DxDirect Gene Expression User Manual ver 1
Contents
1. Therefore 15 0 mL of DirectMix B is sufficient for approximately 1 000 reactions 2 Calculate the volumes of the 1 uM daughter tube L probes and TC probes that should be added to obtain the desired final concentrations A good starting point is an in reaction concentration of 200 pM L probe per gene and 400 pM TC probe per gene 3 Add the calculated volume of each probe into an appropriately sized container 4 Add the appropriate volume of diluent 1X TE 16 For Research Use Only Not for Use in Diagnostic Procedures Contact Information For technical questions regarding the DxDirect Assay and Probe Design contact DxTerity Technical Support Telephone 424 772 DXT9 e mail techsupport dxterity com For ordering or general questions regarding DxDirect or DxCollect products contact DxTerity Customer Support Telephone 424 772 DXT8 e mail customersupport dxterity com Dxlerity 19500 S Rancho Way Suite 116 Rancho Dominguez CA 90220 United States of America Phone 310 537 7857 Email info dxterity com 17 For Research Use Only Not for Use in Diagnostic Procedures2. DirectBeads in Preparation for Addition to Samples Number of reactions N E aa Volume per Volume Total Volume P Reaction uL Required uL to Add uL Dispense into a 1 5 mL microtube Total Volume 8 35 or 8 microtube strip 13 For Research Use Only Not for Use in Diagnostic Procedures DxDirect Assay Worksheet Amplification Setup Pre Dispense of PCR Master Mix in Preparation for Addition to Beads ten De Coton Volume per Volume Calculated Volume 5 Reaction uL Required uL to Add uL Dispense into a 1 5 mL microtube T tal Volume 28335 or 8 microtube strip Preparation of Formamide Size Standard mixture Volume per Volume Calculated Volume Reaction uL Required uL to Add uL Formamide 17 5 17 5XNX1 2 GeneScan 600 LIZ Size O5XNX12 Item Description Standard v2 0 Total Volume 18 18XNX1 2 Di i 1 5 mL mi SPER SE IMC 2m microtube Total Volume 8 35 or 8 microtube strip Reagent Volumes for DxDirect Reactions includes 20 extra volume Volume for Volume for Volume for Volume for Volume for A A DirectMix B C NA PCR Master Mix CE Loading Master Mix Le Reagents 24 48 72 96 DirectReact DirectMix A DirectMix B DirectMix C M270 Beads DirectTaq DirectPrime Formamide LIZ Size Standard 1 reaction reactions reactions 0 5 uL 14 4 uL 28 8 uL 43 2 uL reactions reactions 1728 uL 576 uL 576 uL 576 uL 576 uL 2016 uL 57 6 uL For Research Use On
3. 8 C DirectReact Reaction buffer for 200 reactions 2 mL 2 C to 8 C DirectWash Store between 2 to 25 C Description Storage DirectWash Wash buffer for 200 reactions 2x125 mL 2 C to 25 C 4 For Research Use Only Not for Use in Diagnostic Procedures Equipment and Reagents Required DirectMix A User supplied DirectMix B User supplied l PCR Thermal Cycler with a temperature range from 4 0 C to PCIR TREE EEES NO GS 99 9 C maximum block ramp rate of 3 9 C sec Vortex Mixer Any model Centrifuge for 96 well Plates Any model Adjustable Volume Pipettes single Riy madel and 8 channel Life Technologies DynaMag 96 well Side Skirted Magnetic Plate CAT 120 27 96 well 0 2 mL PCR plates 8 well strip covers or plate tape Lab Consumables p p P p seals and reagent reservoirs Recommended Capillary Electrophoresis Platforms Polymer Applied Biosystems 8 amp 16 capillary POP 6 amp POP 7 ABI 3130 array i Handheld Magnetic Capture Plate ABI 3500 and 8 amp 24 capillary ABI 3730 48 capillary array POP 6 amp POP 7 ABI 3730XL 96 capillary array POP 6 amp POP 7 Technical Support For questions regarding the DxDirect Assay contact DxTerity Technical Support 19500 S Rancho Way Suite 116 Rancho Dominguez CA 90220 Telephone 1 310 537 7857 e mail Techsupport dxterity com 5 For Research Use Only Not for Use in Diagnostic Procedures Introduction The DxDirect assay tech
