User manual KRAS·BRAF·PI3K
Contents
1. extracted from colorectal cancer biopsies GENOMICA is not responsible for the results obtained if other types of samples are used 7 WORKING PROTOCOL CLART CMA KRAS BRAF PI3K has been validated using two different protocols for sample pre treatment 7 1 Sample pre treatment Preprocessing FFPE sections formalin fixed paraffin embedded tissue should be placed on a glass slide for the examination by the pathologist Each sample must be processed using a new sterile scalpel The pathologist will perform a study of each cut by staining it with hematoxylin and eosin H amp E The staining will help to define and verify the tumor area being indicated as a percentage of tumor cells Afterwards follow the instructions below e By a percentage of tumoral cells 5096 and a small area take 6 cuts of 10 um each e By a percentage of tumoral cells gt 50 and greater area take 2 4 cuts of 10 um each e Incase of larger tissue amount regardless of the percentage take 1 cut of 10 um e Inthe event of endoscopic biopsies very small take 10 cuts of 10 um each Place all the obtained cuts in a 1 5 mL tube Select between these two protocols the one that best suits PROTOCOL 1 Add 1 ml Xilen to the tube containing the sample and vortex it during 5 sec Incubate at RT during 10 min Centrifuge the tube at 13 200 rpm during 5 min 1 2 3 4 Discard supernatant 5 Add 1ml 96 99 Ethanol and vortex it for 5 sec 6 Centrif2. 20 200 uL and 200 1000 uL for the visualization laboratory e Thermoblock Thermomixer compatible with 96 well plates and adjustable shaking at 20 C 25 C and 502C e Vortex e Vacuum system desirable 5 RECOMMENDATIONS AND HANDLING PROCEDURES Read carefully before starting the assay in order to avoid contamination 5 1 General recommendations 10 1 This assay should be performed in two physically separated areas in order to avoid sample contamination with the previously amplified product Separate working materials should be available in each area pipettes tips tubes grids gloves etc which should never be used outside these areas 1 Pre PCR area DNA extraction and sample preparation are performed in this area Sample manipulation must be carried out within a biosafety cabinet BSC 2 Post PCR area Amplification and visualization of the amplified product are carried out in this area The material of this area should never come into contact with the material of the extraction area Avoid entering the pre PCR area after having worked in the visualization area 2 Always use gloves It is recommended to change gloves quite frequently and it is mandatory to change gloves before start working in each of the aforementioned areas New gloves must always be used when DNA is added to the amplification tubes 3 Clean working areas laboratory cabinets hoods grids pipettes thoroughly with a 10 diluted bleach
3. Red tube Mix 1 BRAF 5 uL Mix 1 PI3K 5 uL Mix 2 PI3K 5 uL Mix it up carefully in order to not touch the array and place the microplate again on the autoclart Press the knob to continue the visualization process 20 17 Once the visualization process is finished the autoclart will beep indicating the end of the run Please carefully remove the microplate and proceed with the reading step on the CAR WARNING Once the visualization phase is finished on the autoclart the arrays should immediately be read on the CAR otherwise false negatives might appear caused by intensity lost 18 CAR CLINICAL ARRAY READER place the plate normally on the tray and the CAR will take and analyse the arrays automatically 8 READING OF THE RESULTS The processing of data obtained from each analysis is carried out automatically The reading and analysis system CAR will provide a report indicating the results 9 INTERPRETATION OF THE RESULTS Each gene has its own extraction control to make sure that there is enough genomic material to carry out the test For the correct interpretation of the results the sample must be processed with all the amplification tubes corresponding to each gene to be analysed and visualised placed in the same array As described in point 7 1 a negative control of extraction must be included to verify that the samples have not suffered contaminations during the processes of extraction amplificati
