yl pipet tip Use the 2 ml processing tubes included in the PAXgene Blood RNA Kit D Be sure to only pipet the required volume as indicated in Table 5 Table 5 Volume of reagents required in processing tubes for the microcentrifuge tube slots Volume of reagent required for the indicated number of samples yl Number of Proteinase Elution buffer samples PK DNase incubation mix BR5 2 126 187 23 DNase RNFD 164 RDD 313 3 170 260 33 DNase I RNFD 228 RDD 399 4 213 334 42 DNase I 292 RDD 486 5 256 407 51 DNase I 356 RDD 572 6 299 481 60 DNase I RNFD 421 RDD 658 7 342 554 69 DNase RNFD 485 RDD 745 8 386 628 78 DNase RNFD 549 831 9 429 701 88 DNase RNFD 613 918 10 472 774 97 DNase RNFD 678 1004 12 558 921 115 DNase RNFD 806 RDD 1177 Table 6 Microcentrifuge tube slots Position A B C Content Proteinase PK DNase incubation mix Elution buffer BR5 Vessel Processing tube PT Processing tube PT Processing tube PT Use the 2 ml
54. 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 40 PAXgene Blood RNA Kit Handbook 04 2008 e Current data shows that reconstituted DNase RNFD can be stored at 2 8 C for up to 6 weeks For long term storage of DNase RNFD remove the stock solution from the glass vial divide it into single use aliquots use the 1 5 ml microcentrifuge tubes MCT supplied with the kit there are enough for 5 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 e When reconstituting and aliquoting DNase RNFD ensure that you follow the guidelines for handling RNA Appendix A page 52 0201014 Procedure 1 Centrifuge the PAXgene Blood RNA Tube BRT for 10 minutes at 3000 5000 x g using a swing out rotor D Ensure that the blood sample has been incubated in the PAXgene Blood RNA Tube BRT for a minimum of 2 hours at room temperature 15 25 in order to achieve complete lysis of blood cells I The rotor must contain tube adapters for round bottom tubes If other types of tube adapter are used the tubes may break during centrifugation 2 Remove the supernatant by decanting or pipetting Add 4 ml RNase free water RNFW to the pellet and close the tube using a fresh secondary BD Hemogard
55. the corresponding replicate tubes using the manual protocol Relative transcript levels of EY FOS and IL1B were determined by real time duplex RT PCR using 185 rRNA as an internal standard Possible differences of transcript levels between RNA prepared from paired blood samples using both extraction protocols automated and manual protocol were calculated by the AAC method Individual AAC values for all sample pairs 4 replicates x 8 donor pools x 3 kit lots x 3 operators 288 pairs for each gene are plotted as single dots with means larger dots and standard deviations black bars for all samples shown The dashed lines indicate the 3x total precision of the assays FOS 2 34 1 93 Cj PAXgene Blood RNA Kit Handbook 04 2008 29 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 BRT cat no 762165 e Ethanol 96 100 purity grade p a e Pipets 10 pl 4 ml e Sterile aerosol barrier RNase free pipet tips e Graduated cylinder e Centrifuge capable of attaining 3000 5000 x g and equipped with a swing out rotor and buckets to hold PAXgene Blood RNA Tubes BRT e Vortex mixer e Crushed ice e Permanent pen for labeling For the manual protocol
56. ually check that the lysate is completely transferred to the spin column PSC To prevent damage to columns PSC and tubes PT do not exceed 20 000 x 1 Some samples may flow through the PAXgene Shredder spin column PSC without centrifugation This is due to low viscosity of some samples and should not be taken as an indication of product failure Carefully transfer the entire supernatant of the flow through fraction to a fresh 1 5 ml microcentrifuge tube MCT without disturbing the pellet in the processing tube Add 350 pl ethanol 96 100 purity grade p a Mix by vortexing and centrifuge briefly 1 2 seconds at 500 1000 x to remove drops from the inside of the tube lid D The length of the centrifugation must not exceed 1 2 seconds as this may result 9 9 in pelleting of nucleic acids and reduced yields of total RNA Pipet 700 pl sample into the PAXgene RNA spin column PRC red placed in a 2 ml processing tube PT and centrifuge for 1 minute at 8000 20 000 x g Place the spin column PRC in a new 2 ml processing tube PT and discard the old processing tube PT containing flow through Pipet the remaining sample into the PAXgene RNA spin column PRC and centrifuge for 1 minute at 8000 20 000 x g Place the spin column PRC new 2 ml processing tube PT and discard the old processing tube PT containing flow through D Carefully pipet the sample into the spin column PRC and visually che