4. DxDirect Multiplexed Gene Expression Analysis Direct from DxCollect RNA Stabilized Blood Compatible with DxTerity DxCollect Blood RNA Collection Tubes User Manual DxTerity HCTILy 1 For Research Use Only Not for Use in Diagnostic Procedures For Research Use Only Not for Use in Diagnostic Procedures Information in this document is subject to change without notice Limited License Subject to the DxTerity terms and conditions that govern your use of DxTerity products DxTerity grants you a nonexclusive nontransferable nonsublicensable license to use this DxTerity product only in accordance with the manual and written instructions provided by DxTerity with no rights for resale commercial use or reformulation You understand and agree that except as expressly set forth in the DxTerity terms and conditions no right or license to any patent or other intellectual property owned or licensable by DxTerity is conveyed or implied by this DxTerity product In particular no right or license is conveyed or implied to use this DxTerity product in combination with a product not provided licensed or specifically recommended by DxTerity for such use The license provided specifically excludes use for diagnostic applications Trademarks DxTerity DxDirect and DxCollect are registered trademarks of DxTerity Diagnostics Inc Applied Biosystems 3500 3500XL 3730 and 3730XL are registered trademarks of Thermo Fish
5. er Inc All other trademarks are the property of their respective owners Citing DxDirect and DxCollect in Publications When describing a procedure for publication using this product please refer to it as the DxDirect Gene Expression Assay from DxTerity Copyright 2014 DxTerity Diagnostics Inc All rights reserved 2 For Research Use Only Not for Use in Diagnostic Procedures Table of Contents USE LIMIN S sussie EEE A EEE EE E E EE E i 4 DxDirect Reagent System Contents and Storage Conditions sessessesessescoessessesoesoecssecescoesoessessesosssesee 4 Equipment and Reagents Required but not Supplied csccscscscsscscscsccccscsccccccscsceccccscscececcscscesessecs 5 Recommended Capillary Electrophoresis Platforms cccscsccscssscsccscsceccccscsccccccscsceccccccsceseccecscecessecs 5 Technical Support sssssssssessssessssscorscsosesssisosssasssssecnsssssussssissossscssssesssscssssdssssess sonsesesisssissssssssonsssassosss 5 HAE OGUICTION IEEE A E E E A E O A E suns E E E E E E S 6 HOW Dx DIRECE WOT Kocen err E E E E E 6 Detaled Procedure esros eer ossmecesosanmnnosesaucenecanteanecenersuacen scones 9 Lome P o ACO ee EE E stanbocdnien doaseecutaceutensietateconetetccdes 9 2 Hybridization and LigatiON sscississsisserrsicnsserurests tanii asn isens NEEN Nar ONEA NEANS TREET ANEUS E 9 ST p AOC EEE EE EE E TEE E E E E EE E E E ea 10 A O E E E E E 10 DECU eee A E ee E EEE 11 DxDirect Assay Workshee
6. erated from the assay are too low then the S L probe concentration can be increased up to 800 pM In the case of signals that are too high the S L probe concentrations can be lowered to as low as 50 pM If a further decrease in signal is desired an S Probe Attenuator SA Probe can be used for further information see DxDirect Use of Attenuated S Probes to Modulate Signal Magnitude for Specific Genes Application Note available from DxTerity Technical Support The concentrations of matched S and L probes should always be the same In the case of attenuators the concentration of an L probe for any given gene should be equal to the sum of the concentration of the respective S probe plus SA probe In addition it is also possible to modulate all signals in a given sample by changing the CE injection time This results in a reduction of raw signals proportional to the change in injection time For example decreasing the CE injection time from 30 seconds to 15 seconds will result in a 50 decrease in the peak heights of all peaks including the LIZ internal Size standard Quantification of DxDirect Probes Upon receiving your probe sets it is recommended that you confirm the concentration for each oligo by UV absorbance analysis Measure the absorbance value of the probes at 260 nm and calculate the concentration of the stock probe concentration using the extinction coefficient value provided with the oligos 15 For Research Use Only Not
