DNF-491-1000 - Tablazat.hu
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1. la _ Gel Prime Separation Method DNF 491T33 HS Total R 7 Full Conditioning Gel prime to buffer Gel selection Gel 1 8 0 4 Row A Row Voltage Injection Voltage Vacuum Injection Pressure Rinse Tray Marker V Sample Injection Voltage Injection Voltage Pressure Figure 9 DNF 491T33 tRNA method Rev DNF 491 2015APRO9 16 Advanced Analytical Technologies Inc V Full Conditioning Gel Prime V Perform Prerun E Rinse Tray Marker Row A 7 Dips Gel prime to buffer Gel selection Gel 1 Voltage 120 25 kV Time Marker Injecti Voltage Injection Vacuum Injection V Rinse Tray Marker F Sample inject Voltage Injection Vacuum Injection Separation Row A v Voltage 5 00 Pressure 20 5 v Row A 12 00 Voltage 20K Time 120i kw Voltage 70 06 V Full Conditioning Gel Prime V Perform Prerun E Rinse Gel prime to buffer 120 kV Time Voltage Gel selection Gel 1 Tray Marker Row A Dips Marker Injection Voltage Inje2. Use fresh sample and or ladder Make sure the diluent marker is stored at 20 C and keep on ice before use Use a new vial of diluent marker Clean working area and equipment with RNaseZap Always wear gloves when preparing sample ladder Use new sample ladder aliquot and diluent marker Verify sample was within concentration range specified for the High Sensitivity RNA Analysis kit Prepare sample at higher concentration OR Repeat experiment using increased injection time and or injection voltage Verify sample was correctly added and mixed to sample well Prepare a new rinse buffer plate with 240 uL well 0 25XTE buffer Flush array with 0 5 N NaOH solution and repeat experiment See Appendix G Capillary Array Cleaning of the Fragment Analyzer User Manual for details B Sample signal drops abruptly at the end of separation Sample concentration too high and out of range Verify sample was within concentration range specified for the High Sensitivity RNA Analysis kit Rev DNF 491 2015APR09 22 Advanced Analytical Technologies Inc C Missing 25S or 28S ribosomal peak missing 6000 nt fragment in ladder No rinse buffer in Marker plate row A wrong rinse buffer Dirty array inlet Aging array 1 Use a fresh rinse buffer plate with 240 uL well 0 25XTE buffer 2 Flush array with 0 5 N NaOH solution and repeat experiment See Appendix G Capillar
3. b 50 mL for 12 Capillary instruments or 50 mL volumes BD Falcon 352070 available from Fisher 14 432 22 or VWR 21008 940 11 Clean graduated cylinder for measurement of Separation Gel volume and dilution of 5X 930 Inlet Buffer and 5X Conditioning Solution 12 96 well plate centrifuge for spinning down bubbles from sample plates 13 Vortexer Handling IMPORTANT RNA samples and RNA ladders are very sensitive to RNase contamination which can lead to experimental failure To minimize RNase contamination wear gloves when working with RNA samples and reagents and when handling accessories that will come in contact with the RNA sample Use certified RNase free plastics and disposable consumables It is also recommended to work in a separate lab space if possible and decontaminate the pipettes and work surface to avoid cross contamination Safety When working with chemicals always follow high safety guidelines such as wearing a suitable lab coat disposable gloves and protective eyewear For more information about the specific reagents please refer to the appropriate material safety data sheets MSDSs that can be obtained from the product supplier Fragment Analyzer Start Up Instrument Preparation Gel Preparation 1 Store the RNA Separation Gel at 4 C upon arrival 2 The Intercalating Dye is supplied as a 20 000X concentrate in DMSO and should be stored at 20 C NOTE For this assay the Intercalatin
4. 2488 om 1 t t i t I i t i t t t I 00 00 00 00 03 00 00 06 00 00 09 00 00 12 00 00 15 00 00 18 00 00 21 00 00 24 00 00 27 00 00 30 00 00 33 00 00 36 00 00 40 04 Time HH MM SS Figure 16 Rat kidney mRNA sample result using the Fragment Analyzer system with the DNF 491 High Sensitivity RNA Analysis Kit mRNA assay The mRNA Property Summary for each mRNA sample is displayed when in the mRNA analysis mode and reports the rRNA Contamination of ribosomal RNA in the total concentration Figure 17 mRNA Property Summary rRNA Contamination 10 4 Figure 17 mRNA Property Summary mRNA analysis mode 21 Rev DNF 491 2015APRO9 Advanced Analytical Technologies Inc Troubleshooting The following table lists several potential assay specific issues which may be encountered when using the DNF 491 High Sensitivity RNA Analysis Kit and suggested remedies For a full list of instrument specific troubleshooting information refer to the Troubleshooting and Maintenance Guide for the Fragment Analyzer system Issue Cause Corrective Action A Sample and or ladder signal too weak or degraded Sample and or ladder degraded Diluent marker degraded Sample ladder and or diluent marker are contaminated Sample concentration is too low and out of range Sample not added to Diluent Marker solution or not mixed well Rinse buffer is not fresh or a wrong rinse buffer is used Array was contaminated
