Sharpvue™ Human miRNA Primer Array
Contents
1. check the qPCR products Check the purity of the primers by electrophoresis or use PAGE purified primers if the bands are diffused One may also use phenol chloroform extraction and ethanol precipitation methods to treat the primers before the experiment Signal in the blank No Template Control sample e There may be contamination of the positive samples in the qPCR reaction system if the Tm of the melting curve of the blank control is the same as the positive control Eliminate sample application error first If the situation still persists replace the PCR grade water and or primers and or use a new Sharpvue 2X Universal qPCR Master Mix High Rox If the Tm of the melting curve of the blank control is lower than the positive control the qPCR reaction may have produced nonspecific amplification such as primer dimers Prepare the qPCR reaction mix on ice and increase the temperature of fluorescence detection If this does not work redesign the primers No signal Ct or late appearing signal Double peaks and multiple peaks in the melting curve of the positive control In the absence of other primers present in the reaction double or multiple peaks in the melting curve of the positive control indicate that the qPCR reaction produced nonspecific amplification fragments Prepare the qPCR reaction mix on ice optimize the qPCR reaction conditions for example by increasing the annealing temperature decreasing the primer conc2. with the NIH guidelines developed for recombinant DNA and genetic research Use of any part of the Product constitutes acceptance of the above terms Limited Warranty Signalway Biotechnology warrants that the Product meets the specification described in the accompanying Product Datasheet If it is proven to the satisfaction of Signalway Biotechnology that the Product fails to meet these specifications Signalway Biotechnology will replace the Product In the event a replacement cannot be provided Signalway Biotechnology will provide the purchaser with a refund This limited warranty shall not extend to anyone other than the original purchaser of the Product Notice of nonconforming products must be made to Signalway Biotechnology 30 days of receipt of the Product Signalway Biotechnology s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price Signalway Biotechnology s liabilitydoes not extend to any damages arising from use or improper use of the Product or losses associated with the use of additional materials or reagents This limited warranty is the sole and exclusive warranty Signalway Biotechnology does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose Signalway Biotechnology is committed to providing our customers with high quality products If you should have any questions or co
3. A Signalway Sharpvue Human miRNA Primer Array Performance optimized with Sharpvue miRNA First Strand Kit and Sharpvue 2X Universal qPCR Master Mix Sharpvue Human miRNA Primer Array Set v1 0 384 well __ Cat No SW K1001 Sharpvue Human miRNA Primer Array Set v1 0 96 well Cat No SW K1007 User Manual Sharpvue Human miRNA Primer Array Description Real Time PCR Technology uses fluorescent material or probes conjugated with fluorophores in the PCR reaction system and monitors the whole process of PCR reaction by accumulation of fluorescence signal then qualities and quantities the unknown samples Compared to chip technology and the common PCR the Real Time PCR is fast precise quantitative with high throughput and no chance of contamination so it is internationally recognized the standard method to quality the nucleosidase molecular Sharpvue Human miRNA Primer Array use a new non toxic dye EvaGreene for detecting provide a comprehensive solution to your miRNA functional analyses for both profiling of large numbers of miRNAs and quantitation of individual ones with extremely high sensitivity specificity linearity dynamic range and reproducibility Signalway Biotechnology released most advanced and comprehensive miRNA expression profiling assays covering 1700 human miRNAs from latest miRBase release of v17 0 and provided two different knids of Arrays as 384 well Array from A to E 96 well Array fr
