For miRNA expression profiling
Contents
1. zn m o o o 112131415 6171 8 9 20 44 72 3 14 15 16 17 18 19 20 27 22 23 24 Figure 4 To load a 384 well format PCR Array add 10 ul of the Experimental Cocktail from each numbered sample into the staggered wells with the same number as indicated in the figure 19 3 Performing Real Time PCR Detection NOTE Be sure to follow the manufacturer s instructions for the proper operation and maintenance of your real time instrument a CAREFULLY but tightly seal the PCR Array with the optical thin wall 8 cap strips Formats A and D or with the optical adhesive film Formats C E F and G NOTE Be sure that no bubbles appear in any of the wells of the PCR Array To remove bubbles tap the plate gently on the bench top or centrifuge the plate briefly b Place the plate on ice while setting up the PCR cycling program below c Place one plate in your real time thermal cycler If recommended by your instrument s user manual use a compression pad with the optical film sealed plate formats NOTE PCR Arrays containing experimental cocktail may be stored at 20 C wrapped in aluminum foil for up to one week until ready to run This recommendation is particularly useful for the set of four 96 well Genome PCR Arrays d Enter and run the appropriate program for your real time instrument below If prompted by your instrument software select Absol2. 17 22 25 26 Il Background and Introduction MicroRNA miRNA first discovered in C elegans in early 1990s are a class of naturally occurring small RNA molecules generally 17 to 30 nucleotides long processed from much larger stem loop structures and pre miRNA Mature miRNA is recognized by and guides the RNA Induced Silencing Complex RISC to specific messenger RNA transcript sequences causing either translation repression or mRNA degradation This phenomenon adds even more complexity to the regulation of gene expression Each miRNA can regulate multiple mRNA targets and a single target mRNA may be regulated by multiple miRNA sequences Researchers are currently correlating miRNA expression profiles to biological phenotypes in attempts to better understand miRNA based regulation of gene or mRNA expression The RT miRNA PCR Array is designed to analyze miRNA expression using real time reverse transcription PCR or qRT PCR the most sensitive and reliable method for nucleic acid expression analysis currently available The arrays take advantage of a SYBR Green real time PCR detection system designed for high performance to analyze the expression of many mature miRNA sequences simultaneously Each 96 or 384 well array plate contains a panel of primer sets for a thoroughly researched set of 88 or 376 relevant pathway or disease focused miRNA plus four housekeeping assays and two RNA and PCR quality controls T
3. 4x96 format RT miRNA PCR Arrays o Wells P23 and P24 of the 384 well RT miRNA PCR Arrays The two sets of duplicate control wells and also test for inter well intra plate consistency The 384 well format of the pathway focused RT miRNA PCR Arrays includes four replicates of the same 96 well format in which each two by two set of wells gray labeled wells 1 4 in Figure 2C contains the same primer set represented by the corresponding 96 well designations black labeled Custom RT miRNA PCR Arrays have your specified layout and the product information enclosed with the array reiterates your layout and the genes included Materials Provided NOTE The format of the RT miRNA PCR Array is indicated by the last letter of the catalog number Be sure that you have the correct PCR Array format for your instrument before starting the experiment Format For Real Time Instruments Plate ABI standard blocks 5700 7000 7300 7500 7700 7900HT 96 block Bio Rad iCycler iQ 5 MyiQ Chromo4 MJ Research Eppendorf MasterCycler ep RealPlex OMM Stratagene Mx3005P Mx3000P Takara TP 800 C ABI 7500 FAST block 7900HT FAST block StepOnePlus 96 well Bio Rad CFX96 Opticon and Opticon 2 MJ Research D Stratagene 4000 APANE ABI 7900HT 384 well block E Bio Rad CFX384 el F Roche LightCycler 480 96 well block 96 well G Roche LightCycler 480 384 we
