TaqMan Assays Shipped at Ambient Temperature Reduce Environmental Impact and Retain Their Quality and Stability

Transcription

1 WHITE PAPER TaqMa s Shippig TaqMa s Shipped at Temperature Reduce Evirometal Impact ad Retai Their Quality ad Stability ABSTRACT I order to miimize the adverse evirometal impact of packagig ad shippig products o dry ice, Life Techologies ivestigated the feasibility of shippig its TaqMa products at ambiet temperature. This report describes stability ad performace testig of three classes of assays (TaqMa Gee Expressio s, TaqMa MicroRNA s, ad TaqMa SNP Geotypig s) after subjectig them to simulated summer ambiet shippig coditios. Fuctioal ad stability testig demostrated that TaqMa products that uderwet simulated summer ambiet shippig coditios performed as well as assays shipped o dry ice. By shippig at ambiet coditios, the eed for expaded polystyree (EPS) coolers ad dry ice is elimiated ad the fuel cosumptio ad greehouse gas emissios from trasportig the product are sigificatly reduced. INTRODUCTION The adverse evirometal impact of shippig refrigerated or froze products is tremedous. The aual carbo footprit to maufacture the EPS ad covert it ito coolers for our TaqMa products is approximately 100 tos (CO 2 -equivalets). It takes early 300 barrels of crude oil equivalets ad 400 MWhr of power aually to make the EPS coolers that are used to ship Life Techologies geomic assay products [1]. Additioally, 75,000 liters of water are cosumed i the maufacture of the EPS coolers [1]. A average of 7 pouds of dry ice is added to each cooler to esure the product is delivered froze to our customers, further icreasig the mass ad dimesios of each package. Factorig i the umber of shipmets ad average distace traveled per package ad the fact that most packages are shipped via air, the aual total carbo footprit for trasportig froze oligoucleotides is i excess of 100 tos (CO 2 -equivalets) [2]. Life Techologies has bee systematically evaluatig ovel ways to miimize the impact of shippig products o dry ice, ad the CO 2 footprit left by these products durig distributio. Oe way to achieve this is to ship products at a temperature cosistet with its demostrated stability. By avoidig the cooler ad refrigerat, products could be shipped i smaller boxes, which improves the carrier s freight desity (less fuel ad emissios per box) ad reduces the amout of packagig materials requirig disposal or recyclig. This eables Life Techologies to elimiate a aual total of early 26,000 kg (70,000 ft 3 ) of EPS from ladfills ad icierators ad replace it with recyclable corrugated paper packagig. By combiig the elimiatio of EPS ad reductio i impact attributed to trasportig the added weight of the dry ice, we reduce the aual total carbo footprit from product delivery by over 200 tos (CO 2 -equivalets) [1,2]. For may years, TaqMa s have bee shipped as preformulated liquid products o dry ice [3]. Extesive accelerated stability studies (4 weeks at 37 C) followed by real-time fuctioal testig showed that TaqMa products are stable for at least 5 years whe stored at 20 C. For this reaso, the products were shipped uder coditios aliged with their log-term stability temperature. Additioal freeze/thaw stability data have show that TaqMa products remai stable for multiple freeze/thaw cycles without compromisig their fuctioal performace. This paper describes the results from fuctioal ad stability studies carried out after TaqMa products were exposed to established summer shipmet profiles. These experimets demostrate that by shippig our TaqMa products uder ambiet coditios, ot oly ca we supply researchers with the same superior quality product they are used to receivig, but we ca reduce our evirometal footprit i the process. This is a wi for our compay (elimiatig the eed for maagig cold chai trasport), a wi for our customers (miimizig packagig waste), ad a wi for our plaet (reducig resource cosumptio ad total carbo footprit). MATERIALS AND METHODS Products Tested. TaqMa s comprise a preformulated set of ulabeled gee-specific oligoucleotide primers ad fluorescet dye labeled mior groove bidig (MGB) probe(s) provided i liquid form i approved Matrix storage tubes. s were selected to represet the widest rage of performace as well as chemical, sequece, ad structural motifs. I this paper a total of 42 differet TaqMa s were subjected to simulated summer ambiet shippig coditios ad subsequetly measured for physical itegrity ad fuctioal performace. These assays were selected from five product lies that fell ito three represetative TaqMa products, based o their formulatios (Table 1).

