MiRNA Plate Assay Kits for Different Cancers Signosis
Contents
1. and incubate the plate at 50 C overnight Ensure the numbers and letters on the plate are clearly visible from under foil seal by pressing the foil down on every single experimental well Put an open container with water in the incubator to keep humidity and prevent evaporation from experimental wells 3 Invert the plate over an appropriate container and expel the contents forcibly and wash the plate 3 times by adding 200p1 of pre warmed 1x Plate Hybridization Wash Buffer 4 Complete removal of liquid at each wash by firmly tapping the plate against clean paper towels 5 Add 200u1 of Block buffer incubate for 15 minutes at room temperature with gentle shaking 6 Invert the plate over an appropriate container to remove block buffer 7 Add 100 ul of diluted streptavidin HRP conjugate to each well and incubate for 30 min at room temperature with gentle shaking 8 Wash the plate 3 times with 1X Detection wash buffer Complete removal of liquid at each wash by firmly tapping the plate against clean paper towels 9 Freshly prepare the substrate solution For the whole plate Iml Substrate A 1ml Substrate B 8ml Substrate dilution buffer 10 Add 95 ul substrate solution to each well and incubate for 1 min 11 Place the plate in the luminometer and read Set integration time to 1 second with no filter position For the best results read the plate within 5 20 minutes Signosis Inc 1700 Wyatt Drive Suite 10 12 Sa2. D Signosis MiRNA Plate Assay Kits for Different Cancers Catalog Number MA 0105 Introduction MicroRNAs miRNAs regulate up to 30 of mammalian gene expression Aberrant expression of miRNAs has shown to associate with human cancers Profiling of miRNA expression displays a set of unique miRNAs in a variety of cancers Signosis developed a panel of miRNA plate assay kits to analyze these miRNA molecules Principle of the assay Signosis proprietary miRNA plate array is a plate based detection In the assay one miRNA molecule is flanked by a capture oligo and a biotinated detection oligo through two bridge oligos One of the bridge oligos is partially hybridized with the miRNA molecule and the capture oligo and another one with the miRNA and the detection oligo The hybrid is captured onto plate through hybridization with an immobilized oligo and detected by a streptavidin HRP conjugate and chemiluminecscent substrate This hybrid structure is sensitive to the sequence of the miRNA molecule One nucleotide difference can prevent the formation of the hybrid and therefore miRNA isoform can be differentiated which normally is hard to do with Northern blot In addition the sensitivity of the assay is higher than miRNA Northern blot assay Materials provided with the kit One 96 well white plate 4 C Streptavidin HRP conjugate 4 C Plate hybridization buffer RT 5x Plate hybridization wash buffer RT Block buffer RT 5x Det
3. ection wash buffer RT Substrate A 4 C Substrate B 4 C Substrate dilution buffer RT 8 different miRNA oligo mixes For Research Use Only 123456789012 Pafe oo oe oe oe l Pafe ee ee ee ee y Pafe ee oe T CO HHO LOL LL E an a ee ee oe ee an on oe ne Chemiluminescent detection with a plate reader Diagram of miRNA plate array Material required but not provided Hybridization incubator Shaker Plate reader for chemiluminescent detection ddH20 RNAase free Reagent preparation before starting experiment Warm up Plate hybridization buffer and Hybridization Wash buffer at 45 C before use Dilute 30ml of 5x Plate Hybridization wash buffer with 120 ml of dH O before use Dilute 40ml of 5x Detection wash buffer with 160 ml of dH O before use Dilute 1000 times of streptavidin HRP with block buffer before use at Step 7 Signosis Inc e 1700 Wyatt Drive Suite 10 12 Santa Clara CA 95054 Tel 408 747 0771 Fax 408 864 2182 Assay procedure 1 Warm up the plate to room temperature and arrange the appropriate number of the wells of the plate based on your experiment by removing the top foil sealing film with a blade Keep the unused well sealed Make fresh 30X dilution of each oligo mix with RNase free water Mix the following items in one well 2ul 5 ul RNA 0 2ug 2 ug 100 ul Plate hybridization buffer 4 ul oligo mix 4ul Biotin Detection Oligo 2 Seal the wells with foil film securely
4. nta Clara CA 95054 Tel 408 747 0771 Fax 408 864 2182
Download Pdf Manuals
Related Search