7. etic plate for at least 30 seconds Repeat L 9 Remove all supernatant and remove the plate from magnetic plate p C 10 Add 180 uL DirectWash to each well and re suspend the beads by pipetting 3 times L 11 Prepare the PCR Master Mix 1 1 mix of DirectPrime and DirectTaq L 12 Remove all supernatant from the plate and add 10 uL of PCR Mastermix to each well of the reaction plate cap wells and vortex for 5 to 10 seconds L 13 Centrifuge the reaction plate and perform PCR Thermal Cycler Protocol PCR Hot Start C 2m 3 Step Cycling L 14 Run the final products on CE according to the manufacturer s instructions 8 For Research Use Only Not for Use in Diagnostic Procedures Detailed Procedure 1 Sample Preparation DxDirect has been optimized to work with DxCollect RNA Blood Collection Tubes Blood samples collected in DxCollect RNA Blood Collection Tubes are stable at room temperature for up to 7 days prior to processing If blood samples are to be stored longer than 7 days then the DxCollect Tubes should be stored in a 20 C freezer until processing Prior to using frozen samples place the tubes on the bench top for at least 60 minutes to bring samples to room temperature Once at room temperature vortex the tubes gently and proceed with the procedure 2 Hybridization and Ligation Prior to starting the procedure prepare both DirectMix A and DirectMix B please refer to Appendix A and follow t
8. for Use in Diagnostic Procedures Creation of Daughter Tubes Dilute 100 uL of 10 UM stock probe with 900 ul of diluent to create 1 uM working stocks of all probes For S probes use a diluent of 1X TE 1 mM DTT For TC and L probes use a diluent of 1X TE Formulation Directions for DirectMix A NOTE The probes in DirectMix A are at a concentration 6 67 fold higher than in the final mix Therefore if the desired final in assay concentration of a probe is 100pM the probe should be formulated in DirectMix A at a concentration of 667 pM 1 Determine the volume of DirectMix A required 15uL of DirectMix A is required for a single reaction Therefore 15 0 mL of DirectMix A is sufficient for approximately 1 000 reactions 2 Calculate the volumes of the 1 uM daughter tube S probes and SA probes if required that should be added to obtain the desired final concentrations A good starting point is an in reaction concentration of 200 pM S probe per gene 3 Add the calculated volume of each probe into an appropriately sized container 4 After all probes have been added and before the diluent is added heat activate the S probe mix in a thermal cycler for 2 minutes at 95 C 5 Transfer the S probes back to the container used for formulation and add the appropriate volume of diluent 1X TE 1 mM DTT Formulation Directions for DirectMix B 1 Determine the volume of DirectMix B required 15 uL of DirectMix B is required for a single reaction
9. he directions 1 Dispense the appropriate amount of DirectReact reagent into a labeled 1 5 mL microcentrifuge tube for the total number of tests to be performed 15 uL per reaction 2 Dispense the appropriate amount of DirectMix A into a labeled 1 5 mL microcentrifuge tube for the total number of tests to be performed 15 uL per reaction 3 Mix DirectMix B DirectMix C by combining 15 uL of DirectMix B and 5 uL DirectMix C per reaction of both reagents into a labeled 1 5 mL microcentrifuge tube Alternatively the reagents can be dispensed into 8 well strip microfuge tubes to facilitate pipetting with a mutli channel pipette into the 96 well reaction plate 4 In a96 well PCR plate dispense 15 uL of DirectReact per well into the appropriate number of wells Keep in mind the total number of wells will depend on the number of replicates per sample Per well a Add 50 ul of DxCollect stabilized blood sample into the appropriate wells and mix by pipetting 2 3 times b Add 15 uL DirectMix A and 20 uL DirectMix B C mixture to all reaction wells c Mix the reactions in each well by pipetting up and down 5 times with a multi channel pipette set at 20 uL 5 Seal the 96 well reaction plate and place the plate in a PCR thermal cycler pre programmed to perform one cycle each of the following 3 incubation steps 9 For Research Use Only Not for Use in Diagnostic Procedures Thermal Cycler Protocol Ligation 3 Target Captu