4. collection optional 5 Start the program and place the tubes in the thermocycler when the block has exceeded 909C This way possible unspecified amplifications due to incubation below hybridization temperature are minimized The duration of the amplification ranges between 90 150 minutes depending on the thermocycler The amplified product must be visualized within a maximum of 5 days to avoid its degradation and kept at 42C 7 4 Visualization of the product amplified in CLART Strip CS Specific recommendations before starting visualization THE PROTOCOL DESCRIBED BELOW SHOULD ALWAYS BE USED IN THE POST PCR AREA DO NOT TAKE THE AMPLIFIED PRODUCT IN THE PRE PCR AREA 1 Turn on the CAR CLINICAL ARRAY READER before starting the whole procedure The self calibration of the equipment takes a few minutes and it is also necessary to introduce the name of the sample in the program before the reading The device must be ready at the moment of reading to avoid unnecessary waiting that would produce an excessive exposure to developer 16 Make sure that before the hybridization begins the thermomixer temperature has been 509C for at least 60 minutes At room temperature the SH solution hybridization solution forms crystals so is need before using pre warm up at 502C until becomes homogeneous and it must be maintained at 502C until it is going to be added PREPARE THE WASH SOLUTION BEFORE EACH ASSAY DO NOT REUSE PREVIOUSLY
5. not been contaminated during the extraction amplification and visualization processes which might lead to a false positive result 7 3 Amplification reaction Amplification specific recommendations e Work in the pre PCR area always using a cabinet and following the recommendations mentioned in section 5 1 e During the process keep tubes separate and refrigerated e Exclusive use of standard thermal cyclers only with speed ramp of cooling heating up to 39C per second The maximum cooling heating block ramp rate is 3 C Do not use 15 fast thermal cyclers In some of those the ramp rate can be adequate according these needs 1 Thaw the necessary number of amplification tubes according to the number of samples and gene s to analyse Thawing the tubes at 42C 2 Centrifuge the amplification tubes for a few seconds so that all liquid can get to the bottom of the tubes in case you don t have microcentrifuge adaptors available for the tubes you can use larger tubes after having cut their cap off 3 Add 5 10 uL see point 7 2 of the extracted DNA to each amplification tube just after having check for DNA concentration and purity and mix several times with the micropipette Keep the tubes refrigerated at any time 4 Program the following temperature cycles on the thermocycler 959C 15 min 949C 15 sec 629C 60 sec 729C 10 min 1 cycle 40 cycles 49C continuously until tube
6. of Pathological Anatomy 12 de Octubre University Hospital Madrid Spain The presence of each one of the mutations that were detected with the kit was analyzed in 408 biopsy extracts For each sample the result was considered true if it there was concordance between the reference technique and CLART CMA KRAS BRAF PI3K In case of discordances between both techniques the result offered by sequencing was considered as valid 23 Mutation N 408 Sensitivity Specificity PPV NPV G12A 96 00 99 48 92 31 99 74 G12C 23 G12D 68 G12V 52 G12R 100 00 99 75 87 50 100 00 V600K 2 100 00 100 00 100 00 100 00 Wild type 132 NA 98 51 NA 97 06 Table 2 Diagnostic sensitivity and specificity of the CLART CMA KRAS BRAF PI3K technique for each mutation PPV Positive predictive value NPV Negative predictive value NOTE Due to the low prevalence there has been no positive samples for the following mutations E542K E545D E545K and H1047R T herefore failed to identify the diagnostic sensitivity of the kit for these mutations In all cases the analytical sensitivity was evaluated by DNA recombinant plasmids Diagnostic specificity The technique has been validated with a number of 132 negative samples giving rise to a specificity close to 10096 for all point mutations Reproducibility and diagnostic repeatability The data obtained are the following Reproducibility 94 4 n 67 samples and repeatability 94 49