4. Business Development 29851 Willow Creek Road Eugene OR 97402 Tel 541 465 8300 Fax 541 335 0504 The purchase of this product includes a limited non transferable license under specific claims of U S Patent Nos 6 174 670 6 569 627 and 5 871 908 owned by the University of Utah Research Foundation or Evotec Biosystems GmbH and licensed to Idaho Technology Inc and Roche Diagnostics GmbH to use only the enclosed amount of product according to the specified protocols No right is conveyed expressly by implication or by estoppel to use any instrument or system under any claim of U S Patent Nos 6 174 670 6 569 627 and 5 871 908 other than for the amount of product contained herein Patent Pending 2011 QIAGEN all rights reserved M eS es www giagen com Australia Orders 1 800 243 800 Fax 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800 28 10 10 Fax 0800 28 10 19 Technical 0800 28 10 11 Belgium Orders 0800 79612 Fax 0800 79611 Technical 0800 79556 Brazil Orders 0800 557779 Fax 55 11 5079 4001 Technical 0800 557779 Canada Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 86 21 3865 3865 Fax 86 21 3865 3965 Technical 800 988 0325 Denmark Orders 80 885945 Fax 80 885944 Technical 80 885942 Finland Orders 0800 914416 Fax 0800 914415 Technical 0800 914413 France Orders 01 60 920 926 Fax 01 60 920 925 Technical 01 6
5. air where it will contaminate and confound the results of future real time PCR experiments See also the Note on Preparing a Workspace Free of DNA Contamination 21 D Data Analysis AAC Method NOTE PCR Array Data Analysis Web Portal Access our free PCR Array Data Analysis Web Portal from the following address http www sabiosciences com pcrarraydataanalysis php The PCR Array Data Analysis Web Portal automatically performs the following calculations and interpretation of the control wells upon including threshold cycle data from a real time instrument The PCR Array Data Analysis Web Portal presents the results in a tabular format a scatter plot a three dimensional profile and a volcano plot when replicates are included 1 Change all C values reported as greater than 35 or as N A not detected to 35 At this point any C value equal to 35 is considered a negative call 2 Examine the threshold cycle values of the control wells a Reverse Transcription Control RTC Any impurities in your RNA sample that affect the reverse transcription of the miRNA External RNA Control ERC built into the RT miRNA First Strand Kit also affect the reverse transcription of your miRNA sequences of interest Calculate AC AVG AVG If this value is less than 5 then no inhibition is apparent If this value is greater than 5 then evidence of impurities that inhibited the reverse transcription phase of the procedure is evi
6. 100 96 well Genome V2 0 MAH 200 MAM 200 X PF aoe ng SMAIL og oral 384 well Genome V2 0 MAH 200 MAM 200 E G 300 ng small 3 0 ug total b Use the same amount of RNA in this reaction for every sample c Lower amounts of small RNA than 50 ng will increase the false negative rate of the miRNA Array method d Do not use more than 400 ng of small RNA 4 ug of total RNA per reaction to avoid expending reagents and inhibiting the reaction 2 For each RNA sample combine the following in a sterile PCR tube Small RNA 50 ng to 400 ng or 0 5 4 ug total RNA miRNA RT Primer amp ERC Mix 1 0 uL 5x miRNA RT Buffer 2 2 0 uL miRNA RT Enzyme Mix 1 0 uL miRNA Nucleotide Mix 1 0 uL RNase free H20 to a final volume of 10 0 uL 3 Mix the contents gently with a pipettor followed by brief centrifugation 4 Incubate at 37 C for 2 hours 5 Heat at 95 C for 5 minutes to degrade the RNA amp to inactivate the reverse transcriptase 6 Chill on ice for at least one minute and add 90 ul of RNase free H20 to each 10 of cDNA synthesis reaction Mix well 7 Hold the finished First Strand cDNA Synthesis Reaction on ice until the next step or store overnight at 20 C 16 C Performing Real Time PCR NOTE The use of RT SYBR Green qPCR Mastermixes is critical for obtaining the most accurate results from the RT miRNA PCR Arrays Be sure to use the correct master mix for your instrument before continuing with this pro
