1. Open the ABI 7900 Fast System SDS Software on the desktop of the computer that is connected to the ABI 7900HT system.

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1 Prtcl Sheet ABI 7900HT (fr SDS Sftware 2.1, 2.3 and 2.4) instrument setup instructins fr RT 2 Prfiler PCR Arrays Imprtant pints befre starting Please read the handbk supplied with the RT 2 Prfiler PCR Array, paying careful attentin t the Safety Infrmatin and Imprtant Ntes sectins, befre beginning this prcedure. The RT 2 Prfiler PCR Array is intended fr mlecular bilgy applicatins. This prduct is nt intended fr the diagnsis, preventin, r treatment f a disease. Please make sure the system has been prperly calibrated fr the regin f interest (ROI), backgrund, and pure dye. (Refer t the manufacturer s Installatin and Maintenance manual fr the calibratin prcedures if needed). Please make nte f the installed SDS Sftware versin n yur instrument. Set up a PCR prtcl template file n the ABI 7900HT Fast System SDS sftware as fllws. Prcedure Creatin f PCR prtcl template 1. Open the ABI 7900 Fast System SDS Sftware n the desktp f the cmputer that is cnnected t the ABI 7900HT system. 2. Select File New, the New Dcument dialg bx will appear. Select Assay SDS 2.1: Abslute Quantificatin (Standard Curve) SDS 2.3 : Standard Curve (AQ) SDS 2.4 : Standard Curve (AQ) Select Cntainer SDS 2.1: 384-well Clear Plate SDS 2.3: 96 r 384 wells Clear Plate SDS 2.4: 96 r 384 wells Clear Plate Select Template SDS 2.1: Blank Dcument SDS 2.3: Blank Template SDS 2.4: Blank Template The field fr Barcde is ptinal. Sample & Assay Technlgies

2 Click OK Under the Setup tab n the center-right, select Add Detectr buttn frm bttm. In the Detectr Manager windw, SDS2.1 Select SYBR fr the reprter dye Click Cpy T Plate Dcument buttn Click Dne buttn (See Figure 1). Chse ROX fr the Passive Reference bx. SDS2.3 Click New In the Add Detectr Dialg Bx, Enter: SYBR in the Name Field Select SYBR fr the Reprter Click OK Click nce n the newly added Detectr Click Cpy T Plate Dcument buttn Click Dne buttn Chse ROX fr the Passive Reference bx. SDS2.4 Click New In the Add Detectr Dialg Bx, Enter: SYBR in the Name Field Keep Grup field as Default Keep Descriptin and AIF Assay ID empty Select SYBR fr the Reprter Select Nn Flurescent as Quencher Click the square with clr beside Clr, then select yur preferred clr in Select Detectr Clr/Swatches page (usually Green fr SYBR). Click OK t clse that page. Click OK Click nce n the newly added Detectr Click Cpy T Plate Dcument buttn Click Dne buttn Chse ROX fr the Passive Reference bx. ABI 7900 HT instrument setup instructins fr RT 2 Prfiler PCR Arrays (Oct-12)

3 SYBR ROX Figure 1. Plate setup dye selectin. Select the Instrument tab, then the Thermal Prfile tab. Highlight Stage 1 (50 C fr 2:00 minutes) by right clicking the muse and dragging it acrss Stage 1. Delete Stage 1 by clicking the Delete buttn. Then cntinue t edit the Thermal Prfile as fllws: Enter 95.0 C fr 10:00 minutes fr Stage 1; fr Stage 2, enter 95.0 C fr 0:15 (15 secnds) fllwed by 60.0 C fr 1:00 minute with Repeats 40 (40 cycles). Click the Add Dissciatin Stage buttn; the pre-set dissciatin stage will be added as Stage 3. Fr SDS Sftware 2.1: Uncheck the bx next t 9600 Emulatin. Fr SDS Sftware 2.3/2.4: select Standard mde. Click Add Dissciatin Stage fr dissciatin curve detectin. Put 10 µl fr Sample Vlume. Then select File Save As t save the template file. Save the file as SDS 7900HT Template Dcument (*.sdt) with the filename RT2 Prfiler PCR Array Prtcl 7900HT Template (click Save). Alternatively, dwnlad the PCR prtcl template file (RT 2 Prfiler PCR Array Prtcl Template-ABI7900.sdt) frm SABisciences website ( ABI 7900 HT instrument setup instructins fr RT 2 Prfiler PCR Arrays (Oct-12)