7. of a low density microarray platform CLART Clinical Arrays Technology The platform is based on a principle that is very simple but at the same time economical and effective It consists of a microarray printed at the bottom of a microtiter plate well which simplifies the entire hybridization and visualization process when compared to classic microarray systems Figure 1 displays a CLART Strip or CS of 8 wells Figure 1 CLART Strip CS platform in the form of an 8 well strip The CLART CMA KRAS BRAF PI3K detection system is based on the precipitation of an insoluble product in those microarray areas in which hybridization of amplification products with specific probes takes place During PCR amplified products are labelled with biotin After amplification these products are hybridized with their respective specific complementary probes that are immobilised in specific and well known microarray areas Afterwards they are incubated with a streptavidine peroxidase conjugate The conjugate is bound through streptavidine with the biotin present in the amplified products which are bound to their specific probes and the peroxidase activity prompts the appearance of a non soluble product in the presence of the o dianisidine substrate which precipitates on the microarray areas where hybridization occurs Figure 2 Labelled products Probes on the Biotin Hybridization Incubation with conjugate monet hae Pa m
8. 6 n 51 samples The reproducibility and repeatability series have been set starting with the extraction of the biopsy up to visualization in the array of the amplified material 24 11 REFERENCES Molecular Mechanisms of resistance to Cetuximab and Panitumumab in Colorectal Cancer A Bardelli amp S Siena 2010 J Clin Oncol 28 1254 61 Biomarkers Predicting Clinical Outcome of Epidermal Growth Factor Receptor Targeted Therapy in Metastatic Colorectal Cancer S Siena A Sartore Bianchi F Di Nicolantonio J Balfour A Bardelli 2009 J Natl Cancer Inst 101 1308 24 Effects of KRAS BRAF NRAS and PI3KCA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy refractory metastatic colorectal cancer a retrospective consortium analysis W De Roock B Claes D Bernasconi J De Schutter B Biesmans G Fountzilas et al 2010 Lancet Oncol 11 753 62 KRAS BRAF PI3KCA and PTEN mutations implications for targeted therapies in metastatic colorectal cancer W De Roock V De Vriendt N Normanno F Ciardiello S Tejpar 2011 Lancet Oncol 12 594 603 25
9. AL ARRAY READER which allows the reading and automatic interpretation up to 12 CS that means a total amount of 96 samples This platform is manufactured exclusively for GENOMICA kits use only e SAICLART software developed by GENOMICA for image processing e CLART CMA KRAS Software It is specific for CLART CMA KRAS designed and validated by GENOMICA Installed and ready to use Figure 3 CAR CLINICAL ARRAY READER 4 MATERIALS REQUIRED BUT NOT PROVIDED Below you can find a list of all materials required but not provided 4 1 Reagents and materials Distilled water Disposable gloves Filter tips or positive displacement pipettes Crushed ice container 1 5 mL autoclaved Eppendorf tubes 1 5 mL tube grids 0 5 mL 0 2 mL tube holder 4 2 Equipment e autoclart Figure 3 which enables the automatic visualization of up to 12 CS that means a total amount of 96 samples Figure 4 autoclart e Microcentrifuge e UV visible spectrophotometer e Standard thermal cycler cooling heating ramps up to 32C second Maximum cooling heating block ramp rate is 3 C e Laminar flow chamber for the extraction laboratory e Three adjustable micropipettes ranging from 1 20 uL 20 200 uL and 200 1000 uL for the extraction laboratory e One adjustable micropipette ranging from 1 20 pL to add the genetic material to the amplification tubes e Three adjustable micropipettes ranging from 1 20 uL
10. CLART gt GEN MICA CLART CMA KRAS BRAF PI3K DETECTION AND GENETIC IDENTIFICATION OF POINT MUTATIONS IN 3 OF THE GENES BELONGING TO THE EGFR PATHWAY ASSOCIATED TO COLORECTAL CANCER KRAS BRAF AND PI3K FOR IN VITRO DIAGNOSIS CLART CMA KRAS BRAF PI3K CLART CMA is under protection of 2 patent families corresponding to International PCT Patent Applications WO2013092839 and WO2014044828 These 2 patent families comprise national and regional members in different territories Europe USA Canada Brazil Mexico and China CLART CLART Strip CAR SAICLART and AUTOCLART are registered Trademarks of GENOMICA For more information please refer to the web site www genomica com ial GENOMICA S A U Parque Empresarial Alvento Edificio B Calle V a de los Poblados 1 12 planta 28033 Madrid Spain www genomica com Version 8 July 2015 TABLE OF CONTENTS 1 GLOSSARY 2 PROTOCOL DESCRIPTION 3 KIT COMPONENTS AND STORAGE 3 1 Amplification reagents 3 2 Visualization reagents 3 3 Other components 4 MATERIALS REQUIRED BUT NOT PROVIDED 4 1 Reagents and materials 4 2 Equipment 5 RECOMMENDATIONS AND HANDLING PROCEDURES 5 1 General recommendations 5 2 Precautions for visualization 6 SAMPLES 7 WORKING PROTOCOL 7 1 Sample pre treatment 7 2 Material extracted 7 3 Amplification reaction 7 4 Visualization of the amplified product 7 4 1 Manual visualization 7 4 2 autoclart visualiz