2 Note: TaqMa Copy Number s, Custom TaqMa Copy Number s, crna s, ad pri-mirna assays were ot icluded i this testig as the tested assay products were cosidered to be similar eough to be represetative of all product lies. TaqMa CNV Referece Gee ad Edogeous kits were ot icluded i this study, but are beig ivestigated to go ambiet i the ear future. Creatig Replicates. To help elimiate maufacturig lot variability whe creatig the replicates, idividual tubes represetig the various assays were take from ivetory stock, pooled, ad aliquoted ito Matrix tubes at the same fill volume as specified for the maufactured product. Six replicate tubes ad six replicate cotrol tubes were prepared for each assay, for a total of 252 test sample tubes ad 252 cotrol sample tubes (matched replicate sets). The cotrol tubes were kept at 20 C for the duratio of the testig. Simulated Shippig Temperature Exposure. To simulate temperatures icurred durig shippig, samples were placed i a cyclig evirometal chamber (temperature ad humidity cotrolled) programmed to reproduce a worst-case 144 hr (6 day) summer temperature profile (sequetially ra two times for a total of 288 hr) (Figure 1), previously established from testig profiles established at Amge [4]. This profile mimics temperature extremes ecoutered from over 2,500 shipmets durig summer moths betwee the latitudes of 59.9 orth ad 37.8 south. Due to a failure i the chamber cotrol system, a additioal 3-day room temperature (~25 C) icubatio was added to the cycle as idicated i the graph (Figure 1) [4]. Samples were exposed to the 144 hr temperature cycle twice to accout for iter-compay shipmets before beig delivered to our customers. Testig of witer ambiet coditios was ot cosidered due to the extesive, positive historical data o freeze/thaw ad low-temperature stability of TaqMa Gee Expressio ad SNP s (data ot show). [5]. To esure ambiet/liquid product will arrive itact, we redesiged the packagig for orders of 16 tubes or less. To simulate the damage-producig pressure, motios, ad forces typically ecoutered i the shippig ad distributio eviromet, the package was tested accordig to the ISTA 3A Test Protocol [5]. Aalytical Testig Volume ad Cocetratio. Volume ad cocetratio for all test sample tubes were measured before ad after the ambiet shippig simulatio. Volume was measured by gravimetric aalysis ad cocetratio was measured spectroscopically at OD 260. Stability/Itegrity. Structural itegrity chages i test samples compared to cotrols was measured by reverse phase HPLC (RP-HPLC). Samples were aalyzed usig a Agilet HPLC1100 System with a quaterary pump system. HPLC colums used were Trasgeomic OligoSep HC ( micro, porous, alkylated polymer (DVB), 4.6 x 50 mm, P/N NUC )). Mobile phases used were 0.1 M TEAA (triethylamie acetate) i water ad 0.1 M TEAA i 75% acetoitrile/25% water. Fuctioal Performace TaqMa Gee Expressio s. Sixty matched test ad cotrol tubes from 10 TaqMa Gee Expressio s (20X formulatio) ad 36 matched test ad cotrol tubes from 6 TaqMa Gee Expressio s (60X formulatio) were fuctioally tested. Reactios were set up followig the stadard product protocol for a 20 μl reactio volume with 4 replicates for each tube. Te aograms of cdna sythesized from Uiversal Huma RNA (Stratagee) was used as the template for all reactios. PCR reactios were coducted with TaqMa Uiversal PCR Master Mix (P/N ) ad ru o a 7900HT Real-Time PCR System usig uiversal cyclig coditios (95 C, 10 mi; 95 C, 15 sec; 60 C, 1 mi for 40 cycles). S 2.3 software was used to geerate the amplificatio plots ad determie C t values (autobaselie; threshold set at 0.2). C t variability ad o-template cotrol (NTC) C t values were calculated usig JMP 8 statistical software. TaqMa MicroRNA s. Thirty-six matched test ad cotrol sample tubes from 6 TaqMa MicroRNA s were processed. The RT step was performed usig a TaqMa MicroRNA Reverse Trascriptio Kit (P/N ). TaqMa Uiversal PCR Master Mix, No UNG was used for qpcr (P/N ). A mixture of 5 g each Huma Brai ad Huma Lug Total RNA (Ambio ) was used as the template i 15 μl RT reactios. The RT reactio products were used i 10 μl PCR reactios at a fial dilutio of 1:15. qpcr reactios were ru, with 4 replicates for each assay tube, o a 7900HT Real-Time PCR System usig uiversal cyclig coditios (95 C, 10 mi; 95 C, 15 sec; 60 C, 1 mi for 40 cycles). S 2.3 software was used to geerate the amplificatio plots ad determie C t values (autobaselie; threshold set at 0.2). C t ad NTC variability was calculated usig JMP 8 statistical software. Hadlig. I additio to the summer ambiet temperature cycle, the products ad packagig were subjected to reduced pressures ad drop ad vibratioal tests as detailed uder Iteratioal Safe Trasit Associatio (ISTA 3A) simulated performace protocols Figure Hr Summer Temperature Profile Used to Simulate Shippig Temperatures. The summer temperature profile was used to mimic average high temperature extremes betwee the latitudes of 59.9 orth ad 37.8 south (profile derived from the Amge protocol as described). A cotroller failure iduced a 3-day icrease i exposure to ormal room temperatures, further icreasig the strigecy of the challege.

