CMA KRAS·BRAF·PI3K
Contents
1. Q61H 3 100 00 100 00 100 00 100 00 Q61L 2 100 00 100 00 100 00 100 00 V600E 30 100 00 99 47 93 75 100 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 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 4 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 21 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 Rooc2. to be used exclusively with its diagnostic kits e Anadaptor support that should be placed on the CAR tray before the scanning e SAICLART CMA KRAS BRAF PI3K software developed and validated by GENOMICA CAR Clinical Array Reader GEN MICA 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 Saline solution 0 9 NaCl 4 2 Equipment Microcentrifuge UV visible spectrophotometer Thermal cycler Laminar flow chamber for the extraction laboratory Three adjustable micropipettes ranging from 1 20 pL 20 200 uL and 200 1000 uL for the extraction laboratory One adjustable micropipette ranging from 1 20 ul to add the genetic material to the amplification tubes Three adjustable micropipettes ranging from 1 20 pL 20 200 uL and 200 1000 LL for the visualization laboratory Thermoblock Thermomixer compatible with 96 well plates and adjustable shaking at 20 C 25 C and 502C Vortex Vacuum system desiderable 5 RECOMMENDATIONS AND HANDLING PROCEDURES Read carefully before starting the assay in order to avoid contamination 5 1 General recommendations 1 This assay should be performed in two phy
3. 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 C until its analysis It is important to include a negative control in every run to verify that the samples have 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 1 Thaw the necessary number of amplification tubes according to the number of samples and gene s to analyse Do not use temperatures above 259C for thawing 13 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 Ke
4. and shaking at 1 400 rpm Day 2 1 Add 10ul Proteinase K and vortex briefly 2 Incubate overnight at 569C shaking at 1 400 rpm Day 3 1 Centrifuge 1 minute at 13 200 rpm 2 Add 2 ul glycogen at 20 mg ml Optional It s indicated for samples with very little prior extracted material 3 Collect supernatant 4 Homogenize the sample with the pipette tip in case of remaining tissue 5 Move the supernatant to a tube provided by the extraction kit Qiagen specifically for extracting FFPE samples 6 Place 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 12 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 Not exceed 150 ng of amount of DNA and 10 ul of volume add to each PCR amplification tube The following table shows all the possibilities Extracted DNA ng ul 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 If the concentration is below 15ng ul the sample should be re extracted 2
5. 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 and should be stored at 49C 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 42C 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 42C e Adaptor and lid for 8 well strips microtiter plate 3 3 Other components For the capture and subsequent processing of the image a reader unit running a tailor made software and the plate adaptor are required e CAR Clinical Array Reader it allows for the automatic reading and interpretation of up to 12 CS i e up to a maximum of 96 samples It is distributed by GENOMICA
6. cuts of 10 um each e Incase of larger tissue amount regardless of the percentage take 1 cut of 10 um e I nthe event of endoscopic biopsies very small take 10 cuts of 10 um each Place all the obtained cuts in a 1 5 mL tube 11 Select between these two protocols the one that best suits PROTOCOL 1 1 Add 1 ml Xilen to the tube containing the sample and vortex it during 5 sec 2 Incubate at RT during 10 min 3 Centrifuge the tube at 13 200 rpm during 5 min 4 Discard supernatant 5 Add 1ml 96 99 Ethanol and vortex it for 5 sec 6 Centrifuge the tube at 13 200rpm for 2 min 7 Discard supernatant 8 Incubate aprox 15 min at 562C 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 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 2 Heat the tube containing the sample in the thermomixer for 5 min at 752C without shaking 3 Add 190ul of G2 lysis buffer EZ1 DNA Tissue Kit Qiagen to each sample 4 Incubate 5 minutes at 759C shaking at 1400 rpm 5 Set the thermomixer temperature at 569C 6 Add 25 ul Proteinase K previously prepared according user s manual of the above mentioned kit 20 mg ml 7 Incubate overnight at 569C
7. 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 solve this the corresponding tubes must be amplified again before continuing with the process 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 n cases in which the readings of absorbance of the replicate probes 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 How
8. 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 7 Avoid foaming when adding any reagent 8 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 6 SAMPLES The CLART CMA KRAS BRAF PI3K kit has been designed and validated to be used with DNA 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 Byapercentage 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
9. the thermomixer for 1 hour at 509 C shaking at 550 rpm previously this termomixer has to be prewarm at 509C at least for 30 minutes and make sure that the thermomixer reaches 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 1
10. wells prepare 10 mL of diluted wash solution by adding 1 mL of TL solution to 9 mL of distilled water 3 Prewash of the CS Before beginning the assay it is necessary to wash the strips by adding 200 uL 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 4 Hybridization 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 15 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 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
11. 5 pL of high affinity CJ solution Discard the diluted TL Solution without leaving any residues of the solution and add 100 uL of diluted CJ solution to each CS well Incubate for 30 exact minutes in the thermomixer at 202C 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 16 7 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 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 8 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 9 Discard the complete TL solution using a pipette or a vacuum system The array must remain dry for the reading 10 CAR Clinical Array Reader Carry out the steps described in point 7 3 Place the adaptor in the CAR tray and the plate on top Once the tray is closed the readin