5. Ensure the fluid line is positioned at the bottom of the conical tube to avoid introducing air bubbles which can cause pressurization errors 7 When adding RNA Separation Gel to the instrument update the solution levels in the Fragment Analyzer instrament control software From the Main Menu select Utilities Solution Levels A menu will be displayed to enter in the updated fluid levels Figure 1 a Solution Levels Check the fluid volumes before proceeding Ensure that the waste is empty and that the gel and conditioning solutions are full Record the solution volumes here Volume mL Solutions Gel 1 50 0 RNA Gell Gel 2 50 0 NaOH Conditioning Solution 50 0 Waste 0 0 Figure 1 Solution Levels menu 8 When switching applications e g between NGS and RNA kits prime the appropriate gel fluid line after loading fresh gel dye mixture From the Main Menu of the Fragment Analyzer instrumental control software select Utilities Prime Select the desired fluid line s Conditioning Gel 1 or Gel 2 and press OK to purge the fluid line with fresh gel Figure 2 6 Rev DNF 491 2015APR09 Advanced Analytical Technologies Inc ad Prime talak Fluid Selected Cycles 1 E Conditioning Neal Gel Fill Rate 300 al s E Gei2 Empty Rate 300 pL s E ok Cancer Figure 2 Prime menu Inlet Buffer Preparation Store the 5X 930 dsDNA Inlet Bu
6. C and keep on ice before use 2 The total RNA input sample MUST be within a total concentration range of 50 pg uL to 5000 pg L for optimal assay results If the concentration of the sample is above this range dilute with RNase free water mRNA Sample Preparation 1 Heat denature the RNA samples at 70 C for 2 min if needed and immediately cool to 4 C and keep on ice before use 2 The mRNA input sample MUST be within a total concentration range of 250 pg uL to 5000 pg uL for optimal assay results If the concentration of the sample is above this range dilute with RNase free water Sample Plate Preparation 1 The total input RNA sample concentration MUST be within a range of 50 pg uL to 5000 pg L total RNA or 250 pg uL to 5000 pg uL mRNA for optimal assay results If the concentration of the sample is above this range pre dilute the sample with RNase free water prior to performing the assay 2 The above RNA sample concentrations assume the sample is in water If salt is present some loss of sensitivity may be observed and slight adjustments may need to be made to the sample injection conditions IMPORTANT Avoid total input RNA sample concentrations above the specified limits Overloading of RNA sample can result in saturation of the CCD detector and poor results The peak heights for RNA smears should lie in an optimal range between 20 2000 RFUs The peak heights for individual RNA fragments in total RNA should lie in an
7. CV 1 Results using RNA Ladder as sample and 33 55cm capillary array Storage Conditions Store at 2 8 C See Sree once Store at Room Temperature DO NOT FREEZE DO NOT FREEZE RNA Separation Gel ee eee Intercalating Dye oo ceases 5X 930 dsDNA Inlet High Sensitivity RNA Buffer Diluent Marker BF 1 Blank Solution 0 25X TE Rinse Buffer NOTE Always thaw RNA Ladder and RNA Diluent Marker on ice and keep them on ice Ensure all other reagents are completely warmed to room temperature prior to use 3 Rev DNF 491 2015APRO09 Advanced Analytical Technologies Inc Additional Materials and Equipment Required Hardware Software and Reagent Available from AATI 1 Hardware Fragment Analyzer 12 capillary or 96 capillary CE system with LED fluorescence detection e 12 Capillary Array Cartridge Fluorescence 22 cm effective 47 cm total length 50 um ID part A2300 1250 2247 OR e 12 Capillary Array Cartridge Fluorescence 33 cm effective 55 cm total length 50 um ID part A2300 1250 3355 OR e 12 Capillary Array Cartridge Fluorescence 55 cm effective 80 cm total length 50 um ID part A2300 1250 5580 OR e 6 Capillary Array Cartridge Fluorescence 33 cm effective 55 cm total length 50 um ID part A2300 9650 3355 OR e 6 Capillary Array Cartridge Fluorescence 55 cm effective 80 cm total length 50 um ID part A2300 9650 5580 Software o Fragment Analyze
8. ID aati Database SysLog sdf l Eile Admin Utilities Help Operation Run Status in Pan falee ialo Sample Tray 1 Capillary Wel Sample ID 32 D SampD1 A OOOOOOOONOOOO D2 SampD2 B OOCOOO0000N0 ND Ios sampD3 ILILILILIL Fi j EET D OOOOOOOOOOOO s is EIOOOOOC0O000000 n FIOOOOOOOO0O0O0O O jE ed iam To start running OOOOCO0000000 pr Sea 11OOOO0000000NO 8 08 SampD8 the queue press 1234567 8 9101112 9 D9 SampD9 Treyname Enter Tray Name Here 10 p10 SampD1o the Play button u pu SampD11 2 D12 SampD12 Load from File Save Tray Save Selected Row Reset Row Reset tray Run Selected Row Add to queue Edit method SEPARATION Method DNF 47 agment 1 6000bp mthds Tray Samp tray 1 A Gel 1 Run Entire Tray Add to queue Edit method SEPARATION Method DNF 473 33 agment 1 6000bp mthds Tr amp tray 1 B Gel 1 ERG ry Capillary Array Conditioning SEPARATION Method DN GS Fragment 1 6000bp mthds Tray Samp tray 1 C Gel 1 Add to queue Edit method SEPARATION Method DNI GS Fragment 1 6000bp mthds Tray Samp tray 1 D Gel 1 i 0 0kKV Sr 00uA 0 0 PSI LED Off Vent Closed K Waste Closed J Stage Buffer H Tray1H Figure 5 Main Screen after selection of samples to the run queue 13 Rev DNF 491 2015APRO9 Advanced Analytical Technologies Inc 15 Once an experime