11. PREPARED SOLUTIONS OR RESIDUES Clean the thermocycler with a 1096 diluted bleach solution before starting the denaturation programme Place the amplification tubes in the thermocycler during the process that should never exceed 10 min During the visualization it is not necessary to use filtered tips but it is necessary to use a different tip for each well and change it every time a reagent is added even if it is TL It is necessary though to use filtered tips during the addition of amplified products to the CS well In case of using vacuum pumps equipped with 8 tip comb for aspirating solutions discard the combs after each use or decontaminate them with a 1096 diluted bleach solution after every assay Make sure the pump aspirates properly and does not leave traces at the bottom of the well Aspirate the different solutions completely without touching the array 7 4 1 Manual visualization 4 Denaturation Use the thermocycler to denature the PCR products For this step place the amplification tubes in the thermocycler and incubate at 959C for 8 min Remove the tubes from the 952C incubation and place them immediately in a container at 42C It is advised not to exceed 10 min time of denaturation Diluted TL solution preparation For each CS strip a total of 8 wells prepare 10 mL of diluted wash solution by adding 1 mL of TL solution to 9 mL of distilled water Prewash of the CS Before beginning the assay it is necessary to
12. S P d S Conjugate NP e Specific precipitation Development reaction a M Figure 2 Diagram of the visualization method Probes immobilized on the surface capture their complementary biotin labelled amplified products With the help of biotin they bind to the conjugate in this case streptavidine HRP HorseRadish Peroxidase The o dianisidine substrate by the action of the HRP produces a precipitate on the area where hybridization occurs 3 KIT COMPONENTS AND STORAGE The CLART CMA KRAS BRAF PI3K kit contains enough reagents for the analysis of 8 or 24 clinical samples The reagents included in the kit have been grouped into various packages depending on the temperature at which they should be stored When storage recommendations are observed all reagents should remain stable until kit s expiration date 3 1 Amplification reagents They are shipped and should be stored at 209C Ready to use amplification tubes They contain 45 uL of reaction mixture Only thaw on ice the exact number of amplification tubes that will be used and keep the rest at 209C For the analysis of the KRAS GENE 3 amplification tubes are provided Mix 1 White tube Mix 2 Green tube Mix 3 Red tube With these three tubes mutations G12A G12C G12D G12R G12S G12V G13D Q61H and Q61L are determined For the analysis of the BRAF GENE 1 amplification tube is provided Mix 1 Yellow tube With this tube point mutations V6OOE
13. and V60OK are determined For the analysis of the PI3K GENE 2 amplification tubes are provided Mix 1 White tube Mix 2 Blue tube With these tubes point mutations E542K E545D E545K and H1047R will be determined Note The kit package includes a self adhesive and irreversible temperature indicator the appearance of a reddish colour on the visualization window indicates that at a certain moment products have exceeded the storage temperature of 20 C and they should not be used 3 2 Visualization reagents The visualization kit is shipped at 49C This visualization kit should be stored at 49C except Strips and hybridization solution which should be stored at room temperature WARNING Upon arrival the CLART Strip CS as well as the hybridization solution SH must be kept at room temperature e CS strips including all specific probes They are provided in a sealed thermal envelope Store it closed at room temperature protected from direct light and high temperatures e SH Hybridization Solution Store at room temperature e DC Conjugate Diluent Store at 49C e CJ Conjugate Store at 42C Centrifuge once before use e RE Development Solution Store at 42C and protected from light e TL Wash Buffer Store at 49C e Adaptor and lid for 8 well strips microtiter plate 3 3 Other components The following equipment and accessories are required for the capture and subsequent image processing e CAR CLINIC