12. 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 Amplification 24 KRAS tests Ref CS 0412 24 CS 0412 8 24 BRAF tests Ref CS 0512 24 CS 0512 8 24 PI3K test Ref CS 0612 24 CS 0612 8 CLART CMA KBP Array Detection 24 CMA KBP Array tests Ref CS 0712 24 CS 0712 8 CLART CMA is under protection of patent family corresponding to European Patent Application N9 11382397 5 CLART and CLART Strip are registered Trademarks of GENOMICA For more information please refer to the web site PI3K analysis is for research use only GENOMICA S A U Alcarria 7 28823 Coslada Madrid Spain Tel 34 91 674 89 90 Fax 34 91 674 89 91 Version 3 May 2013 TABLE OF CONTENTS 1 GLOSSARY 2 PROTOCOL DESCRIPTION 3 KITCOMPONENTS 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 8 READING OF THE RESULTS 9 INTERPRETATION OF THE RESULT
13. S 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 Store at room temperature 20 C 8 C Store at 42C to 82C 4 C 18 C Store at 309C to 182C 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 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 of a low density microarray platform CLART Clinical Array Technology The platform is based on a principle that is very simple but a
14. c 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 100 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 19 e Department of Pathology Clinical Research Department Copenhagen University Hospital Hvidore Denmark e Department 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 the CLART CMA KRAS BRAF PI3K In case of discordances between both techniques the result offered by sequencing was considered as valid 20 Mutation N 408 Sensitivity Specificity PPV NPV G12A 25 96 00 99 48 92 31 99 74 G12C 23 86 96 100 00 100 00 99 23 G12D 68 98 53 99 71 98 53 99 71 G12V 52 92 31 100 00 100 00 98 89 G12R 7 100 00 99 75 87 50 100 00 G12S 25 96 00 100 00 100 00 99 74 G13D 39 92 31 100 00 100 00 99 19
15. dine HRP HorseRadish Peroxidase of the HRP produces a precipitate on the area where o dianisidine substrate by the action hybridization occurs Y y Labelled product 43 eroe Biotin e wa nt ON X Y y M rs M Og 35 33 ff H d Conjugate n 20o0Dosmis opoogo 2ooooog n 2OCHD E D Precipitation M T DD of substrate Probes immobilized on the surface 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 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 V600E and V600K are determined For the analysis of the PI3K GENE 2 amplification tubes
16. ep the tubes refrigerated at any time 4 Program the following temperature cycles on the thermocycler 1 cycle 959C 15 min 94 C 15 sec dB ues 629C 60 sec 1 cycle 729C 10 min 49C continuously until tube collection optional 5 Start the program and place the tubes in the thermocycler when the block has exceeded 902C 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 49C if it is to be used immediately or at 209C if it will not be used for several days 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 2 Make sure that before the hybridization begins the thermomixer temperature has been 509C f
17. ever 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 18 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 of copies of recombinant Amount of Point mutation DNA from cell plasmid per PCR lines reaction 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 1ng Analytical specificity Specificity experiments were carried out with 13 recombinant plasmids and cell lines observing that an unspecifi
18. g is performed 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 amplification and visualization which would give rise to a false positive result The internal 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 17 The internal control of amplification 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 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
19. k 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 22
20. or at least 30 minutes 3 Warm up the SH hybridization solution in the thermomixer 4 PREPARE THE WASH SOLUTION BEFORE EACH ASSAY DO NOT REUSE PREVIOUSLY PREPARED SOLUTIONS OR RESIDUES 14 5 Clean the thermocycler with a 10 diluted bleach solution before starting the denaturation programme Place the amplification tubes in the thermocycler during the process that should never exceed 10 min 6 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 7 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 10 diluted bleach solution after every assay Make sure the pump aspirates properly and does not leave traces at the bottom of the well 8 Aspirate the different solutions completely without touching the array VISUALIZATION 1 Denaturation Use the thermocycler to denature the PCR products For this step place the amplification tubes in the thermocycler and incubate at 952C for 8 min Remove the tubes from the 95 C incubation and place them immediately in a container at 42C It is advised not to exceed 10 min time of denaturation 2 Diluted TL solution preparation For each CS strip a total of 8
21. sically 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 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 a
22. t the same time economical and effective It consists in a microarray printed at the bottom of a microtiter plate well CLART Strip CS Figure 1 which simplifies the entire hybridization and visualization process when compared to classic microarray systems 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 their respective specific complementary probes that are immobilised in specific and well known microarray areas Afterwards they are then 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 these products are hybridized with Figure 2 Probes on the array eC tbt L4 gt E a Hybridization Incubation with conjugate Figure 2 Diagram of the visualization method capture their complementary biotin labelled amplified products With the help of biotin they bind to the conjugate in this case streptavi
23. utoclaved 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 5 2 Precautions for the extraction and addition of extracted material to the amplification 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 502C 2 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 3 It is recommended to add all solutions to the wall of the CS well never directly at the bottom 10 4 Itis convenient not to add the SH solution hybridization solution until the denatured products of PCR are ready 5 The array must not remain dry 6 Following incubation with the CJ solution it is very important to wash
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