9. 0 mL Part DNF 265 0500 Intercalating Dye 30 uL x 2 Part DNF 600 U030 5X 930 dsDNA Inlet Buffer 300 mL dilute with sub micron filtered water prior to use Part DNF 355 0300 5X Capillary Conditioning Solution 100 mL dilute with sub micron filtered water prior to use Part DNF 475 0100 High Sensitivity RNA Diluent Marker 4 mL x 6 Part DNF 387 0004 Store at 20 C High Sensitivity RNA Ladder 25 ng uL 15 uL x 2 dilute with RNase free water prior to use Part DNF 386 U015 New Volume 0 25X TE Rinse Buffer 250 mL Part DNF 497 0250 BF 1 Blank Solution 8 mL Part DNF 301 0008 Eppendorf LoBind 0 5 mL tubes package of 50 New Kit Item Applications Total RNA High Sensitivity Assay 50 pg uL 5000 pg L input sample concentration mRNA High Sensitivity Assay 250 pg uL 5000 pg uL input sample concentration 2 Rev DNF 491 2015APRO09 Advanced Analytical Technologies Inc Specifications Specifications Sample Volume Required 2 uL 12 Capillary 11 1 well RNA Ladder Number of Samples per Run 96 Capillary 95 1 well RNA Ladder Total Electrophoresis Run Time 31 min 22 47 Array 40 minutes 33 55 Array 70 minutes 55 80 Array Sizing Accuracy 20 Sizing Precision 20 CV Limit of Detection S N gt 3 50 pg uL 250 pg uL Quantitative Range per smear 50 pg uL 5000 pg uL 500 pg L 5000 pg L Quantification Accuracy 30 Quantification Precision 20
10. A total of 9 peaks should be observed 3 Start with the preloaded Global Configuration and modify the parameters as needed to fit the data during data processing 4 Note that if a pre dilution was performed prior to the experiment the Dilution Factor setting should be changed to accurately reflect the final sample concentration 5 For additional information on processing data refer to the PROS7ze 2 0 User Manual Fragment Analyzer Shut Down Storage Instrument Shut Down Storage The instrument automatically places the capillary array in the Store position against Capillary Storage Solution 12 Capillary Systems Row H of the buffer tray 96 Capillary Systems Sample 3 after each experiment no further action is required If the instrument is to be idle for more than one day turn off power to the system to preserve lamp lifetime Typical Separation Results Total RNA Sample Figure 12 shows the typical result for the High Sensitivity RNA Ladder from a 33 cm effective 55 cm total short capillary array The initial concentration of the ladder is 2 ng uL final concentration of the ladder after mixing with DM is 0 2 ng uL A total of 9 peaks should be observed with the sizes annotated as in Figure 12 The first peak corresponds to the 20 nt lower matker peak LM The size calibration of the High Sensitivity RNA Ladder should be fitted with a point to point curve fitting algorithm in the PROS7ze 2 0 software The High Se
11. NA Concentration ng uL RNA Quality Number 70 Figure 14 RNA Property Summary Total RNA analysis mode mRNA Sample Figure 15 shows the typical result for the High Sensitivity RNA Ladder using the method for mRNA from a 33 cm effective 55 cm total short capillary array The initial concentration of the ladder was 2 ng uUL final concentration of the ladder after mixing with DM was 0 2 ng L A total of 9 peaks should be observed with the sizes annotated as in Figure 15 The first peak was the 20 nt lower marker peak LM The size calibration of the High Sensitivity RNA Ladder should be fitted with a point to point curve fitting algorithm in the PROS7ze 2 0 software 20 Rev DNF 491 2015APRO9 Advanced Analytical Technologies Inc D12 RNA ladder 2 ng uL RFU 2242 z i t i j t I t i i I 00 00 00 00 03 00 00 06 00 00 09 00 00 12 00 00 15 00 00 18 00 00 21 00 00 24 00 00 27 00 00 30 00 00 33 00 00 36 00 00 40 04 Time HH MM SS Figure 15 Representative High Sensitivity RNA ladder result using Fragment Analyzer system with the DNF 491 High Sensitivity RNA Analysis Kit mRNA assay Figure 16 shows the typical results for a rat kidney mRNA sample from a 33 cm effective 55 cm total short capillary array The data was normalized to the lower marker and the size was calibrated to the High Sensitivity RNA Ladder run in parallel to the sample D1 Rat Kidney mRNA 3 8 ng uL RFU
12. New Ladder Preparation Protocol and Reagent Storage Condition Refer to red highlighted ADVANCED ANALYTICAL High Sensitivity RNA Analysis Kit User Guide DNF 491 0500 DNEF 491 1000 For use with the Fragment Analyzer Automated CE System Fragment Analyzer Software Version 1 0 2 PROSize 2 0 Software Version 1 3 Revised April 9 2015 Advanced Analytical Technologies Inc Ph 515 296 6600 2711 South Loop Drive Suite 4150 Fax 515 294 7141 Ames IA 50010 Wwww aati us com Rev DNF 491 2015APRO9 Advanced Analytical Technologies Inc High Sensitivity RNA Analysis Kit 500 Samples Part DNF 491 0500 Kit Components RNA Separation Gel 240 mL Part DNF 265 0240 Intercalating Dye 30 uL Part DNF 600 U030 5X 930 dsDNA Inlet Buffer 125 mL dilute with sub micron filtered water prior to use Part DNF 355 0125 5X Capillary Conditioning Solution 50 mL dilute with sub micron filtered water prior to use Part DNF 475 0050 High Sensitivity RNA Diluent Marker 4 mL x 3 Part DNF 387 0004 Store at 20 C High Sensitivity RNA Ladder 25 ng uL 15 uL dilute with RNase free water prior to use Part DNF 386 U015 New Volume 0 25X TE Rinse Buffer 250 mL Part DNF 497 0250 BF 1 Blank Solution 8 mL Part DNF 301 0008 Eppendorf LoBind 0 5 mL tubes package of 50 New Kit Item High Sensitivity RNA Analysis Kit 1000 Samples Part DNF 491 1000 Kit Components RNA Separation Gel 50