3 TaqMa DME ad SNP Geotypig s. Matched test ad cotrol samples were processed for 10 TaqMa Drug Metabolism (DME) Geotypig s (20X formulatio) ad 10 TaqMa SNP Geotypig s (80X formulatio). The SNP assays were diluted to 20X workig stock solutios with TE before use. TaqMa DME ad SNP Geotypig s were performed followig the maufacturer s recommeded protocol. Templates for these tests cosisted of 20 distict geomic DNA samples (Coriell Cell Repositories, html), cotaiig homozygous ad heterozygous targets for all assays tested. The DNAs were ormalized, dispesed oto 384-well plates, ad dried dow. NTC reactios were icluded. Reactio mixes were prepared for 10 μl reactios usig TaqMa Uiversal PCR Master Mix, No UNG. PCR was performed usig the GeeAmp PCR System SNP PCR was ru usig uiversal cyclig coditios while DME PCR was ru usig more PCR cycles ad loger extesio time (50 cycles x 90 secods). Edpoit data were collected usig the Applied Biosystems 7900HT Real-Time PCR System. FAM dye, VIC dye, ad FAM :VIC dye R ratios ad allelic discrimiatio (cluster) plots were geerated usig S 2.3 software. JMP 8 software was used to calculate variability for both R-NTC ad FAM ad VIC R ratios. Statistical Aalysis. The umber of replicate tubes for each assay (test ad cotrol) was predetermied by the sample size calculator of JMP 8 software usig prior fuctioal performace data with α = 0.01 ad power = For each of the selected assays, 12 tubes were combied ad split back ito 12 tubes (6 cotrol ad 6 test samples). Each assay was ru with 6 replicates for cotrol ad 6 replicates for test (12 tubes for each assay) as predetermied by JMP 8 software. For the qpcr ru, each tube was ru with 4 techical replicates, thus each cotrol assay ad test assay had 24 data poits. We used the SAS JMP statistical package to test for sigificat differeces betwee test ad cotrol assays. For TaqMa Gee Expressio ad MicroRNA s, C t variability ad NTC C t values were aalyzed. Variability for both R-NTC ad FAM :VIC R ratios were calculated for TaqMa DME ad SNP Geotypig s. Table 1. TaqMa s Tested. Product 1. TaqMa Gee Expressio s TaqMa Gee Expressio s, ivetoried Custom TaqMa Gee Expressio s, large scale 2. TaqMa MicroRNA s Descriptio 2 oligoucleotide primers 1 MGB probe with FAM dye* 2 oligoucleotide primers 1 MGB probe with FAM dye* TaqMa MicroRNA s 1 oligoucleotide primer 2 oligoucleotide primers 1 MGB probe with FAM dye* 3. TaqMa SNP Geotypig s TaqMa Drug Metabolism Geotypig s TaqMa SNP Geotypig s, large scale * TaqMa mior groove bidig (MGB) probe with FAM dye. Part Number Cocetratio Number of s X X X 20X 2 oligoucleotide primers 1 MGB probe with FAM dye X 10 1 MGB probe with VIC dye 2 oligoucleotide primers 1 MGB probe with FAM dye X 10 1 MGB probe with VIC dye TaqMa MicroRNA s cosist of a reverse trascriptio (RT) primer ad TaqMa. This is the oligoucleotide primer for primig the RT step. Allele-specific TaqMa MGB probes with distict dyes for detectig the alleles for the specific polymorphism of iterest; VIC dye is liked to the 5 ed of the Allele 1 probe, ad FAM dye is liked to the 5 ed of Allele 2 probe. ad cocetratio (data ot show). Stability/Itegrity. RP-HPLC was used to create peak profiles of the oligoucleotide TaqMa compoets (FAM ad VIC dye-labeled MGB probe oligoucleotides ad ulabeled oligoucleotide primers) usig UV/ VIS absorbace detectio. Forty-two matched test ad cotrol tubes, represetig samples from all assays, were aalyzed. A example of the data is show i Figure 2. Test ad cotrol peak profiles were compared. Test Shipped Sample samples were judged as idetical to matched cotrols (o degradatio). For this study, the comparisos of all 42 test samples aalyzed were show to be idetical to their matched cotrols, cofirmig that the simulated summer ambiet shippig protocol did ot affect product itegrity. Fuctioal Performace TaqMa Gee Expressio s. Fuctioal performace for both types of Sample Pael A. Pael B. Figure 2. Simulated Summer Shippig Does Not Affect Oligoucleotide Stability Represetative Data. The effect of simulated summer ambiet shippig o oligoulceotide itegrity was measured by comparig RP-HPLC profiles of paired test ad cotrol samples. The HPLC chromatogram profiles of the testig samples are comparable to the profiles of the cotrol samples. There was o idicatio of probe or primer degradatio i the ambiet-shipped SNP assay C _10 (A) compared to its matched cotrol (B). 6 RESULTS Aalytical Testig Volume ad Cocetratio. Negligible differeces were observed for both volume

