User Manual/Hand book - Applied Biological Materials
Contents
1. of 8 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 alll 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 4 1 Access the miRNA Array Data Analysis Excel file through the ABM website www abmGood com 4 2 Input the C values of all the samples into the Sample Data tab If replicates are performed enter the average C values instead 4 3 The Excel File normalizes the miRNA expression levels for each sample using the 4 en dogenous miRNA controls on each plate 4 4 Under the same tab view the fold difference and any up or down regulations be tween the test compared to the control sample The results data can be sorted based on any of the categories in the excel file 4 5 Visual representation of the fold differences can be seen on the Plate 1 Plate 2 and Plate 3 tabs These pages are useful in identifying which miRNA samples have the biggest positive or negative changes Yellow to Orange to Red to Dark Red Brown sym bolize 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 correspon2. qPCR Reaction Setup Note Prepare 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 GPCR reaction set up for one 384 well qPCR plate 2 1 1 Mix the following components in a 15ml tube Components Volume 2X EvaGreen miRNA qPCR Mastermix 5 0 ml First strand products from 1 2 3 20 ul 5 ul ddH O 1 0 ml Total volume 6ml Note Kit only includes enough Mastermix for two 384 well qPCR plates Supplementary EvaGreen miRNA qPCR Mastermix can be ordered for additional reactions refer page 8 for contact information 2 1 2 Thaw the 384 well plate at room temperature Spin briefly to collect content at the bottom of well There should be 4 ul 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 6 ul of the mixture from 2 1 1 into every well in the provided 384 well QPCR plate Cover the plate with the qPCR plate adhesive films 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 bu
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4. abm User Manual Hand book qPCR miRNA Arrays ABM catalog MA003 human and MA004 mouse Kit Components Cat No MA003 ce eeseeeeeeee Human Whole Genome miRNA qPCR Profiling Kit 20 C The following components are sufficient for 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 ncluding free 2X EvaGreen miRNA qPCR Mastermix 5 ml Cat No MA004 ee eeeeeseees Mouse Whole Genome miRNA qPCR Profiling Kit 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 ncluding free 2X EvaGreen miRNA qPCR Mastermix 5 ml Sufficient for two 384 well plates Supplementary mastermix can be ordered for addi tional reactions please refer to page 8 for contact information Storage Conditions Store at 20 C in a frost free freezer miRNA Profiling Handbook Page 1 of 8 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 translat
5. bbles are present Bubbles remaining at the bottom of the well will interfere with the results miRNA Profiling Handbook Page 5 of 8 Real Time PCR Instrument Parameters 3 Instrument Setup Follow the manufacturer s instructions as detailed for your specific real time instrumenta tion The 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 Ramp Rate Cycle s C s Pre incubation ISIC 10 minutes 4 8 95 C 10 seconds 4 8 Amplification 63 C 15 seconds 25 40 72C 5 seconds 4 8 IRS 5 seconds 4 8 Melting curve i 1 60 C 1 minute 25 Cooling 40 C 30 seconds 2 5 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 6
6. d to the actual array locations Everytime when new sample data is entered remember to click refresh the 4 6 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 A8 This plot is most useful to determine good cut off ranges for the resulting data miRNA Profiling Handbook Page 7 of 8 Contacts Applied Biological Materials Inc Phone 8 30am 4 30 0m PST M F Toll Free 866 757 2414 Local 604 247 2416 Fax 604 247 2414 24Hr Address Suite 8 13520 Crestwood Place Internet www abmGood com Email General Information info abmGood com Order Products order abmGood com Technical Support technical abmGood com Business Development bd abmGood com 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 Mexico Quimica Lavoisier S A de C V Tel 52 333 848 8484 informes lavoisier com mx Puerto Rico AVP Caribe Tel 787 892 0047 Fax 787 264 3816 Ivelez avpcaribe com Taiwan Interlab Co Ltd Tel 886 2 2736 7 100 Fax 886 2 2735 9807 service interlab com tw Japan Cosmo Bio Co Ltd Tel 03
7. e 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 G26 and G270 Applied Biological Material Manda tory Attention The qPCR miRNA arrays are only compatible with cDNA generated from ABM s miRNA cDNA synthesis kits The reverse primer pre loaded into each qPCR miRNA Array specifically anneals to the proprietary miRNA Oligo dT adapter supplied in the miRNA EasyScript CDNA Synthesis Kits G269 amp G270 Subsequently any CDNA generated using other CDNA synthesis kits will not produce amplicon and or yield any valid qPCR data 1 2 1 Add 2 ul of miRNA Oligo dT adapter 10 UM into the PolyA tailing products in the tube from 1 1 1 2 2 Incubate at 65 C for 5 minutes and let cool to room temperature for 2 min utes 1 2 3 Add the following components into the tube from 1 2 2 Components Volume Concentration Final 20u ANTP 10 mM 1 ul 500 UM 5X RT Buffer 4ul 1X RNasin 40 U ul 0 5 ul 20 U per reaction EasyScript RTase 200 U ul 1 ul 200 U per reaction RNase free H2O 1 5 ul Final volume 20 ul Incubate the mixture at 42 C for 15 minutes Immediately stop the reaction by heating at 70 C for 10 minutes Chill on ice Store in 20 C If not proceed to real time qPCR immediately Page 4 of 8 miRNA Profiling Handbook Basic Protocol for miRNA Profiling 2 Real time
8. everse transcribe your experimental small RNA samples into first strand CDNA Then mix the template with our high performance instrument spe cific EvaGreen miRNA qPCR Mastermix and aliquot the mixture into each well of the same miRNA specific array Perform qPCR and determine relative miRNA expression nor malized 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 re search laboratories Page 2 of 8 miRNA Profiling Handbook miRNA Profiling Process RNA Isolation isolate RNA from the sample cells or tissue using Trizol reagent cDNA Synthesis use miRNA CDNA synthesis kit G269 or G270 ABM to synthesize CDNA Real time PCR Reaction Setup mix the cDNA template with high performance 2X EvaGreen miRNA qPCR mastermix 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 miRNA Profiling Handbook Page 3 of 8 Basic Protocol for miRNA Profiling 1 cDNA synthesis 1 1 PolyA tailing Reaction Cat No AM1350 Ambion In a thin well PCR tube combine Components Volume Total RNA Variable 2 ug total RNA or 200 ng small RNA 5X PolyA buffer 2 ul 25 mM MnCl 1 ul 5 mM ATP 1 5 ul PolyA polymerase 0 5 ul RNase fre
9. ion interference or MRNA degradation by binding to the 3 or 5 UTR or open reading frame ORF Alternatively some endog enous miRNA demonstrate potential positive gene regulation through transcriptional ac tivation of related genes This aspect of gene regulation provides complex mechanisms for more specific and controlled expression 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 qPCR Profiling Kit is a thorough and sensitive tool in novatively designed for analyzing miRNA expression using real time quantitative reverse transcription PCR or qRT PCR The arrays simultaneously provide specific and relative comparison of mature miRNAs using high performance EvaGreen real time PCR detec tion In addition to four reliable endogenous controls each 384 well array contains a collection 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 miRNA qPCR Mastermix is specially formulated to ensure the best specificity sensitivity and reproducibility for miRNA expression analysis To use the miRNA qPCR Array r
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