13. Solution Levels A menu will be displayed to enter in the updated fluid levels Figure 1 7 Rev DNF 491 2015APRO9 Advanced Analytical Technologies Inc Instrument Preparation Check the fluid level of the waste bottle and waste tray daily and empty as needed Prepare a fresh 96 DeepWell 1mL Plate filled with 1 0 mL well of 1X 930 dsDNA Inlet Buffer daily 12 Capillary System Row A only 96 Capillary System All Rows Do NOT overfill the wells of the inlet buffer plate 12 Capillary Systems In Row H of the same prepared buffer plate place 1 1 mL well of Capillary Storage Solution AATI GP 440 0100 Row H of the buffer plate is used for the Store location and the array moves to this position at the end of the experimental sequence 96 Capillary Systems In the Sample 3 drawer place a sample plate filled with 100 uL well of Capillary Storage Solution AATI GP 440 0100 Sample 3 is used for the Store location and the array moves to this position at the end of the experimental sequence IMPORTANT Ensure Row H of the buffer tray 12 Capillary Systems or Sample 3 96 Capillary Systems is always filled with Capillary Storage Solution and the capillary array is placed against the Storage Solution when not in use to prevent the capillary tips from drying out and potentially plugging Place the prepared inlet buffer plate into Drawer B top drawer of the Fragment Analyzer Ensure that the plate is l
14. ction Vacuum Injection Row v 5 00 Voltage 20 F Rinse Z Sample Injection Voltage Injection Vacuum Injection V Separation Tray Marker Figure 11 DNF 491T55 tRNA method Rev DNF 491 2015APRO9 17 Advanced Analytical Technologies Inc 4 An Administrator level user has the option to adjust the Gel Selection Prerun settings 5 Rinse settings including Tray Row and Dips Sample Injection settings and the Separation settings For example if the rinse buffer is loaded into a row other than Row A this can be adjusted prior to or while the method is loaded on the experimental queue To apply any adjustments to the method being placed on the experimental queue press the OK button To exit the editor screen without applying any changes press the Cancel button IMPORTANT Any edits made to the experimental method from the Separation Setup or Method Summary screen will only apply to the currently loaded experiment in the queue No changes are made to the original separation method file Processing Experimental Data 1 When processing data the PROSize 2 0 software Version 1 3 and higher will automatically recognize the separation method performed and apply the appropriate matching configuration file from the C PROSize 2 0 Configurations directory a The DNF 491M22 separation method
15. ddition to the default save directory C AATI Data check the Copy results box and select the desired Copy path directory by clicking the button and navigating the desired save directory 8 Any Notes can be entered regarding the experiment they will be saved and displayed in the final PDF report generated by the PROS7ze 2 0 software 9 Once all information has been entered press OK to add the method to the instrument queue press Cancel to abort adding the method 10 Repeat Steps 3 9 for any remaining sample rows to be analyzed 11 On 96 Capillary Systems or in 12 Capillary Systems if the entire 96 well sample tray is to be run using the same experimental method under the Run Entire Tray field press Add to queue A form similar to Figure 4 will be displayed for entering information and adding the run to the instrument queue for the entire 96 well sample tray 12 After a row or tray has been added to the queue the method s will be listed on the main screen under the Method Queue field Figure 5 13 Prior to starting the experiment verify all trays buffer storage rinse waste sample etc have been loaded into their respective drawer locations 14 Press the Play icon to start the sequence loaded into the queue To Pause the queue after the currently running experiment is completed press the button To Clear the run queue of all loaded runs press the button A Fragment Analyzer 2555 User
16. ffer at 4 C upon arrival DO NOT FREEZE Bring the 5X 930 dsDNA Inlet Buffer to room temperature prior to mixing and use In a clean container add 20 mL of the 5X 930 dsDNA Inlet Buffer per 80 mL of deionized sub micron filtered water Agitate to mix The entire bottle can be mixed to 1X concentration and stored at 4 C if desired Capillary Conditioning Solution Preparation i Store the 5X Capillary Conditioning Solution at room temperature upon arrival DO NOT FREEZE In a clean container e g 50 mL or 250 mL conical centrifuge tube add 20 mL of the 5X Capillary Conditioning Solution per 80 mL of deionized sub micron filtered water Agitate to mix The entire bottle can be mixed to 1X concentration and stored at room temperature if desired Once mixed place the 1X Capillary Conditioning Solution onto the instrument and insert the CONDITIONING fluid line Conditioning Solution pump position Ensure the fluid line is positioned at the bottom of the conical tube to avoid introducing air bubbles which can cause pressurization errors The 1X Capillary Conditioning Solution should be added to the system as use demands A typical 12 capillary experiment cycle consumes less than 4 mL a typical 96 capillary experiment consumes less than 35 mL When adding fresh 1X capillary Conditioning Solution to the instrument update the solution levels in the Fragment Analyzer instrument control software From the Main Menu select Utilities