14. ation 8 READING OF THE RESULTS 9 INTERPRETATION OF THE RESULTS 10 TECHNICAL AND OPERATIONAL SPECIFICATIONS 11 REFERENCES 1 GLOSSARY Attention see instructions for use Expiration date In vitro diagnostic medical device IVD Lot LOT 25 C 20 C Store at room temperature 8 C Store at 49C to 89C 4 C 18 C Store at 302C to 189C 30 C 2 PROTOCOL DESCRIPTION CLART CMA KRAS BRAF PI3K detects the presence of the most prevalent point mutations in the Epidermal Growth Factor Receptor EGFR pathway associated to colorectal cancer The point mutations detected by the kit are the following KRAS G12A G12C G12D G12R G12S G12V G13D Q61H Q61L BRAF V600E V600K PI3K E542K E545D E545K H1047R SAMPLE formalin fixed paraffin embedded colorectal biopsies Detection is carried out by the specific amplification of the mutation in the sample originating a variable fragment for each mutation of between 100 200 base pairs The amplification is performed in a variable number of PCR tubes depending on the gene of interest The detection of point mutations for KRAS requires 3 amplification tubes that are differentiated by colour white green and red The detection of point mutations for BRAF requires 1 yellow amplification tube The detection of point mutations for PI3K requires 2 blue and white amplification tubes The detection of the product amplified by PCR is carried out by means
15. e the specific tube at the BIO ROBOT EZ1 in order to proceed with the extraction Card must be paraffin DNA and the elution volume must be set at 50 ul 14 7 2 Extracted Material The extraction method chosen should be one that ensures the following concentration and purity parameters 1 Total amount of extracted DNA add in each PCR amplification tube must be 150ng An excess or a default of DNA can lead to misdiagnosis Measure of DNA must be ranged in 15 30 ng yl If measure data are over this range you must diluted your sample until obtain these values The following table shows all the possibilities Extracted DNA ng l Volume pl of extracted Total amount ng of DNA DNA add to each PCR tube add to each PCR tube 30 5 ul 150 ng 25 ng ul 6 ul 150 ng 21 5 ng ul 7 ul 150 ng 19 ng ul 8 ul 150 ng 17 ng ul 9 ul 150 ng 15 ng ul 10 ul 150 ng In case of obtain values below 15 ng ul the sample should be re extracted 2 The extracted DNA should have the following purity enough to avoid misdiagnosis The ratio between the absorbance at 260nm and the absorbance at 280 nm should be as close to 2 as possible If the purity is not adequate sample should be re extracted 3 The extracted material should be stored at 4 C when processing it immediately after extraction otherwise keep it at 20 2 C until its analysis It is important to include a negative control in every run to verify that the samples have
16. es 509C correctly previously to start the assay see point 5 3 For the correct interpretation of the results it is mandatory to visualise all the tubes of the same sample in the same well even if they are different genes After this incubation remove the plate from thermomixer and aspirate the SH solution of the CS with a pipette or preferably with a vacuum pump The array must be free from solution residues although it must never remain dry Add the next solution immediately After incubation set the thermomixer at 209C and in motion so it may be used later in step 6 Double Wash Add 200 uL of diluted TL solution to each CS well resuspend 10 to 15 times with the multichannel pipette Discard the diluted TL solution with a pipette or preferably with a multichannel vacuum pump without leaving any residues Repeat the procedure This step must be carried out with different tips for each well in both washes If having arrived at this step the thermomixer has not reached 209C the wells are left with TL solution until the thermomixer reaches the temperature Blocking and conjugate It is recommended to centrifuge the high affinity CJ solution for 10 seconds before use Then prepare the diluted CJ solution as follows for each CS strip mix 1 mL of DC solution and 15 pL of high affinity CJ solution Prepare this solution at least 5 minutes before ending the hybridization step Discard the diluted TL Solution without leaving any residues of