10. ick Guide 15 uL DirectReact 50 uL of the DxCollect Blood Ligation 15 uL of the DirectMix A 20 uL of DirectMix B DirectMix C Run Ligation Program on thermal cycler 5 uL pre mixed DirectBeads Run Incubation Program on thermal cycler Place on magnetic capture plate for 2 minutes then aspirate liquid Capture 180 uL DirectWash and resuspend beads Place on magnetic capture plate for 2 minutes then aspirate DirectWash solution Lr 10 uL of PCR Mastermix Run Amplification Program on thermal cycler Detect 18 uL of CE Master Mix into new CE Plate 2 uL of PCR product Detect PCR products by CE 7 For Research Use Only Not for Use in Diagnostic Procedures DxDirect Assay Checklist L 1 Add 15 uL DirectReact to each well of the reaction plate L 2 Add 50 uL of the DxCollect Blood Sample to each well of the reaction plate MIX WELL LI 3 Add 15 uL of the DirectMix A to each well of the reaction plate LI 4 Add 20 uL of pre mixed DirectMix B DirectMix C to each well of the reaction plate MIX WELL LI 5 Incubate the reaction plate in a PCR Thermal Cycler Thermal Cycler Protocol Ligation L 6 Remove the reaction plate and add 5 uL of pre mixed M270 Beads to each well of the reaction plate MIX WELL L 7 Incubate the reaction plate in a PCR Thermal Cycler Thermal Cycler Protocol Product Capture C Step Temperature Duration L 8 Place the plate on the magn
11. ly Not for Use in Diagnostic Procedures Appendix A General Guidelines for Preparation of DirectMix A and B Before performing the DxDirect assay DirectMix A and B must be prepared beforehand DirectMix A and B contain the probes that are used in the assay and are custom designed for your specific assay For designing DxDirect probes L S and Target Capture TC Probe sets please refer to the separate manual on probe design and the use of the AllelelD software Premiere Biosoft DirectMix A contains S probes in a diluent of 1XTE 1mM DTT DirectMix B contains L probes plus TC probes in a diluent of 1XTE buffer When analyzing DxDirect assays on the ABI CE platforms it is preferable that gene specific amplicons generate peak heights of at least 500 Relative Fluorescent Units RFUs Signals below 500 RFU can result in higher technical variability Typically the upper range of signaling for any given fragment should be a peak height of 10 000 to 15 000 RFU The preferred upper limit of peak heights will depend on whether the gene is a normalizer or is expected to be up or down regulated If signals are over 15 000 RFU there is arisk that the peak height will be off scale over 28 000 RFU As a threshold any signals lower than 100 RFU should be excluded from the analysis as background For initial testing the recommended final in assay concentration is 200 pM for S and L probes and 400 pM for TC probes If gene specific signals gen
12. nology combines Chemical Ligation Probe Amplification CLPA technology with a proprietary blood stabilization buffer DxCollect to enable direct from sample analysis of genomic signatures CLPA uses a robust non enzymatic chemical reagent system to enable the direct quantitative analysis of RNA and DNA in denaturing chemical environments like those experienced in stabilized unpurified blood Sample processing and multiplexed end point PCR is performed in a single reaction tube and the final assay amplicon detection is accomplished using capillary electrophoresis instruments How DxDirect Works DxDirect must be used in conjunction with probes designed by the user according to the DxDirect Probe Design User Manual and probe mixtures optimized according to the Assay Optimization Application Note For every gene in the assay a unique probe set is designed consisting of two DNA oligonucleotides known as the Thiol Group Probe S Probe and the Leaving Group Probe L Probe that bind adjacently to one another and capture probes upstream or downstream from the target site refer to Figure 1 Each S and L Probe pair is designed to contain a target specific hybridization sequence and a unique identification sequence that enables size separation by CE All S and L Probes contain upstream and downstream universal primer sequences to allow for multiplex PCR amplification of all ligation products In the DxDirect method refer to Fig