17. g Dye should be used at 2X normal concentration 1 10 000 dilution 3 Bring the RNA Separation Gel and Intercalating Dye to room temperature prior to mixing 4 Mix appropriate volumes of Intercalating Dye and RNA Separation Gel necessary for one day of operation Use the supplied 50 mL conical centrifuge tube to allow a small minimum working volume For larger volumes use a 250 mL conical centrifuge tube and remove the collar of the tube holder in the instrument reagent compartment 5 The volume of RNA Separation Gel required per run varies between 12 Capillary and 96 Capillary Fragment Analyzer systems The volumes required are summarized below 5 Rev DNF 491 2015APRO9 Advanced Analytical Technologies Inc For 12 capillary Fragment Analyzer systems of samples to be analyzed Volume of Intercalating Dye Volume of RNA Separation Gel 11 1 0 uL 10 mL 22 1 5 uL 15 mL 33 2 0 uL 20 mL 44 2 5 uL 25 mL 88 4 5 uL 45 mL A 5 mL minimum volume should be initially added to the tube For 96 capillary Fragment Analyzer systems of samples to be analyzed Volume of Intercalating Dye Volume of RNA Separation Gel 95 4 0 uL 40 mL 190 8 0 uL 80 mL 285 12 0 uL 120 mL 380 16 0 uL 160 mL 475 20 0 uL 200 mL 6 Place the RNA Separation Gel Intercalating Dye mixture onto the instrument and insert into the desired gel fluid line Gel 1 or Gel 2 pump position
18. level operators cannot edit any steps of a queued separation method 2 Administrator level operators can Edit certain steps of the experimental method To open the method editor screen press the Edit link from the Separation Setup screen Figure 4 The method editor screen is displayed showing the steps of the method Figure 6 3 The preloaded optimized steps for the DNF 491M22 Figure 6 DNF 491T22 Figure 7 DNF 491M33 Figure 8 DNF 491T33 Figure 9 DNF 491M55 Figure 10 and DNF 491T55 Figure 11 methods are shown below The general steps of the methods are as follows 1 Full Condition flushing method Automatically enabled Default Gel Selection Gel 1 2 Perform Prerun ENABLED 7 12 kV 30 sec 3 Rinse DISABLED 4 Marker Injection DISABLED 5 Rinse ENABLED Tray Marker Row A Dips 2 This step moves to the Marker tray and rinses the capillary tips twice with 0 25X TE Rinse Buffer 6 Sample Injection ENABLED Voltage Injection 6 12 kV 150 200 sec This step injects the prepared sample plate 7 Separation ENABLED Voltage 7 12 kV 31 70 min This step performs the CE Separation 14 Rev DNF 491 2015APRO09 Advanced Analytical Technologies Inc Full Conditioning C Gel Prime V Perform Prerun 7 Gel prime to buffer Voltage Gel selection Gel 1 7 Tray Marker Row A Voltage Injection Vac
19. ng the plate into the instrument 9 To run the samples in the 12 Capillary System place the plate in one of the three sample plate trays Drawers 4 6 from the top To run the samples in the 96 Capillary System place the plate in one of the two available sample plate trays Drawers 4 5 from the top Load or create the experimental method as described in the following sections 10 Rev DNF 491 2015APRO09 Advanced Analytical Technologies Inc Performing Experiments Running an Experiment 1 To set up an experiment from the Main Menu of the Fragment Analyzer instrument control software select the Operation tab Figure 3 Select the sample tray location to be analyzed 1 2 or 3 by left clicking the Sample Tray dropdown or by clicking the appropriate sample plate tab alternate plate view and choosing the appropriate location 96 Capillary Systems Note that Sample 3 is typically assigned to the Capillary Storage Solution Left click a well of the desired sample plate row with the mouse The selected row will be highlighted in the plate map e g Row A in Figure 3 Enter the sample name if desired into the respective Sample ID cell by left clicking the cell and typing in the name Alternatively sample information can be imported from txt or csv file by selecting the Load from File option A Fragment Analyzer 2555 User ID aati Database SysLog sdf Te ea eo eh eee File Admin Utilities Help Operation Run Stat