4 ID Pael A. (C t ) p Value Pass/Fail Hs _m Pass Pael B. Figure 3. TaqMa Gee Expressio (60X). The effect of simulated summer ambiet shippig o assay fuctioal performace was evaluated by performig real-time qpcr of paired test ad cotrol samples. ID: Hs _m1. (A) Amplificatio plot for cotrol sample stored at 20 C (blue, = 24). (B) Amplificatio plots for ambiet test sample (gree, = 24) overlaid with cotrol sample (blue, = 24). The table below the plot shows the mea differece betwee the average C t of 24 samples (6 cotrol ad test tubes, 4 qpcr replicates of each tube). The box plot ad circle plot show the distributio of the C t values ad a visual display of the results of the variace aalysis (studet s t-test). TaqMa Gee Expressio s, at 20X ad 60X cocetratio, was assessed by fuctioal aalysis of qpcr amplificatio results. A statistical evaluatio of C t variability ad C t value for the NTC test was performed betwee the test ad cotrol assays. All 60 matched test ad cotrol sample tubes from the 10 TaqMa Gee Expressio s ad all 36 matched test ad cotrol sets from the 6 TaqMa Gee Expressio s were tested. Represetative results for matched pairs (test ad cotrol) are show i Figure 3, with results for all assays provided i Appedix 1 ad 2. The data showed that the fuctioal performace as measured by C t value is equivalet betwee ambiet-shipped assays ad cotrol assays for both TaqMa Gee Expressio products tested. All 20X formulated assays showed o statistical differece (p >0.05) (Figure 3). I additio, the mea differece i C t betwee the test ad cotrol was <0.1 for all assays tested. Two of the 60X formulated assays had p <0.05; however, the mea differece betwee the test ad cotrol tubes was <0.15. The NTC testig showed that all assay tubes, cotrol ad test, showed C t >38. TaqMa MicroRNA s. Fuctioal performace for TaqMa MicroRNA s was assessed by qpcr followed by calculatio of C t variability ad C t value for the NTC. These tests also icluded a RT step prior to PCR amplificatio (to test the RT primer icluded i these assay products). Represetative results (matched test ad cotrol pairs) are show i Figure 4, with results for all assays provided i Appedix 3. The data showed that the fuctioal performace is equivalet betwee ambiet-shipped samples ad cotrol samples for MicroRNA s as measured by the C t value. Two assays showed statistical differeces (p <0.05); however, the mea differeces betwee test ad cotrol C t are less tha 0.2. NTC for all tubes tested gave C t >38. Pael A. ID (C t ) Pass/Fail Pael B. Amplificatio plots from test ad cotrol assay tubes for TaqMa Gee Expressio s ad TaqMa MicroRNA s displayed idetical profiles, ad whe overlayed were idistiguishable <.0070 Pass Figure 4. TaqMa MicroRNA. The effect of simulated summer ambiet shippig o assay fuctioal performace was evaluated by performig real-time qpcr followed by RT-PCR of paired test ad cotrol samples. ID: 391. (A) Amplificatio plot for cotrol sample stored at 20 C (blue, = 24). (B) Amplificatio plots for ambiet test sample (gree, = 24) overlaid with cotrol sample (blue, = 24). The table below the plot shows the mea differece betwee the average C t of 24 samples (6 cotrol ad test tubes, 4 qpcr replicates of each tube). The box plot ad circle plot show the distributio of the C t values ad a visual display of the results of the variace aalysis (studet s t-test). TaqMa SNP ad TaqMa DME Geotypig s. Fuctioal performace for TaqMa Geotypig s was assessed by measurig the differece i the ed poit ΔR (R-NTC) value betwee the test