17. es of an array are very different from each other 10 TECHNICAL AND OPERATIONAL SPECIFICATIONS 10 1 Control of known interferences False negatives are one of the drawbacks in the detection by genomic amplification due to either an inadequate quality of the extracted DNA due to insufficient sample quantity DNA degradation inadequate storage or DNA loss during extraction or to the presence of DNA polymerase inhibitors in the samples that are to be processed alcohol salts etc With the CLART CMA KRAS BRAF PI3K kit these false negatives have been eliminated by adding the different internal controls that are indicative of the efficiency of the amplification reaction of the different tubes However to avoid these interferences the indications appearing in the sections 5 6 and 7 of this manual must be followed 10 2 Technical specifications Processing parameters Analytical sensitivity Analytical sensitivity has been determined by the amplification of serial dilutions of recombinant plasmids for each one of the mutations detected by the kit Each one of them has the amplified product inserted including the part that is complementary to the specific detection probes This sensitivity has also been determined by means of the amplification of serial dilutions of commercial cell lines that contain the mutation to be determined The visualisation was done in CS giving rise to the following results Table 1 No
18. nd follow the instructions described on the screen Close the door and press the knob Select Run Program at the main menu Select the assay CMA KRAS test among those listed Select the well of the strip where the run should start A1 or E1 in case the first 4 wells have already been processed Select the number of samples to be processed With the autoclart the user can process from 4 up to 96 samples per run In any case samples must be multiples of four Confirm that the number of samples and the start up well A1 or E1 are correct Place the tips rack full on its position Load the array microplate in the holder Make sure that the catch in fastened in order to clamp the plate down 10 Check that both the tip waste and the liquid waste containers are empty and on its proper position 11 Fill the DI bottle with 250 ml distilled water 12 Add each of the reagents to its specific container The autoclart calculates the specific volumes required according to the amount of samples indicated 19 TL Washing buffer The volume showed in the display indicates the diluted washing buffer required In order to prepare the diluted washing buffer please dilute the TL reagent provided 1 10 into distilled water CJ Conjugate It s recommended to shortly spin the CJ before use The display shows the final volume of diluted CJ to add meaning that each mL indicated on the display should be prepared as follows 1 ml of DC C
19. of copies of recombinant Amount or Point mutation DNA from cell plasmid per PCR i lines reaction 22 G12A G12C G12D G12R G12S G12V G13D Q61H V600E V600K E542K E545D E545K H1047R Table 1 Relationship of the number of copies of recombinant plasmid cell line nanograms necessary to obtain a sensitivity of 100 in the detection of each one of the microorganisms 1000 1 ng Analytical specificity Specificity experiments were carried out with 13 recombinant plasmids and cell lines observing that an unspecific detection of other mutations different to what is sought to be determined is not produced Therefore it is considered that the technique reaches an analytical specificity of 10096 Diagnostic utility parameters In order to determine the diagnostic parameters of the kit a comparative assessment of the CLART CMA KRAS BRAF PI3K kit was carried against the reference technique TheraScreen For this evaluation we collaborated with the following laboratories e Department of Pathological Anatomy Vall de Hebr n University Hospital Barcelona Spain e Department of Pathology Clinical Research Department Copenhagen University Hospital Hvidore Denmark e Department
20. on and visualization which would give rise to a false positive result The control of extraction of genomic DNA is necessary for the confirmation of a true negative result as it informs us of the presence of the patient s DNA in the sample although there has been no amplification of any mutation The internal control of amplification it will allow us to distinguish between cases of PCR reaction inhibition and those in which no DNA was found in the sample There are three possibilities that give rise to a NOT ANALYSED result e Non valid extraction The presence of inhibitors or a mechanic failure in the extraction of the sample does not allow the amplification of mutations and or of the controls of amplification and extraction to solve this problem the entire process must be repeated e Non valid amplification The absence of amplification in one of the tubes and the presence of amplification in other tubes will indicate that a correct extraction has been carried out but that there has been a failure in the amplification of one of the tubes to 21 solve this the corresponding tubes must be amplified again before continuing with the process e Non inclusion of the gene in the analysis When any of the three genes is not included in the analysis it appears in the report as not analyzed There is a possibility that gives rise to an UNCERTAIN result e n cases in which the readings of absorbance of the replicate prob