20. nsitivity RNA Ladder is run in parallel to the samples for use in calculating the size and concentration of the samples 3144 CIZ RNA ladder 2 ng ul 2269 1 1 t t i j t t I 1 00 00 00 00 03 00 00 06 00 00 09 00 00 12 00 00 15 00 00 18 00 00 21 00 00 24 00 00 27 00 00 30 00 00 33 00 00 36 00 00 40 04 Time HH MM SS Figure 12 Representative High Sensitivity RNA ladder result using Fragment Analyzer system with the DNF 491 High Sensitivity RNA Analysis Kit 19 Rev DNF 491 2015APRO09 Advanced Analytical Technologies Inc Figure 13 shows the typical results for a chicken spleen total RNA sample from a 33 cm effective 55 cm total short capillary array The data was normalized to the lower marker and the size was calibrated to the High Sensitivity RNA Ladder run in parallel to the sample ee C1 Chicken Spleen tRNA 5 4 ng uL 2472 al t t j t t I t i i 00 00 00 00 03 00 00 06 00 00 09 00 00 12 00 00 15 00 00 18 00 00 21 00 00 24 00 00 27 00 00 30 00 00 33 00 00 36 00 00 40 04 Time HH MM SS Figure 13 Chicken spleen total RNA sample result using the Fragment Analyzer system with the DNF 491 High Sensitivity RNA Analysis Kit The RNA Property Summary is displayed for each total RNA sample when in the Total RNA analysis mode This includes the total RNA concentration the 28S 18S ratio Eukaryotic mode and the RNA Quality Number RQN Figure 14 RNA Property Summary R
21. nt has been loaded onto the queue the user can view or edit the method Administrator level only can edit a method by pressing the Method Summary field To remove the method from the queue press the X button to view the stepwise details of the method press the double down arrow icon 16 The user may add a Pause or Prime step into the queue by right clicking the mouse while over the queue and selecting Insert Pause or Insert Prime 17 The order of the experimental queue can be rearranged by dragging down individual entries Purther information regarding the Method Queue operation is provided in the Fragment Analyzer User Manual 18 Once started the instrument will perform all the programmed experiments in the Method Queue uninterrupted unless a pause step is present Note that additional experiments can be programmed and added to the Method Queue at any time while the instrument is running if desired After completion of the last queued experiment the instrument stage will automatically move to the Store location 12 Capillary Systems Row H of the inlet buffer tray containing the Capillary Storage Solution 96 Capillary Systems Sample 3 location Viewing and Editing Experimental Methods 1 A User level operator can View the steps of the experimental method by pressing the View link on the Separation Setup screen or by pressing the Method Summary option once a method has been loaded onto the experimental queue User
22. o the 18 uL of diluent marker swirl the pipette tip while pipetting up down to further mix B After adding 2 uL of sample or ladder to the 18 uL of diluent marker place a plate seal on the sample plate and vortex the sample plate at 3000 rpm for 2 min Any suitable benchtop plate vortexer can be used Ensure that there is no well to well transfer of samples when vortexing The plate should be spun via a centrifuge after vortexing to ensure there are no trapped air bubbles in the wells C After adding 2 uL of sample or ladder to the 18 uL of diluent marker use a separate pipette tip set to a larger 20 uL volume and pipette each well up down to further mix D Use an electronic pipettor capable of mixing a 10 uL volume in the tip after dispensing the 2 uL sample volume Some models enable using the pipette tip for both adding and mixing 7 After mixing sample RNA Ladder and Diluent Marker Solution in each well centrifuge the plate to remove any air bubbles Check the wells of the sample plate to ensure there are no air bubbles trapped in the bottom of the wells The presence of trapped air bubbles can lead to injection failures 8 For best results run the plate as soon as possible If the sample plate will not be used immediately cover the sample plate with RNase free cover film store at 4 C and use within the same day Spin the plate again if any bubbles developed in the sample wells Be sure to remove the cover film before placi
23. oaded with well A1 toward the back left on the tray Place an empty 96 DeepWell 1mL Plate into Drawer W second from top of the Fragment Analyzer This plate serves as the capillary waste tray and should be emptied daily Alternatively the supplied open reservoir waste plate may be used Prepare a fresh sample plate filled with 240 pL well of 0 25X TE Rinse Buffer daily 12 Capillary System Row A only 96 Capillary System All Rows Place the prepared 0 25X TE Rinse Buffer plate into Drawer M third from top of the Fragment Analyzer Ensure that the plate is loaded with well A1 toward the back left on the tray Sample Ladder Preparation General Information 1 The recommended 96 well sample plate for use with the Fragment Analyzer system is a semi skirted PCR plate from Eppendorf 951020303 Please refer to Appendix C Fragment Analyzer Compatible Plates and Tubes in the Fragment Analyzer User Manual for a complete approved sample plate list The system has been designed to operate using these dimensions styles of PCR plates Plates with similar dimensions may be used but note that capillary damage may occur with the use of poor quality PCR plates IMPORTANT Contact AATI if a different vendor or style of PCR plate is to be used in order to verify compatibility The use of PCR plates with different dimensions to the above recommended plate could possibly damage the tips of the capillary array car