4 Figure 2. Plate setup setting thermal prfile. Perfrming real-time PCR detectin 3. If the thermcycler is ff, press the pwer buttn t switch n the instrument. Wait fr the instrument t bt and display the Pwer status light. Switch n the cmputer cnnected t the thermcycler. 4. Make sure that there are n bubbles in any well, and that the reactin mix is psitined at the bttm f each well. If nt, centrifuge the plate at ~1000 g fr 60 secnds. 5. Open the ABI 7900HT Fast System SDS Sftware 6. Select File New. In the New Dcument Wizard dialg bx, select Brwse t lad the RT2 Prfiler PCR Array Template file. Then click Finish. This will lad the previusly saved setup t the new plate dcument. T set up a sample plate (See Figure 2), click the square buttn in the upper left crner f the full plate layut panel (between the letter A and 1 ) t select all wells. Once selected, the lwer full plate table panel will be highlighted in yellw and the bx next t SYBR n the left panel will be checked. 7. Save the new dcument under a new filename as SDS 7900HT Dcuments (*sds). ABI 7900 HT instrument setup instructins fr RT 2 Prfiler PCR Arrays (Oct-12)

5 Buttn fr selecting all wells SYBR Figure 3. Plate setup selecting sample. 8. Select the Instrument/Real-Time tab. Click Cnnect/Cnnect t Instrument t cnnect the cmputer t the thermal cycler. Click Open t pen the plate tray and place yur plate in the precisin plate hlder with A1 in the tp left crner. Click Clse t lad the plate. Click Start/Start Run t begin the PCR run. Wait 30 secnds t 1 min fr the initial priming, then the run shuld start. The estimated run time will then appear n the screen. After the PCR run 9. When the PCR run is cmplete, a small dialg bx stating The run cmpleted successfully will appear n the screen. Click OK, this will clse the bx. 10. Select Analysis Analysis Settings, the Analysis Settings dialg bx will appear. Select the radi buttns next t Autmatic Baseline and Manual C T (mst f time 0.2 is fine, please refer 5) fr manual selecting threshld), click OK. 11. Click the Result tab. Display Data as Rn vs. Cycle. If yu have nt selected all wells, click the square buttn in the upper left crner f the diagram f the plate (between the letter A and 1 ) t select all wells. The selected wells will be highlighted in yellw in the lwer left panel. 12. Select Analysis Analyze. It will calculate the data and generate the C T (lwer-left panel). 13. Fllw the prcedures belw t select the baseline and threshld manually (see Figures 4 and 5): (Please use the same threshld fr all yur plates in ne study). ABI 7900 HT instrument setup instructins fr RT 2 Prfiler PCR Arrays (Oct-12)

6 T define the Baseline, use the Linear View f the amplificatin plts. Duble click n Y-axis. The windw fr Display Settings will appear. Fr Amplificatin Plt Prperties, select Aut Scale fr bth the Y and X-Axes. Select Linear view fr Y-Axis, then click OK. With the linear plts, determine the cycle number at which the earliest amplificatin can be seen. Using the red sliding bars n X-axis t set the Manual Baseline t start frm cycle number 2 thrugh tw cycle values befre the earliest visible amplificatin. T define the Threshld Value, use the Lg View f the amplificatin plts. Duble click n Y-axis. The windw fr Display Settings will appear. Fr Amplificatin Plt Prperties, select Aut Scale fr bth the Y and X-Axes. Select Lg view fr Y-Axis and click OK. With the lg plts, place the threshld line abve the backgrund signal but within the lwer third f the linear phase f the amplificatin plt. The values fr C T will be displayed in the lwer left panel fr each well. T exprt the result t an Excel spreadsheet, select File Exprt Results Table and save the file as Tab-delimited Text file (*txt). This file frmat can be pened in the Micrsft Excel Prgram. Select all samples Linear view Setting the baseline Figure 4. Setting the baseline. ABI 7900 HT instrument setup instructins fr RT 2 Prfiler PCR Arrays (Oct-12)

7 Select all samples Linear phase Lg view Set the threshld C T values Figure 5. Setting the threshld. The RT 2 Prfiler PCR Array is intended fr mlecular bilgy applicatins. This prduct is nt intended fr the diagnsis, preventin, r treatment f a disease. QIAGEN handbks can be requested frm QIAGEN Technical Service r yur lcal QIAGEN distributr. Selected handbks can be dwnladed frm Material safety data sheets (MSDS) fr any QIAGEN prduct can be dwnladed frm Trademarks: QIAGEN (QIAGEN Grup); ABI 7900 HT (Applied Bisystems, Inc.); SYBR (Life Technlgies Crpratin), Excel (Micrsft Crpratin). Oct QIAGEN, all rights reserved. France The Netherlands Australia Austria 0800/ Belgium Canada China Denmark Finland Germany Hng Kng Ireland Italy Japan Krea (Suth) Luxemburg Nrway Singapre Spain Sweden Switzerland UK USA Sample & Assay Technlgies