21. onjugate Diluent and 5 ul CJ reagent Vortex the diluted solution in order to mix it properly up RE Developer Add the RE volume indicated on the display 13 Denaturation Use the thermocycler to denature the amplification tubes Place the amplification tubes in the thermocycler when this has reached 952C and incubate the tubes for 8 min Remove the tubes from the 952C incubation and place them immediately on ice Denature the amplification product before setting the visualization reagents in the autoclart 14 SH Hybridization solution Ready to use Add the specified volume in the container once it has been tempered WARNING It is important to add the HS at this point If the HS is added in a previous step its temperature might decrease affecting to the intensity of the probes and it might lead to false negative results 15 Close the door and press the knob to start the program The device will start priming the system Then it will perform the pre washes of the CS and add the Hybridization Solution Once finished these steps the device will beep as a signal for the user to add the samples on the CS The autoclart will keep beeping until the user opens the door 16 For the adition of the samples on the CSs please carefully remove the plate from autoclart unit and add each of the following volumes of the denatured products from the same sample to the same well Mix 1 KRAS 5 uL Mix 2 KRAS 5 uL Mix 3 KRAS 10 uL
22. solution after every sample batch processing it is mandatory to disinfect all working areas in case of contamination For thermocyclers and thermomixers it is advised to clean them before and after used in these same conditions 4 Always use filter tips and positive displacement pipettes to avoid contamination due to micropipettes Different sets of pipettes should be used in each area 5 Use disposable and autoclaved laboratory material 6 Never mix reagents from two different vials even if they belong to the same lot 7 Close reagent tubes immediately after use in order to avoid contamination 8 Discard the micropipette tip after pipetting 9 GENOMICA is not responsible for the results obtained with the kit if other samples different to the ones indicated are used 11 5 2 Precautions for the extraction and addition of extracted material to the amplification tube 1 Always wear gloves 2 Clean working surfaces of cabinets with a 10 diluted bleach solution 3 Turn on the laminar flow and UV light at least 20 minutes before extraction Turn off the UV light when it is working inside the cabinet 4 The preparation of the samples before extraction must be made inside the cabinet 5 3 Precautions for visualization 1 Before starting the assay is recommended to verify the THERMOMIXER measuring the temperatures which will be used during the assay 20 C 25 C and 509C For it using a thermocouple in direct contact to themomi
23. the solution and add 100 uL of diluted CJ solution to each CS well Incubate for 30 exact minutes in the thermomixer at 209C shaking at 550 rpm After this incubation remove the plate and discard the solution rapidly with a pipette or a multichannel vacuum pump Once finished the incubation set the thermomixer at 259C and in motion so it may be used later in step 8 Triple Wash Add immediately 200 uL of diluted TL solution to each CS well mixing it 10 to 15 times with the multichannel pipette and discard the solution completely with the pipette or 18 8 9 10 vacuum pump Repeat the procedure two more times It is very important to avoid any residues of the CJ solution since they could react with the RE Solution generating an unspecified signal Development with RE solution Remove the diluted TL solution completely and add 100 uL of RE solution to each CS well and incubate for 10 minutes at 25 2 C in the thermomixer without shaking previously make sure that the thermomixer reaches 259C Warning It is very important to use the thermomixer without shaking Discard the complete TL solution using a pipette or a vacuum system The array must remain dry for the reading CAR CLINICAL ARRAY READER Carry out the steps described in point 7 3 place the plate normally on the tray and the CAR will take and analyse the arrays automatically 7 4 2 autoclart s visualization Uu d U M H2 Switch on the autoclart unit a