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8. and one gene specific primer for each miRNA sequence e RT miRNA PCR Arrays are provided to assess the expression of 88 pathway focused miRNAs or the complete miRNA Genome 528 Human miRNA sequences 440 Mouse miRNA sequences as annotated by the Sanger miRBase Release 14 Control Elements The product specification sheet included with your cataloged RT miRNA PCR Array contains a list of the pathway focused and housekeeping genes on the array Housekeeping Assay Panel The small nuclear RNA housekeeping assay panel HK1 HK4 including SNORD 48 47 44 as well as U6 normalizes qPCR Array data Wells H5 to H8 of the 96 Well amp 384 well 4x96 format RT miRNA PCR Arrays Wells P17 to P20 of the 384 well RT miRNA PCR Arrays MAH 3100 MAM 3100 Reverse Transcription Control RTC Duplicate Reverse Transcription Controls RTC test the efficiency of the RT miRNA First Strand Kit cat no 331401 reaction with a primer set detecting the template synthesized from the kit s built in miRNA External RNA Control ERC o Wells H9 and H10 of the 96 Well amp 384 well 4x96 format RT miRNA PCR Arrays o Wells P21 and P22 of the 384 well RT miRNA PCR Arrays Positive PCR Control PPC Duplicate Positive PCR Controls PPC to test the efficiency of the polymerase chain reaction itself using a pre dispensed artificial DNA sequence and the primer set that detects it o Wells H11 and H12 of the 96 Well amp 384 well
9. warranties of merchantability or fitness for a particular purpose SABiosciences Corporation shall not be liable for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product LIMITED LICENSE STATEMENTS Use of this product is covered by one of more of the following US patents and corresponding patent claims outside the US 5 079 352 5 789 224 5 618 711 6 127 155 5 677 152 claims 1 to 23 only 5 773 258 claims 1 and 6 only 5 407 800 5 322 770 5 310 652 5 994 056 6 171 785 and claims outside the US corresponding to US Patent No 4 889 818 The purchase of this product includes a limited non transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research No right under any other patent claim such as apparatus or system claims in US Patent No 6 814 934 and no right to perform commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration is conveyed expressly by implication or by estoppel This product is for research use only Diagnostic uses under Roche patents require a separate license from Roche Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA This pr
10. 0 920 930 Offers 01 60 920 928 Germany Orders 02103 29 12000 Fax 02103 29 22000 Technical 02103 29 12400 Hong Kong Orders 800 933 965 Fax 800 930 439 Technical 800 930 425 Ireland Orders 1800 555 049 Fax 1800 555 048 Technical 1800 555 061 Italy Orders 800 789 544 Fax 02 334304 826 Technical 800 787980 Japan Telephone 03 6890 7300 Fax 03 5547 0818 Technical 03 6890 7300 Korea South Orders 080 000 7146 Fax 02 2626 5703 Technical 080 000 7145 Luxembourg Orders 8002 2076 Fax 8002 2073 Technical 8002 2067 Mexico Orders 01 800 7742 639 Fax 01 800 1122 330 Technical 01 800 7742 436 The Netherlands Orders 0800 0229592 Fax 0800 0229593 Technical 0800 0229602 Norway Orders 800 18859 Fax 800 18817 Technical 800 18712 Singapore Orders 1800 742 4362 Fax 65 6854 8184 Technical 1800 742 4368 Spain Orders 91 630 7050 Fax 91 630 5145 Technical 91 630 7050 Sweden Orders 020 790282 Fax 020 790582 Technical 020 798328 Switzerland Orders 055 254 22 11 Fax 055 254 22 13 Technical 055 254 22 12 B e UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 Go USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN b Sample amp Assay Technologies