4. entration or increasing the fluorescence detection temperature not more than the Tm value of the expected product If this does not work redesign the forward primer e Not enough PCR cycles For good sensitivity one should generally set up more than 35 PCR cycles but more than 45 cycles may result in too much background signal e The amount of template used may not be enough or the template may be degraded Use the highest concentration possible of diluted template samples to set up the qPCR At the same time avoid freezing and thawing the samples repeatedly e The amplification efficiency is low and the qPCR reaction conditions are not optimal Redesign the primers and optimize the reaction conditions Limited Use License and Warranty Following terms and conditions apply to use of all miRNAs and Packaging Kit the Product If the terms and conditions are not acceptable the Product in its entirety must be returned to Signalway Biotechnology within 5 calendar days A limited End User license is granted to the purchaser of the Product The Product shall be used by the purchaser for internal research purposes only The Product is expressly not designed intended or warranted for use in humans or for therapeutic or diagnostic use The Product must not be resold repackaged or modified for resale or used to manufacture commercial products without prior written consent from Signalway Biotechnology This Product should be used in accordance
5. id form 20 C Stable for at least 12 months the primer in the every well of the plate provided as powder form 1 2 3 19 20 Wearing a lab coat disposable gloves and protective goggles are recommended when handling chemicals IMPORTANT NOTES PCR arrays require special laboratory practices to avoid false positive amplifications The high throughput and repetition of these arrays can lead to amplification of a single DNA molecule 1 All the step touch the plate with clean gloves 2 Pick up array plate from the freezer on the desktop at room temperature for 5 minute 3 Centrifuge the plate at 2000 rpm for 2 minute 4 Open the microplate Sealer carefully Procedure This Array Plate can be uesed on ABI7000 7300 7700 7900HT 7900HT Fast and StepOnePlus Required Reagents Total RNA Sharpvue miRNA First Strand Kit Forward primer and reverse primer for PCR amplification customer provide or ordered from Signalway Biotechnology Shanrvue 2xUniversal qPCR Master Mix High Rox Cat No 9000007 Nuclease Free Water 1 RNA Sample Preparation This assay starts with total RNA which must includes miRNA Customers may get purified total RNA with a kit from Qiagen or other manufacturers 2 Poly A Tailing and Reverse Transcription a Set the following components on ice Add the following reagents into an RNase free reaction tube which has been pre cooled on ice The final volume should be 10u Comp
6. l profile as follows 4 Set the one of three kinds of thermal profile as follows or followed manual instruction of Manufacture Set Detector as SYBR Passive Reference as ROX i Two step fast cycling protocol Cycling Step Temperature Enzyme hot activation 96 C Denaturation 96 C Annealing amp Extension 60 C ii Three step fast cycling protocol Cycling Step Temperature Enzyme hot activation 96 C Denaturation 96 C Annealing amp Extension 60 C Cycling Step Temperature Enzyme hot activation 96 C Denaturation 96 C Annealing amp Extension 60 C Holding Time 2 minutes 5 seconds 30 seconds Holding Time 2 minutes 5 seconds 30 seconds HoldingTime 2 minutes 15 seconds 60 seconds Number of Cycles 1 40 Number of Cycles 1 40 Number of Cycles 1 40 5 Dissociation Curve Measurement optional default program in ABI 7900 96 C for 15 seconds 60 C for 15 seconds 96 C for 15 seconds 6 Analyze the experiment Refer to the getting started guides for your real time PCR system to analyze the experiment The general process for analyzing the data from gene expression assays involves the following procedures a View the amplification plots b Set the baseline and threshold values Example Objective let 7 family Equipment ABI 7900 Key Product Features Universal RT primer to minimize variations in RT step Highly specific only measure miRNAS not precursor miRNAS The pro
7. ncerns about any Signalway Biotechnology products please contact us at 866 998 6722 Signalway Biotechnology 11406 Enclave Lake LN Pearland TX 77584 Wwww swbio com Tel 866 998 6722 Fax 713 436 0887 Email info swbio com