24. uge the tube at 13 200rpm for 2 min 13 7 8 Discard supernatant Incubate aprox 15 min at 569C in order to allow the remaining supernatant evaporates dried pellet 9 From this step in advance please follow the protocol outlined in the QlAamp DNA FFPE Tissue Kit user s manual Qiagen The elution volume must be set at 50 ul PROTOCOL 2 Day 1 1 oO uU A W Day 2 Day 3 Centrifuge the tube containing the sample for 1 minute at 13 200 rpm in order to get all FFPE cuts at the bottom of the tube Heat the tube containing the sample in the thermomixer for 5 min at 752C without shaking Add 190ul of G2 lysis buffer EZ1 DNA Tissue Kit Qiagen to each sample Incubate 5 minutes at 759C shaking at 1400 rpm Set the thermomixer temperature at 569C Add 25 ul Proteinase K previously prepared according user s manual of the above mentioned kit 20 mg ml Incubate overnight at 569C and shaking at 1 400 rpm Add 10ul Proteinase K and vortex briefly Incubate overnight at 569C shaking at 1 400 rpm 10 Centrifuge 1 minute at 13 200 rpm 11 Add 2 ul glycogen at 20 mg ml Optional It s indicated for samples with very little prior extracted material 12 Collect supernatant 13 Homogenize the sample with the pipette tip in case of remaining tissue 14 Move the supernatant to a tube provided by the extraction kit Qiagen specifically for extracting FFPE samples 15 Plac
25. wash the strips by adding 200 pL of diluted TL solution to each well Mix it with the multichannel pipette 10 to 15 times taking into account that the surface of the array must not be touched It is advised to carry out this wash while the amplified samples are being denatured and maintain the wash solution in the well until samples are going to be added Discard the diluted TL solution with a pipette or preferably with a vacuum pump The array must be free from solution residues although it must never remain dry Add the next solution immediately Hybridization 17 Before using the SH solution it must be heated at 509C until the complete dilution of the salts Once the PCR products have been denatured add 100 uL of SH solution prevent foaming to each CS well Next add the denatured PCR product from each mix to the same array well according to the following volumes Mix 1 KRAS 5 uL Mix 2 KRAS 5 uL Mix 3 KRAS 10 uL Red tube Mix 1 BRAF 5 uL Mix 1 PI3K 5 uL Mix 2 PI3K 5 uL Use one array per sample patient Mix it several times being careful not to touch the bottom of the well It is recommended to load each strip independently and separately from the rest to avoid contaminations Cover the microtiter plate with the plastic lid provided and incubate in the thermomixer for 1 hour at 509 C shaking at 550 rpm previously this termomixer has to be prewarm at 509C at least for 60 minutes and make sure that the thermomixer reach
26. xer plate 2 The amplification product must be denatured only one time Don t use for visualization a PCR product which has been denatured more than one time If you have this necessity you must do aliquots previously to denaturalized step 3 Avoid the pipette tip or the vacuum system touching the bottom of the well since this could damage the probes printed at the well s bottom 4 Itis recommended to add all solutions to the wall of the CS well never directly at the bottom 5 Atroom temperature the SH solution hybridization solution forms crystals so is need before using pre warm up at 509C until becomes homogeneous Not to add the SH solution until the denatured products of PCR are ready therefore SH solution must be maintained at 502C until it is going to be added 6 The array must not remain dry 7 Following incubation with the CJ solution it is very important to wash the microarray thoroughly in order to avoid any residues that could react with the RE solution resulting in a non specific precipitation that could lead to false interpretations of the result 8 Avoid foaming when adding any reagent 9 When visualizing the image in the reader ensure that position markers appear and that there are no bubbles fibers or spots interfering with the reading Otherwise clean the outer face of the well with cellulose paper 12 6 SAMPLES The CLART CMA KRAS BRAF PI3K kit has been designed and validated to be used with DNA
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