5 versus cotrol sample tubes for FAM - ad VIC -labeled probes averaged across the 20 gdna samples. The FAM :VIC R ratio was also calculated. All 60 matched pairs of test ad cotrol samples for both products were assayed. Represetative results for 4 assays from each (matched test ad cotrol pairs) are show i Figures 5 ad 6, with results for all assays provided i Appedix 4 ad 5. Compariso of ΔR ad FAM :Vic R ratios showed that most assays had o statistical differece betwee the test ad cotrol for these measuremets. Oly a few assays showed a differece (p <0.05) for ΔR; however, the mea differece betwee cotrol ad test for DME assays was less tha 5% ad for SNP Geotypig s was less tha 3%. Cluster plots for the test ad cotrol assays for DME ad SNP assays were plotted together. The profiles were very similar, showig good clusters, ad the test assays were idistiguishable from the cotrol. FAM, VIC, ad FAM :VIC clusters were well separated ad clearly defied for both test ad cotrol samples (Figure 6). I additio, there was o differece i the geotypig calls. CONCLUSIONS The data described i this paper demostrate that ambiet shippig of TaqMa Gee Expressio, TaqMa MicroRNA, TaqMa DME Geotypig, ad TaqMa SNP Geotypig products has o effect o their quality, itegrity, ad fuctioal performace. After elimiatig factors such as maufacturig lot variability by poolig ad re-splittig samples, aalytical testig (icludig measuremets of volume, cocetratio, ad purity) showed that the samples were ot impacted whe shipped uder simulated summer ambiet shippig coditios. For the TaqMa Gee Expressio ad TaqMa MicroRNA products, fuctioal assays compared test ad cotrol amplificatio plots, C t variability, ad NTC C t values. All of the test samples were essetially idetical to the cotrol samples ad fell withi specificatios for these parameters. Likewise, fuctioal data cosistig of cluster plots, R variability, ad FAM :VIC R ratios for the test sample tubes compared to cotrol sample tubes from TaqMa DME ad SNP Geotypig products also fell well withi performace specificatios for these parameters. We will o loger be shippig i EPS (also kow as Styrofoam ) coolers, ad dry ice is o loger eeded. Thus, exteral packagig will be reduced i size by over 92%. For orders comprisig 16 tubes or less we will be usig a recyclable plastic holder; orders 17 ad larger will cotiue to be shipped i Matrix racks. The ambiet packagig, from the corrugate outer box to the plastic ad paper cotaiers, are fully recyclable. The results preseted here validate the chage to ambiet shippig, ad provide the researcher cofidece that whe shipped ambiet, TaqMa s will exhibit o statistically sigificat differece i fuctio or stability compared to dry ice shipped products. I additio to esurig our customers will cotiue to receive the highest quality possible, this study eables us to sigificatly reduce the evirometal impact of trasport of these products. Life Techologies cosumptio of o-reewable raw materials will decrease by over 300 barrel equivalets of oil every year ad reduce our water utilizatio by over 75,000 liters. Our customers will see a reductio of 26,000 kg (76,000 ft 3 ) of EPS waste. Our plaet will see a reductio of CO 2 emissios by over 200 tos every year. Fially, the packagig used to deliver the products, from the corrugate outer box to the plastic ad paper cotaiers, are fully recyclable. Please reuse the cotaiers whe possible ad, whe you caot reuse, please recycle. Figure 5. TaqMa DME (20X). The effect of simulated summer ambiet shippig o assay fuctioal performace was evaluated by performig PCR o paired test ad cotrol samples. ID: C _10. The red, dark blue, ad gree dots i this overlaid cluster plot represet data poits from the cotrol samples for Allele X, Allele Y, ad heterozygote XY geotypes, respectively. The test samples are show as the turquoise dots for correspodig Allele X, Allele Y, ad heterozygote XY geotype clusters. Figure 6. TaqMa SNP (80X). The effect of simulated summer ambiet shippig o assay fuctioal performace was evaluated by performig PCR o paired test ad cotrol samples. ID: C _20. The red, dark blue, ad gree dots i this overlaid cluster plot represet data poits from the cotrol samples for Allele X, Allele Y, ad heterozygote XY geotypes, respectively. The test samples are show as the turquoise dots for correspodig Allele X, Allele Y, ad heterozygote XY geotype clusters.

