AmoyDx TM RET 14 Mutations Detection Kit

Transcription

1 For: ADx-RE0 AmoyDx TM RET Mutations Detection Kit Detection of rearrangement mutations of RET gene Instructions For Use Instructions Version: B. Date of Revision: April 0 Store at -0± o C Website: Tel: sales@amoydx.com Fax: Address: F, Kechuang Building, 89 Haicang Wengjiao Road, Xiamen, 0, China /

2 For: ADx-RE0 Background RET is a proto-oncogene which encodes a transmembrane tyrosine kinase receptor. Rearrangements of the RET gene have been identified in thyroid papillary carcinoma (PTC), also known as RET/PTC. These rearangements result in the formation of chimeric genes having the tyrosine kinase domain of RET fused with the end sequences of different constitutively expressed genes. The AmoyDx RET Mutations Detection Kit is a highly selective and sensitive real-time PCR-based test to detect any of RET rearrangement mutations. AmoyDx s patented technology allows detection of % mutant DNA in a background of 99% normal DNA, while ensuring that false negatives are minimized. The assay uses messenger RNA (mrna) as starting material. RET mrna is reverse transcribed to cdna using reverse transcriptase that is supplied with the kit. The level of RET gene expression can be quantitated using the standard solutions with known concentrations of the RET gene supplied with the kit. Primers and probes for determining the level of expression of beta-actin are also included to allow normalization across various samples. Intended Use AmoyDx RET Mutations Detection Kit is designed to accurately identify the rearrangement mutations listed in Table. This kit is for research use only. Table. Details of rearrangement mutations in RET gene Name Rearrangement Type Rearrangement Site GenBank RET-M RET-M RET-M RET-M RET-M RET-M RET-M RET-M8 RET-M9 RET-M0 RET-M RET-M RET-M RET-M HOOK-RET CCDC-RET (RET-PTC) PRKARA-RET (RET-PTC) NCOA-RET (RET-PTC/) NCOA-RET (RET-PTC/) NCOA-RET (RET-PTC/) GOLGA-RET (RET-PTC) htif-ret (RET-PTC) RFG-RET (RET-PTC) KTN-RET (RET-PTC8) KIAA8-RET (RET-PTC9) ELKS-RET PCM-RET RFP-RET Exon -Exon Exon -Exon Exon -Exon Exon -Exon Exon -Exon Partial Exon -Exon Exon -Exon Partial Exon 9-Exon Exon -Exon Exon 9-Exon Exon 0-Exon Exon -Exon Exon 9-Exon Exon -Exon DQ00. M. L0. S. N.A Kit Contents This kit contains sufficient reagents to carry out 8 tests (Table ). Table. Kit Contents Reagents Supplied Volume Tube No. Channel #: RET-M Reaction Mix 00 µl/tube FAM #: RET-M Reaction Mix 00 µl/tube FAM #: RET-M,, Reaction Mix 00 µl/tube FAM #: RET-M, 0, Reaction Mix 00 µl/tube FAM #: RET-M, Reaction Mix 00 µl/tube FAM #: RET-M8, 9, Reaction Mix 00 µl/tube FAM Website: Tel: sales@amoydx.com Fax: Address: F, Kechuang Building, 89 Haicang Wengjiao Road, Xiamen, 0, China /

3 For: ADx-RE0 #: RET-M Reaction Mix 00 µl/tube FAM #8:RET External Control Reaction Mix 00 µl/tube β -actin RET-exon- #9:RET Taq DNA Polymerase 0 µl/tube / #0 :RET Reverse Transcription Reaction Mix 000 µl/tube / #:M-MLV Reverse Transcriptase µl/tube / RET Quantitative Standard(0 Copies/µL) 00 µl/tube / RET Quantitative Standard(0 Copies/µL) 00 µl/tube / RET Quantitative Standard(0 Copies/µL) 00 µl/tube / RET Quantitative Standard(0 Copies/µL) 00 µl/tube / FAM HEX/VIC Equipment and Reagents Not Supplied With Kit. Compatible PCR instruments are: Stratagene Mx000P, Stratagene Mx00P.. Sterile, nuclease-free tubes.. Dedicated pipette and filtered pipette tips for handling DNA and RNA samples.. Sterile, nuclease-free HO. Shipping and Storage The kit requires cold-chain-transportation. The shelf-life of the kit is eight months when the kit is stored immediately upon receipt at -0± o C in a constant- temperature freezer and protected from light. Specimen Material Human mrna must be extracted from materials prior to use. If it is not used immediately, it should be stored below -0 o C for no more than six months. The RNA quality is essential and we recommend use of Qiagen RNA extraction kits. The OD value of RNA samples should be measured using the spectrophotometer after extraction. The Thermo Fisher NanoDrop 000 /000 spectrophotometer is recommended. The A0/A80 value should be between.9 and.. Technological Principles The kit uses novel, proprietary primers and probes in a real-time PCR assay to detect RET rearrangement mutations in human genomic RNA. These mutant RET genes can be amplified by the specific primers, and detected by the novel probes. Protocol. Reverse Transcription. () For each RNA sample, transfer µl RET Reverse Transcription Reaction Mix and µlm-mlv Reverse Transcriptase to a sterile centrifuge tube; mix well by pipeting gently up and down. () Add µl of sample RNA into the appropriate centrifuge tube. The total amount of RNA should be within 0. ~ μg. () Incubate the tubes at o C for one hour. () Heat the tubes at 9 o C for five minutes, then transfer them onto ice. The resulting cdna solutions are used for PCR amplification.. PCR amplification. () Accordingtotheratioof.µLReactionMixto0.µLRET Taq DNA Polymerase per sample, transfer the appropriate amount of Reaction Mix and RET Taq DNA Polymerase into a sterile tube. () Mix well by pipeting up and down and centrifuge for seconds. () Transfer 8 µl of the above master mix into PCR reaction wells. () Add µl of sample cdna, µl of Mixed Standard and µl ddho(no-template control) to the appropriate PCR reaction wells. () An example of a plate layout is shown in Table. Website: Tel: sales@amoydx.com Fax: Address: F, Kechuang Building, 89 Haicang Wengjiao Road, Xiamen, 0, China /