24. optimal range between 100 20 000 RFUs 9 Rev DNF 491 2015APRO09 Advanced Analytical Technologies Inc 3 Using a fresh RNase free 96 well sample plate pipette 18 uL of the High Sensitivity RNA Diluent Marker DM Solution to each well in a row that is to contain sample or RNA Ladder Fill any unused wells within the row of the sample plate with 20 uL well of BF 1 Blank Solution 4 Pipette 2 uL of each denatured RNA sample into the respective wells of the sample mix the contents of the well using the pipette by aspiration expulsion in the pipette tip 5 RNA Ladder The RNA Ladder must be run in parallel with the samples for each experiment to ensure the accurate quantification Thaw the denatured 2 ng uL working concentration RNA Ladder on ice Pipette 2 uL of denatured RNA Ladder into the 18 uL of Diluent Marker DM Solution in the designated ladder well a 12 Capillary System Well 12 of each row to be analyzed b 96 Capillary System Well H12 6 Mix the contents of the well using the pipette by aspiration expulsion in the pipette tip or use one of the mixing methods suggested in the following Important Sample Mixing Information When mixing sample with diluent marker solution it is important to mix the contents of the well thoroughly to achieve the most accurate quantification It is highly suggested to perform one of the following methods to ensure complete mixing A When adding 2 uL of sample or ladder t
25. perimental Method file The available methods are sorted by kit number and are linked to the directory containing methods for the currently installed capillary array length e g 22cm 33cm or 55cm Select the following method a Select DNF 491M22 HS mRNA mthds when the 22 cm effective 47 cm total ultra short capillary array is installed for mRNA b Select DNF 491T22 HS Total RNA mthds when the 22 cm effective 47 cm total ultra short capillary array is installed for Total RNA c Select DNF 491M33 HS mRNA mthds when the 33 cm effective 55 cm total short capillary array is installed for mRNA d Select DNF 491T33 HS Total RNA mthds when the 33 cm effective 55 cm total short capillary array is installed for Total RNA e Select DNF 491M55 HS mRNA mthds when the 55 cm effective 80 cm total long capillary array is installed for mRNA f Select DNF 491T55 HS Total RNA mthds when the 55 cm effective 80 cm total long capillary array is installed for Total RNA 5 Select the appropriate Gel line being used for the experiment Gel 1 or Gel 2 using the dropdown 6 The Tray Name can be entered to identify the sample plate The Folder Prefix if entered will amend the folder name normally a time stamp of HH MM SS from the start of the CE run 12 Rev DNF 491 2015APR09 Advanced Analytical Technologies Inc 7 To copy the experimental results to another directory location in a
26. r instrument control software Version 1 0 2 or higher e PROSize 2 0 data analysis software Version 1 3 or higher Reagents e Capillary Storage Solution 100 mL AATI GP 440 0100 Equipment Reagents to Be Supplied by User 10 RNase free water for diluting sample and High Sensitivity RNA Ladder Sub micron filtered DI water system for dilution of 5X 930 Inlet Buffer and 5X Capillary Conditioning Solutions RNaseZap Ambion AM9782 or equivalent product Single channel pipettes for use in 2 uL and 18 uL volumes and 12 channel pipettes for use in 20 uL volume with RNase free pipette tips Additional Eppendorf DNA LoBind tubes 0 5 mL Eppendorf 022431005 as needed Thermal cycler for sample denaturing RNase free 96 well PCR sample plates Please refer to Appendix C Fragment Analyzer Compatible Plates and Tubes in the Fraguent Analyzer User Manual for a complete approved sample plate list Fisherbrand 96 DeepWell 1mL Plate Natural Polypropylene Fisher 12 566 120 Inlet Buffer and Waste plate Reagent Reservoir 50 mL VWR 82026 355 or similar for use in pipetting Inlet Buffer plates sample trays Conical centrifuge tubes for prepared Separation Gel Dye mixture and or 1X Capillary Conditioning Solution a 250 mL for 96 Capillary instruments or larger volumes Corning 430776 available from Fisher 05 538 53 or VWR 21008 771 4 Rev DNF 491 2015APRO9 Advanced Analytical Technologies Inc
27. t contact AATI Technical Support by phone at 515 296 6600 or by email at support aati us com 23 Rev DNF 491 2015APRO9 Advanced Analytical Technologies Inc Notes 24 Rev DNF 491 2015APRO09 Advanced Analytical Technologies Inc
28. tridge 2 Remove the High Sensitivity RNA Diluent Marker from 20 C and keep it on ice before use Vortex the tube briefly to mix the content Spin the tube after mixing to ensure liquid is at the bottom of the tube 8 Rev DNF 491 2015APRO9 Advanced Analytical Technologies Inc High Sensitivity RNA Ladder Preparation IMPORTANT Upon arrival of the ladder it is recommended to divide the ladder into 3 uL aliquots Store aliquots in the provided Eppendorf LoBind 0 5 mL tubes at 70 C or below 1 Thaw a 3 uL 25 ng uL ladder aliquot on ice 2 Spin down the contents and mix by pipetting the solution up and down with a pipette tip set to a 2 uL volume Transfer 2 uL of the 25 ng uL Ladder to a fresh Eppendorf LoBind 0 5 mL tube Heat denature the ladder at 70 C for 2 min immediately cool to 4 C and keep on ice 3 Dilute the ladder solution to a working concentration of 2 ng L by adding 23 uL of RNase free water and mixing well Divide the diluted ladder solution into aliquots with working volume typical for one day use or one sample plate Store aliquots in the provided Eppendorf LoBind 0 5 mL tubes at 70 C or below If more than 2 uL of the 25 ng UL is transferred for heat denaturing be sure to add enough RNase free water to dilute the ladder to the working concentration of 2 ng L Total RNA Sample Preparation 1 Heat denature the total RNA samples at 70 C for 2 min if needed and immediately cool to 4