13. or Use in Diagnostic Procedures NOTE Avoid transferring magnetic beads during pipetting They may interfere with the injection process during CE 4 Visually inspect the bottom of the CE plate to ensure that no bubbles are present at the bottom of any sample well If any bubbles are observed re centrifuge the plate 5 Runsamples by CE according to manufacturer s user manual For the ABI 3500 Genetic Analyzer the following settings are recommended Oven temperature 60 C default Sizing standard GeneScan 600 LIZ Size Standard v2 0 12 For Research Use Only Not for Use in Diagnostic Procedures DxDirect Assay Worksheet Ligation and Target Capture Pre Dispense of DirectReact in Preparation for Addition to Samples Number of reactionsN Naor eeerintion Volume per Volume Total Volume P Reaction uL Required uL to Add uL Dispense into a 1 5 mL microtube Total Volume 8 35 or 8 microtube strip Pre Dispense of DirectMix A in Preparation for Addition to Samples Number of reactions N pee ere Volume per Volume Total Volume P Reaction uL Required uL to Add uL Dispense into a 1 5 mL microtube Total Volume 8 25 or 8 microtube strip Pre Dispense of DirectMix B C in Preparation for Addition to Samples Number of reactions N Reaction uL Required uL to Add uL O omms os o san O wewe oo s f n o me tone a Dispense into a 1 5 mL microtube Total Volume 835 or 8 microtube strip Pre Dispense of
14. re 1 At the end of the 55 C incubation remove the plate from the thermocycler and after carefully opening the sealed plate add 5 uL of the DynaBead Magnetic Beads into each reaction using a multi channel pipette Mix the reactions in each well by pipetting up and down 5 times with a multi channel pipette set at 20 uL Return the reaction plate to the thermal cycler and incubate for an additional 15 minutes at 55 C 2 Atthe end of 15 minutes remove the reaction plate and place onto a magnetic bead capture plate for at least 30 seconds During capture fill a reagent reservoir suitable for multi channel pipetting with DirectWash solution 3 Remove all ligation reaction solution from the sample plate using an 8 channel 200 uL pipette set to 180 uL and discard the solution into a biohazard waste container Avoid pipetting out the beads NOTE It is important that all of the reaction mixture is removed from the reaction plate Carry over of the reaction mixture may result in subsequent assay failures Visually inspect the bottom of the plate to ensure that all liquid is removed from all reaction wells If any liquid is visible in a well place the plate back on the magnetic plate and pipette out the remaining liquid 4 After removing the reaction solution visually verify that the beads are on the side of each reaction well If beads are not observed in the wells this indicates loss of the beads ligation reaction product which will result in lo
15. t Ligation and Target Capture ccccscsssccscsceccccscscsccccccscsceccccscscescecccnces 13 DxDirect Assay Worksheet Amplification Setup cccccscscscsccscsceccccscsceccccscscsccccecscsceccccscscesescecsces 14 ADENO A E bac yueus even rs seascecexececkcacncsesesevesuens 15 Contact IMTONMA TION sisesed arae n Een EE EE aa a EEEa eaae 17 4 For Research Use Only Not for Use in Diagnostic Procedures Use Limitations The DxDirect reagents are intended for Research Use Only DxDirect Reagent System Contents and Storage Conditions The components of the DxDirect Reagent System and their recommended storage conditions are listed below The DxDirect Reagent System is designed for use with magnetic plate washers and is available in 2 sizes 200 reactions and 1 000 reactions Kit components have a shelf life of 6 months from date of receipt Store reagents according to labels on boxes Do not pool reagents or mix and match kit components from different lots Reagent Box 1 Store between 30 C and 15 C Description Storage 1 mL 30 C to 15 C DirectPrime Universal amplification primers for up to 40 targets multiplexed assays DirectTaq PCR Reaction Mix containing enzyme for 200 reactions 1 mL 30 C to 15 C Reagent Box 2 Store between 2 C and 8 C Description Storage DirectMix C Proteinase K for 200 reactions 1 mL 2 C to 8 C DirectBeads Magnetic capture beads for 200 reactions 2 mL 2 C to