4 Table. Plate Layout for Standard Curve Method For: ADx-RE0 Assay RET-M RET-M RET-M,, RET-M, 0, RET-M, RET-M8, 9, RET-M External Control NTC NTC NTC NTC NTC NTC NTC NTC () Seal the PCR tubes. () Spin the PCR tubes gently to collect the reagents at the bottom of wells. (8) Place the PCR tubes into the real-time PCR instrument. (9) Carry out real-time PCR using the cycling conditions described in Table. Table. Cycling Parameters Temperature Time Cycles Stage 9 min Stage 9 s 0 s 0 0 s Stage 9 s 0 s Data collection of FAM and HEX/VIC 0 s Data Analysis Qualitative Assessment. The FAM signal indicates the mutation status of samples. In the external control, the internal reference amplifies and detects the expression of the housekeeping-gene (β-actin) indicated by FAM. The HEX/VIC signal in the external control amplifies and detects a region of genomic cdna in the RET gene (RET-exon-).. Ensure the calibration fluorescence is unselected, Select the sample and control positions as a group. Then adjust the Threshold for FAM amplification curves to obtain the Ct values of the samples and controls.. The FAM Ct value of Mixed Standard should be less than, but variation may occur due to different threshold settings on different instruments.. If FAM Ct value, the sample is classed as negative or below the detection limit of the kit. If FAM Ct value <, the sample is classed as mutation positive. Quantitative Assessment (Standard Curve) The absolute concentration of the target gene can be determined by making a graph with the standards provided in the kit. The expression level of the target gene can then be determined by interpolation. () Make a standard curve using the RET rearrangement, RET-exon- and β-actin gene standards. a) X axis: log of the concentration of the standards b) Y axis: Ct values for standards () Record the C value of the sample on RET rearrangement gene standard curve, and the C value of the same sample on β-actin (or RET-exon-) standard curve, then obtain the relative ratio = C/C. Website: Tel: sales@amoydx.com Fax: Address: F, Kechuang Building, 89 Haicang Wengjiao Road, Xiamen, 0, China /

5 For: ADx-RE0 Warnings and Precautions. Please read the instruction carefully and become familiar with all components of the kit prior to use.. Do not exchange and mix up the kit contents with different batches.. The kit and its contents cannot be resold or modified for resale without the written approval of AmoyDx.. Using other sources of reagents is not recommended. Strictly distinguish the reagents from mixed standard to avoid contamination. Otherwise, false positive may be produced.. Do the experiments with attention to prevent exogenous DNA contamination to reagents. It is recommended that users have separate, dedicated pipettes and filter pipette tips to add DNA template and during the preparation of reagents.. To optimize the activity and performance, mixtures should always be protected from light to avoid photo bleaching.. All the chemicals are potential hazard, only trained professionals should use this kit. Please wear suitable lab coat and disposable gloves. The used kit should be disposed of properly. 8. AmoyDx grants customer a non-exclusive and non-transferable license to use AmoyDx technologies. 9. AmoyDx assumes no responsibility for any errors that may appear in this document. The information in this document is subject to change. References. Zitzelsberger H, Bauer V, Thomas G, Unger K, 00. Clinica Chimica Acta. : Mologni L, 0. Curr Med Chem. 8:-.. Grieco M, Santoro M, Vecchio, G et al., 990. Cell 0:-. Website: Tel: sales@amoydx.com Fax: Address: F, Kechuang Building, 89 Haicang Wengjiao Road, Xiamen, 0, China /