User Manual/Hand book - Applied Biological Materials
Contents
1. 4 1 Access the miRNA Array Data Analysis Excel file through the ABM website 4 2 Input the C values of all the samples into the Data amp Results tab If replicates are done enter the average C values instead 4 3 The Excel File will change all the C values entered as greater than 35 or as not de tected to 35 This means C value of 35 is considered tested negative 4 4 The Excel File will normalize the miRNA expression levels for each sample using the 4 endogenous miRNA controls on each plate 4 5 Under the same tab view the fold difference and any up or down regulations be tween the test compared compared to the control sample The results data can be sort ed based on any of the categories in the excel file 4 6 Visual representation of the fold differences can be seen on the Colour Array tab This page is useful in identifying which miRNA samples have the biggest positive or nega tive changes Yellow to Orange to Red to Dark Red Brown symbolize increasing down regulation while Green to Blue to Dark Blue to Black symbolize increasing up regulation The name of miRNA can be easily identified as the well numbers of this array corresoond to the actual array locations 4 7 The fold difference data is also represented in the Scattor plot tab The black line equals 1 and the pink lines equal positive and negative fold changes equivalent to the value indicated cell A5 This plot is most useful to determine go2. tively designed for analyzing miRNA expression using real time quantitative reverse tran scription PCR or qRT PCR The arrays simultaneously provide specific and relative com parison of mature miRNAs using high performance EvaGreen real time PCR detection In addition to four reliable endogenous controls each 384 well array contains a collec tion of individually validated miRNA specific primers designed for the detailed and high throughput analyses of mature miRNA sequences as annotated by the Sanger miRBase Release 16 and are available for human and mouse study Instrument specific EvaGreen qPCR Mastermix is specially formulated to ensure the best specificity sensitivity and repro ducibility for miRNA expression analysis To use the miRNA qPCR Array reverse transcribe your experimental small RNA samples into first strand CDNA Then mix the template with our high performance instrument spe cific EvaGreen qPCR Mastermix and aliquot the mixture into each well of the same miR NA specific array Perform qPCR and determine relative miRNA expression normalized by the provided endogenous controls with your real time instrument and AAC method Our uncomplicated miRNA PCR array is user friendly for routine use in all research labo ratories Page 1 of 9 miRNA Profiling Handbook Notice to the Purchaser This product is for research use only Use in and or for diagnostics and therapeutics is strictly prohibited By opening and using the produ
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4. following are parameters performed on the Roche LightCycler480 but can also apply to other 384 well system 3 1 Leave your plate on ice while setting up the qPCR program detailed below Steps Temperature Duration Pre incubation 29 10 minutes lSseecomes Amplification 30 seconds SU seconds 5 seconds Melting curve 1 minute Cooling 30 seconds Select Reporter dye as SYBR Green Fluorescence 3 2 Calculate the threshold cycle C values for each well using the instrument s soft ware 3 2 1 Select Abs Quant Fit Points for All Samples method under the Analysis tab 3 2 2 Manually define the threshold value by viewing the amplification plots and ad justing the noise band above the background signal For comparable experiments it is recommended that the thresholds are the same across all the miRNA QPCR arrays 3 3 Export the resulting threshold cycle values for all wells into an Excel spreadsheet form 3 4 Analyze the miRNA qPCR data by using the miRNA qPCR Array Data Analysis Excel provided on the ABM website Page 7 of 9 miRNA Profiling Handbook Data Analysis 4 Data Analysis in Microsoft Excel ABM provides a miRNA Array Data Analysis Excel file that requires the input of threshold cycle data from the real time instrument Once the cycle data has been entered for all the samples the excel file will automatically present the results in a tabular format a scat ter plot and a colour array for easy comparison