16. to re suspend the beads with minimal effort 4 6 Cap or seal all reaction wells in preparation for PCR Re suspend the magnetic beads by vortexing the plate at a medium high setting for 5 s to 15 seconds and briefly centrifuge to pellet the beads Visually inspect the bottom of the plate to ensure that all beads are immersed in the PCR Master Mix If any beads adhere to the sides of the wells exposed to air vortex the plate at medium high setting for 5 seconds and centrifuge the plate at medium speed for 5 seconds Transfer the plate to a PCR thermal cycler with a hot lid pre programmed to the following thermal cycling conditions Step Temperature Duration cycles oo so rc o After thermal cycling is completed remove the reaction plate and briefly centrifuge Transfer the reaction plate to the magnetic capture plate to capture the beads for at least 30 seconds 5 Detection 1 Prepare sufficient quantities of CE master mix by combining 0 5 uL of the Size Standard and 17 5 uL of Formamide solutions per reaction Dispense 18 uL of CE Master Mix solution into the appropriate number of wells for the CE analysis plate Transfer 2 uL of final PCR reaction product from each reaction well of the reaction plate to the CE analysis plate while it is still on the magnetic plate Seal the plate with a plate septum Vortex the CE analysis plate at medium speed for 5 seconds and briefly centrifuge 11 For Research Use Only Not f
17. ure 2 S and L Probes hybridize adjacently to one another leading to a chemical LIGATION reaction triggered by proximity of the Leaving L group to the Thiol S group The ligation products are generated directly in the denatured sample CAPTURED on magnetic beads to remove un reacted probes and then AMPLIFIED by PCR directly from the beads The final amplified product is DETECTED by CE based on final amplicon size The assay is quantitative because the amount of each ligation product made during the DxDirect reaction is proportional to the concentration of its RNA target sequence and the relative ratios of ligation products are maintained during PCR amplification since amplification uses only a single PCR primer Quantification is relative to one or more housekeeping genes in the assay Universal Reverse Primer Spacer Hybridization DABSY Sequence Sequence l T 3 M Leaving L Probe Universal Forward Primer Hybridization Spacer Sequence Sequence i j es D Thiol S Probe Figure 1 Design of S and L Probes Leaving group oF rote _ L Probe gm AAA Capture Capture Probe Prob Ligate asin Streptavidin paramagnetic beads Ii Capture Universal Universal Forward Filo 4 Reverse Primer A Oooo Primer Size Standard A CULPA Product Detect Figure 2 Basic workflow of the DxDirect method 6 For Research Use Only Not for Use in Diagnostic Procedures DxDirect Assay Qu
18. w signals 5 Take the reaction plate off the magnetic plate place the plate on a 96 well plate holder and add 180 uL of DirectWash into all reaction wells using a multi channel pipette Gently re suspend the beads by pipetting the solution up and down two times and return the plate to the magnetic capture plate NOTE Avoid introducing bubbles by gently pipetting the bead mixture during resuspension 6 Repeat for a total of 3 washes At the end of the third wash do NOT remove the wash solution from the beads NOTE Be sure to visually confirm the magnetic capture of beads before discarding the supernatant between washes 4 Amplification 1 Prepare the DirectTag PCR Master Mix by combining 5 uL of DirectTag and 5 uL of DirectPrime for a total volume of 10 uL per reaction 10 For Research Use Only Not for Use in Diagnostic Procedures 2 Return the reaction plate to the magnetic capture plate for at least 30 seconds and remove the final wash solution from the reaction plate Visually inspect the bottom of the plate to ensure that all liquid is removed from all wells If any liquid is visible in a well place the plate back on the magnetic plate and aspirate as much remaining wash liquid as possible Remove the reaction plate from the magnetic capture plate and immediately add 10 uL of the prepared DirectTaq PCR Master Mix into each well NOTE Dispensing the DirectTaq PCR Master Mix directly onto the magnetic beads makes it easier
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