6 Appedix 1: TaqMa Gee Expressio s (20X). Statistical compariso of the fuctioal performace. TaqMa Gee Expressio (20X) (C t ) Variace Aalysis Hs _m Hs _m Hs _m Hs _m Hs _m

7 Appedix 1: cotiued TaqMa Gee Expressio (20X) (C t ) Variace Aalysis Hs _m Hs _m Hs _m Hs _m Hs _m

8 Appedix 2: TaqMa Gee Expressio s (60X). Statistical compariso of fuctioal performace. TaqMa Gee Expressio (60X) (C t ) Variace Aalysis Hs _m Hs _m Hs _m Hs _s Hs _s

9 Appedix 3: TaqMa MicroRNA s. Statistical compariso of fuctioal performace. TaqMa Micro RNA (C t ) p value Variace Aalysis < < <

10 Appedix 4: TaqMa DME Geotypig s. Statistical compariso of R values. DME FAM R Variace C _ C _ C _ C _ C _ C _ C _

11 Appedix 4: cotiued DME FAM R Variace C _ C _ DME VIC R Variace C _ C _ C _ C _ C _

12 Appedix 4: cotiued DME VIC R Variace C _ C _ C _ C _ C _D DME FAM/VIC R Ratio Variace C _ C _ C _

13 Appedix 4: cotiued DME FAM/VIC R Ratio Variace C _ C _ DME FAM/VIC R Ratio Variace C _ C _ C _ C _ C _D

14 Appedix 5: TaqMa SNP Geotypig s. Statistical compariso of R values. VA SNP FAM R Variace C _ C _ C _ C _ VA SNP FAM R P-Value Variace C _ C _ C _

15 Appedix 5: cotiued VA SNP VIC R Variace C _ C _ C _ C _ C _ VA SNP VIC R Variace C _ C _

16 Appedix 5: cotiued VA SNP VIC R Variace C _ C _ VA SNP FAM/VIC R Ratio Variace C _ C _ C _ C _

17 Appedix 5: cotiued VA SNP FAM/VIC R Ratio Variace C _ C _ C _

18 NOTES

19 NOTES

20 Authors Hsi-Ru Chag, Jay Crespo, Kathy Lee, Kathlee McGall, Susa MacWhorter, Paul Russell, Jeff Schatz, Doa Seid, Criss Walworth, Joe Yu REFERENCES 1. Data produced usig Compass Comparative Packagig Assessmet olie software tool (v. 1.1) ( 2. Referece data derived from U.S. EPA, Climate Leaders, Greehouse Gas Ivetory Protocol Core Module Guidace (Optioal Emissios From Commutig, Busiess Travel ad Product Trasport) 3. Product Stability Study: TaqMa Gee Expressio s. Publicatio umber: 127WP03-01 ( URLRedirect/idex.htm?xDoD=114115) 4. Cowlad R (2007) Developig ISTA Cold Chai Evirometal Stadards. Paper preseted at the Dimesios.07 Coferece, Orlado, Florida ( 5. ISTA ISTA 3A-2008 Test Protocol: For Research Use Oly. Not for use i diagostic procedures. The trademarks metioed herei are the property of Life Techologies Corporatio or their respective owers. TaqMa is a registered trademark of Roche Molecular Systems, Ic. All other trademarks are the sole property of their respective owers Life Techologies Corporatio. All rights reserved. Prited i the USA. Publicatio O Headquarters 850 Licol Cetre Drive Foster City, CA USA Phoe Toll Free Iteratioal Sales For our office locatios please call the divisio headquarters or refer to our Web site at