5. Gg aom User Manual Hand book miRNA qPCR Profiling Kits ABM catalog MA003 human and MA004 mouse Table of Contents Introduction Notice to the Purchaser Technical Support Kit Components Product Description and Protocols miRNA Profiling Process Basic PCR Protocol for miRNA Profiling Real time PCR Instrument Parameters Data Analysis Contacts miRNA Profiling Handbook ON N 4 8 5 6 Introduction MicroRNA miRNA are highly conserved small non coding RNAs that were first discov ered in C elegans in the early 1990 s miRNAs are on average 21 24 nt long and are processed from lengthier sequences called pri and pre miRNA primary and prema ture miRNA respectively Mature miRNAs interact with RNA Induced Silencing Complex RISC to repress gene expression through translation interference or MRNA degradation by binding to the 3 UTR while some endogenous miRNA also demonstrate potential posi tive gene regulation through transcriptional activation of related genes This aspect of gene regulation provides complex mechanisms for more specific and controlled expres sion A single miRNA may regulate multiple targets and each target MRNA may interact with multiple miRNAs Researchers are presently investigating the functional role of miRNA in an extensive collection of cellular processes including those associated with disease Applied Biological Materials miRNA Profiling Kit is a thorough and sensitive tool innova
6. a workspace free of DNA contamination qPCR reactions should be as sembled in a DNA free environment CDNA sample preparation reaction mixture assem blage and the qPCR process in addition to the subsequent reaction analysis should be performed in separate areas The use of clean automatic pipettors designated for qPCR and aerosol resistant barrier tips are recommended 2 1 Mastermix qPCR reaction set up for one 384 well qPCR plate 2 1 1 Mix the following components in a 15ml tube Components Volume 2x miRNA EvaGreen qPCR Mastermix First strand products from 1 2 3 20ul ddH O Total volume 2 1 2 Thaw the 384 well plate in room temperature Spin briefly to collect content at the bottom of well There should be 4ul of liquid preloaded in each well of the plate Carefully remove the cover film on the 384 well plate before use 2 1 3 Aliquot ul of the mixture into every well in the provided 384 well qPCR plate Cover the plate with the new film provided in the kit Centrifuge the plate to remove any bubbles in the wells Examine the wells visually from underneath to make sure that samples have been added to all wells and that no bubbles are present Bubbles remaining at the bottom of the well will interfere with the results miRNA Profiling Handbook Page 6 of 9 Real Time PCR Instrument Parameters 3 Instrument Setup Follow the manufacturer s instructions as detailed for your specific real time instrumenta tion The
7. ct the purchaser agrees to the fol lowing The components in this kit may not be distributed resold or modified for resale without prior written approval from Applied Biological Materials ABM Inc If you do not agree to the above conditions please return the UNOPENED product to ABM Inc within ten 10 days of receipt for a full refund The information in this document is subjected to change without notice and should not be construed as a commitment by ABM Inc or its affiliated corporations In no event shall ABM Inc or its affiliated corporations be liable for incidental or consequential damages in connection with or arising from the use of this manual and product ABM Inc s products are warranted to meet our QC testing standards at the time of ship ment Notice of problematic products must be made to ABM Inc within 15 days of receipt of the product This product warranty limits ABM Inc s liability to the replacement of the product only Technical Support For more information on ABM products please visit our website http www abmGood com For additional information or technical assistance please call or e mail us at Applied Biological Material Inc Phone 604 247 2416 1 866 757 2414 Fax 604 247 2414 E mail technical abmgood com miRNA Profiling Handbook Page 2 of 9 Kit Components Cat No MAOO3 Human Whole Genome miRNA qPCR Profiling Kits 20 C The following components are sufficient f