8. om Ito 12 However You can also order the individual Assay according to your own demands For a current list of Assays please visit the website www Signalway Biotechnologytech com Related Products Signalway Biotechnology offers comprehensive solutions for studying miRNA and gene expression A careful process of codevelop ment ensures that they work well together and provide robust and reproducible results Sharpvuetm miRNA Assay Product Name miRNA RT Kit Sharpvuetm 2X Universal qPCR Master Mix Human miRNA Assay Primer Sets Human miRNA Primer Array Set v1 0 384 well Human miRNA Primer Array Set v1 0 96 well Sharpvuetm Gene Expression Assay Gene First Strand Kit Sharpvuetm 2 X Universal qPCR Master Mix High Rox Contents and Storage Contents Quantity Individual Sharpvuetm Human 3 3 X miRNA Assay Primer Set Sharpvuerm Human miRNA A B C D E Primer Array Set v1 0 384 well 5 plates Sharpvuetm Human miRNA Primer Array Set v1 0 96 well 20 plates Preparation Description High sensitivity and specificity easy to operate Non toxic EvaGreen based real time quantitative PCR Mix covering 1700 human miRNAs from latest miRBase release of v17 0 two forms of design are 5 and 20 plates Accurate quantification of mRNA expression Non toxic EvaGreen based real time quantitative PCR Mix Storage temperature conditions 20 C Stable for at least 12 months Individual Assay Primer provided as liqu
9. onent Volume ul Total RNA X Sharpvue miRNA First Strand Kit 5x Mix A 2 Cat No 9000005 Sharpvue miRNA First Strand Kit 15x Mix B Cat 0 67 No 9000006 Nuclease free H20 Cat No 9000016 to 10 Total 10 b Mix gently and spin the tube briefly to collect the contents c Transfer the tubes to a thermal cycler Incubate at 37 Cfor 15 minutes 25 C for 15 mins 37 C for 30min d Inactivate the reaction at 85 C for 5 minutes e Store the single stranded cDNA at 20 C or proceed directly to PCR amplification 3 qPCR Set the following components Component Volume ul Forward Reverse miRNA primer sey each 3 33 X 3 RT product from step 1 0 67 Shanrvue 2x qPCR Master Mix Cat No 5 00 9000007 10 Nuclease Free Water Cat No 9000016 1 33 Total 10 00 Component Volume ul Forward Reverse miRNA primer Array 384 well plate RT product from step 1 Shanrvue 2x qPCR Master Mix Cat No 9000007 10 Nuclease Free Water Cat No 9000016 Total Component Forward Reverse miRNA primer Array 96 well plate RT product from step 1 Shanrvue 2x qPCR Master Mix Cat No 9000007 10 Nuclease Free Water Cat No 9000016 Total 0 67 5 00 4 33 10 00 Volume ul 0 0 67 5 00 4 33 10 00 b Seal the plates with qPCR film and mix gently then spin the tube briefly to collect the contents c Transfer the plate to a real time thermal cycler e g ABI 7900 d Set the therma
10. prietary primer design of the Sharpvuetm miRNA Arrays and Assays Distinguishes miRNA family members with single nucleotide mismatches e g let 7 family members only have less than 4 cross reactivity Highly sensitivity detects miRNA in single molecules with 0 995 of linear range and 6 orders of dynamic range Sensitivity and Linearity of Sharpvuerm miRNA Assays Figure 1 demonstrated Sharpvuetm miRNA assays have high sensitivity detecting single miRNA copy Sharpvuetm miRNA assays have excellent linearity with R2 gt 0 99 and wide dynamic range gt 6 log Specificity of Sharpvuetm miRNA Assays Trouble Shooting Guide Poor precision or failed qPCR reactions Abnormal melting curves The fluorescence detection temperature may not be appropriate Adjust accordingly e The set up position for reaction samples in the real time PCR instrument may not be right A djust accordingly e PCR cycle conditions primer concentration and primer sequences may not be appropriate Adjust the primer concentration and annealing temperature If this does not work redesign the primers e The template sample purity may not be adequate Purify the template sample by phenol chloroform extraction and ethanol precipitation If the samples are reverse transcribed cDNA set up the qPCR reaction with a diluted sample as other concentrated reagents in the RT reaction mixture may be interfering with the qPCR e Try to use 0 3 agarose gel electrophoresis to
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