29. us aad Fen B Manually enter Sample ID data OR load from file option of save information by x Save Tray or Save Selected Row 10 11 12 fg o SampA9 SampA10 SampAl Trayname Enter Tray Name Herel Select Row Save Tray Save Selected Row Reset Row Run Selected Row Add to queue Edit method Run Entire Tray Add to queue Edit method Capillary Array Conditioning After entering data Add to queue Edit method select Add to queue Gi 0 0kKV Sf 00uA 0 0 PSI LED Off Vent Closed amp Waste Closed J Stage Buffer H Tray1H Figure 3 Main Screen showing selection of sample row and entering sample information After sample information for the row or plate has been entered under the Run Selected Row field press Add to queue The Separation Setup form will be displayed enabling the user to select the experimental method and enter additional information Figure 4 11 Rev DNF 491 2015APRO9 Advanced Analytical Technologies Inc amp F E Sepaaion seung FF Method DNF 491M22 HS mRNA mthds 7 Edt Get Gets Tray name Tray 1 Folder Prefix E Copy results Figure 4 Separation Setup form to select experimental Method and enter tray folder information 4 Inthe Separation Setup pop up form left click the dropdown and select the appropriate preloaded ex
30. uum Injection Voltage Pressure Row X 50 20 V Rinse Tray Marker F Sample Injecti Voltage Injection Vacuum Injection Separation Voltage Pressure Voltage 70 v Row A 31 0 2 Min 7 Full Conditioning C Gel Prime V Perform Prerun V Gel prime to buffer Rinse Tray Marker Marker Inject Voltage Injection Vacuum Injection Voltage Gel selection Gel 1 V Rinse Tray Marker V Sample Injection Voltage Injection Vacuum Injection Separation Voltage Pressure Voltage Figure 7 DNF 491T22 tRNA method Rev DNF 491 2015APRO9 15 Advanced Analytical Technologies Inc _ Gel Prime Full Conditioning 7 Gel prime to buffer Separation Method DNF 491M33 HS mRNA mthd Gel selection Gel 1 V Perform Prerun Voltage Injection Voltage Vacuum Injection Pressure V Rinse Tray Marker 7 Sample Injection Votage Injection Voltage Pressure Figure 8 DNF 491M33 mRNA method
31. will be processed using the DNF 491M22 HS mRNA configuration file b The DNF 491T22 separation method will be processed using the DNF 491T22 HS Total RNA configuration file c The DNF 491M33 separation method will be processed using the DNF 491M33 HS mRNA configuration file d The DNF 491T33 separation method will be processed using the DNF 491T33 HS Total RNA configuration file e The DNF 491M55 separation method will be processed using the DNF 491M55 HS mRNA configuration file f The DNF 491T55 separation method will be processed using the DNF 491T55 HS Total RNA configuration file NOTE If the preloaded PROS7ze 2 0 software configuration files shown above are not located in the C PROSize 2 0 Configurations directory contact AATI Technical Support to obtain the files UPDATE April 9 2015 The Quantification setting for the ladder Final Conc ng uL should be set to 0 2 to reflect the higher 2 ng uL working concentration RNA Ladder Refer to Product Bulletin PB 2015 002 for instructions on changing the ladder concentration value in PROSize 2 0 software or contact AATI Technical Support 18 Rev DNF 491 2015APRO09 Advanced Analytical Technologies Inc 2 The data should be normalized to the lower marker set to 20 nt and the size and quantification calibrated to calibrated to the RNA Ladder run in parallel to the samples Figure 12 shows the typical result for the High Sensitivity RNA Ladder
32. y Array Cleaning of the Fragment Analyzer User Manual for details 3 Replace the array with a new array If issue persists contact AATI Technical Support Split RNA peak Sample s salt concentration was too high 1 Take steps to lower the salt content in the sample and repeat experiment Peak too broad signal too low and or migration time too long Capillary array needs to be reconditioned Capillary array vent valve is clogged Flush array with 0 5 N NaOH solution and repeat experiment See Appendix G Capillary Array Cleaning of the Fragment Analyzer User Manual for details Clean vent valve with deionized water See Fragment Analyzer User Manual for details No sample peak or marker peak observed for individual sample Air trapped at the bottom of sample plate well or bubbles present in sample well Check sample plate wells for trapped air bubbles Centrifuge plate Insufficient sample volume 2 Verify proper volume of solution was A minimum of 20 uL is added to sample well required Capillary is plugged 3 Check waste plate for liquid in the capillary well If no liquid is observed follow the steps outlined in Appendix G Capillary Array Cleaning of the Fragment Analyzer User Manual for unclogging a capillary array Technical Support For questions with Fragment Analyzer operation or about the DNF 491 High Sensitivity RNA Analysis Ki
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