8. od cut off ranges for the resulting data miRNA Profiling Handbook Page 8 of 9 Contacts Applied Biological Materials Inc Phone 8 30am 4 30pm PST M F Toll Free 866 757 2414 Local 604 247 2416 Fax 604 247 2414 24Hr Address Suite 8 13520 Crestwood Place Richmond BC Canada V6V 2G2 Distributors North America Canada USA Applied Blological Materi als Inc Asia South Korea CMI Biotech Tel 02 444 7101 Fax 02 444 7201 cmibio cmibio com India G Biosciences India Tel 0120 4323330 Fax 0120 4323299 rohit gbiosciences com Europe United Kingdom NBS Biologicals Ltd Tel 44 0 1480 433875 Fax 44 0 1480 459868 info nbsbio co uk Germany BioCat GmbH Tel 49 0 6221 7141516 Fax 49 0 6221 7141529 info biocat com Page 9 of 9 Internet www abmGood com Email General Information Order Products info abmGood com order abmGood com Technical Support technical abmGood com Mexico Quimica Lavoisier S A de C V Tel 52 333 848 8484 informes lavoisier com mx Taiwan Interlab Co Ltd Tel 886 2 2736 7 100 Fax 886 2 2735 9807 service interlab com tw Israel BioConsult Tel 972 0 2 5667043 Fax 972 0 2 5662790 sales bioconsult co il Belgium Gentaur Tel 32 2 732 5688 Fax 32 2 732 4414 ea gentaur com Italy Biosmart s r l Tel 39 0250994515 Fax 39 0250994547 infogbiosmart it Business Development bd abmGood
9. or 3 reactions samples 384 well plate H 1 3 plates 384 well plate H 2 3 plates 384 well plate H 3 3 plates QPCR plate adhesive films 9 pieces 2x EvaGreen qPCR Mastermix smil Cat No MAOOA Mouse Whole Genome miRNA qPCR Profiling Kits 20 C The following components are sufficient for 3 reactions samples 384 well plate M 1 3 plates 384 well plate M 2 3 plates qPCR plate adhesive films 6 pieces 2x EvaGreen qPCR Mastermix 5ml Storage Conditions Store at 20 C in a frost free freezer Page 3 of 9 miRNA Profiling Handbook miRNA Profiling Process Sample Isolation isolate RNA from the sample cells using Trizol reagent CDNA Synthesis use reverse transcriptase to synthesize CDNA from the isolated miRNA Real time PCR Reaction Setup mix the CDNA template with high performance 2x miRNA EvaGreen gPCR mastermix provided and aliquot the mixture into each well Sample Analysis analyze sample using the specified qPCR machine and obtain the CT values for all miRNAs Results Analysis analyze the results you obtained using the provided Excel sheet software ee L uuu u u ee 009000 e e o00 0 00000 0000000000 ee e e o e 0 00 0 0 0 ee miRNA Profiling Handbook Page 4 of 9 Basic Protocol for miRNA Profiling 1 cDNA synthesis 1 1 PolyA tailing Reaction Cat No AM1350 Ambion In a thin well PCR t
10. ube combine Total RNA Variable 2ug total RNA or 200ng small RNA 5x PolyA buffer 2 Ul 25MM MgCl 1 ul 5mM ATP 1 5 ul PolyA polymerase 0 5 Hl RNase free H2O Variable Final Volume 10 ul Incubate for 30 minutes at 37 C Store in 20 C If not proceed to cDNA synthesize immediately 1 2 MIRNA CDNA synthesis Cat No G268 and C270 Applied Biological Material Manda tory Attention The universal primer in every assay of the miRNA QPCR kits is specific for the unique and proprietary sequence incorporated into the CDNA by the miRNA adapter in miRNA cDNA synthesis kit G269 and G270 The miRNA qPCR arrays cannot detect cDNA generated with other sources of first strand synthesis kits 1 2 1 Add 2ul of Oligo dT adapter 10 uM into the PolyA tailing products in the tube from 1 1 1 2 2 Incubate at 60 C for 5 minutes and let it cooled to room temperature for 2 minutes 1 2 3 Add the following components into the tube from 1 2 1 Components Volume Concentration final 20pl ANTP 10mM Tul 500UM 5x RT Buffer Aul 1x RNasin 40U pl 0 5ul 20U per reaction EasyScript RTase 200U ul lul 200U per reaction RNase free H O 1 5 pl Final volume 20ul Incubate the mixture at 42 C for 30 minutes Stop the reaction by heating at 85 C for 5 minutes Chill on ice Page 5 of 9 miRNA Profiling Handbook Basic Protocol for miRNA Profiling 2 Real time qPCR Reaction Setup Note Preparing
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