57. ube BRT was stored at 2 8 C or 20 C or 70 C after blood collection first equilibrate it to room temperature and then store it at room temperature for 2 hours before starting the procedure 5 o 2 i a 2 e Read the safety information on page 8 e Read the guidelines on handling RNA Appendix page 52 e Ensurethat instruments such as pipets and shaker incubator have been checked and calibrated regularly according to the manufacturer s recommendations shaker incubator is required in steps 5 and 20 Set the temperature of the shaker incubator to 55 e Binding buffer BR2 may form a precipitate upon storage If necessary warm to 37 to dissolve Wash buffer 2 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 e If using the RNase Free DNase Set for the first time prepare DNase stock solution Dissolve the solid DNase RNFD 1500 Kunitz units in 550 pl of the DNase resuspension buffer DRB provided with the set Take care that no DNase RNFD is lost when opening the vial Do not vortex the reconstituted DNase RNFD DNase is especially sensitive to physical denaturation Mixing should only be carried out by gently inverting the tube Kunitz units are the commonly used units for measuring DNase defined as the amount of DNase
58. ube paying careful attention to the safety information e Ensure that instruments such as pipets and QlAcube have been checked and calibrated regularly according to the manufacturer s recommendations e Binding buffer BR2 may form a precipitate upon storage If necessary warm to 37 to dissolve Wash buffer 2 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 e lfusing the RNase Free DNase Set for the first time prepare DNase stock solution Dissolve the solid DNase RNFD 1500 Kunitz units in 550 pl of the DNase resuspension buffer DRB provided with the set Take care that no DNase RNFD is lost when opening the vial Do not vortex the reconstituted DNase RNFD DNase is especially sensitive to physical denaturation Mixing should only be carried out by gently inverting the tube e Current data shows that reconstituted DNase RNFD can be stored at 2 8 C for up to 6 weeks For long term storage of DNase RNFD remove the stock solution from the glass vial divide it into single use aliquots use the 1 5 ml microcentrifuge tubes MCT supplied with the kit there are enough for 5 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 Kunitz units are the commonly used units fo
59. ve time loading can be performed during one or both of the 10 minute centrifugation steps steps 3 and 5 in Protocol Automated Purification of Total RNA from Human Whole Blood Collected into PAXgene Blood RNA Tubes BRT page 47 Reagent bottles Carefully fill 4 GlAcube reagent bottles with the reagents listed in Table 3 fill the bottles up to the indicator level on the reagent bottles Label the bottles and lids clearly with buffer names and place into the appropriate position in the reagent bottle rack see Figures 14 and 15 Before every run on the QlAcube make sure that the reagent bottles are filled up to the indicator levels remaining volumes in the original kit buffer bottles should be used to fill the reagent bottles Position the reagent rack with filled reagent bottles onto the GlAcube worktable as shown Figures 14 and 15 D Be sure to remove lids from the bottles before placing onto the worktable D Buffer volumes provided in the PAXgene Blood RNA Kit 50 are sufficient for a maximum of 7 RNA preparation runs on the GlAcube Multiple runs with few samples should be avoided in order allow sufficient buffer volumes for processing the full 50 samples Table 3 Positions in the reagent bottle rack Position Reagent Binding buffer BR2 96 100 ethanol Wash buffer 1 BR3 Wash buffer 2 BR4 leave empty Oo ab wD leave empty Wash buffer 2 BRA is supplied as a concentrate Be
60. y be used solely in accordance with the PAXgene Blood RNA Kit 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 PAXgene Blood RNA Kit 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 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 includ ing 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 compo nents For updated license terms see www preanalytix com 2005 2008 PreAnalytiX GmbH all rights reserved PreAnalytiX PreAnalytiX GmbH Feldbachstrasse CH 8634 Hombrechtikon Switzerland PreAnalytiX Distributors Pr
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