11. 2550 uL 10 0 mL 15 0mL 12 5 mL 17 For 384 well Arrays use the following table Mix the following components in a 15 ml tube or a multi channel reservoir gt EE 0 S 2 2 A 0 6 e gt gt 499 O E O 2 gt 2 o s 94 Efu 8 8u gt o o c o E o E sa 2205 255 L RE os OSE a x o o M Jo ool o ix 2 gt LL 5 ix 3 59s 82 5 5 Bis 8 3 oO 9 2X RT SYBR Green qPCR Mastermix 550 uL 2 0 mL 3 0 mL 2 5 mL Diluted first strand reaction 100 uL 0 1 mL 0 1mL 0 1 mL ddH O 450 uL 1 9mL 29 24mL Total volume 1100 uL 4 0mL 6 0 mL 5 0 mL NOTE This recipe provides an excess volume of ONLY 150 to 600 uL per array plate or array set Very carefully add the cocktail to the RT miRNA PCR Array precisely as described below to insure that each well receives the required volume 18 2 Loading the PCR Arrays a CAREFULLY remove the needed RT miRNA PCR Arrays from their sealed bags 96 Well Pathway Focused Arrays amp 384 well Genome Arrays One plate per sample 96 Well Genome Arrays One set of six plates Human or five plates Mouse per sample 384 Well Pathway Focused Arrays One plate for four samples 384 Well Genome V2 0 Arrays Six Human or Five Mouse plates for four samples b Add the appropriate volume of the correct Experimental Cocktail to e
12. 5 2550 uL uL uL uL V Troubleshooting and FAQs A Troubleshooting 1 Evidence of Poor Reverse Transcription Efficiency The value of AVG CP S AVG CPPS is greater than 5 Double check the A260 A280 and A260 A230 ratios of your RNA samples and be sure to perform the dilutions for spectrophotometry in RNase free Tris pH 8 0 buffer If necessary re purify your RNA samples with a spin column based clean up method such as the miRNeasy Mini Kit cat no 217004 2 Evidence of Poor PCR Amplification Efficiency The average value varies by more than two 2 across the PCR Arrays being compared and or is greater than 22 Different instruments have different levels of sensitivity If an average value of 20 2 is difficult to obtain for your instrument the observed average value should be acceptable as long as it does not vary by more than two cycles between PCR Arrays being compared Be sure that the initial heat activation step at 95 C had been lengthened to 10 minutes from the shorter time in the default program Be sure that all other cycle parameters also have been correctly entered according to the recommendations in this User Manual Also double check the quality of your RNA as described for Evidence of Poor Reverse Transcription Efficiency above 26 B Frequently Asked Questions 1 Will pipetting error affect the PCR Array results The passive reference dyes in the PCR master mixes such as RO
13. Array runs in the same analysis v Export the resulting threshold cycle values for all wells to a blank Excel spreadsheet for use with our Data Analysis Template Excel file 4 Recommended Quality Control a Dissociation Melting Curve Run a melting curve program immediately after the above cycling program and generate a first derivative dissociation curve for each well in the entire plate using your instrument s software No more than one peak should appear in each reaction at temperatures greater than 70 C If your instrument does not have a default melting curve program run the following program instead 95 C 1 min 65 C 2 min OPTICS OFF 65 C to 95 C at 2 C min OPTICS ON If you decide not to obtain the dissociation curve immediately save the plates wrapped in aluminum foil at 20 C as is in case you need to perform this operation at a later point in time for troubleshooting purposes When ready simply warm the plate to room temperature place it into your real time instrument and run the melting program described above NOTE Be sure to visually inspect the plate after the run for any signs of evaporation from any of the wells If evaporation is observed make a note of which wells so that you may qualify your data analysis appropriately NOTE DO NOT open any previously run and stored PCR Array plate Removing the thin wall 8 cap strips or the adhesive film from PCR Arrays releases PCR product DNA into the
14. Designed for Routine Use Brings miRNA expression profiling to almost any lab with a real time PCR instrument How miRNA PCR ARRAYS WORK 1 Convert miRNA to cDNA Ti cDNA 1 miRNA 5 amm 3 1 Polyadenylation Ly X 232 HE Reverse Transcription from Universal RT Primer 5 e 3 mp 5 Universal RT Prim 2 Add cDNA to RT qPCR Master Mix Aliquot Mixture Across PCR Array miRNA Specific Primer 3 5 qPCR using miRNA U S00 Primer and Universal qPCR Primer 3 Run in Your Real Time PCR Instrument Profile Profile 2 0 4 8 12 16 20 24 28 32 36 40 4 Data Analysis 10 i i Ei p Value for Fold Change 1 4 3 2 1 0 1 2 Fold Change Ratio log Breast Tumor Figure 1 Overview of the RT miRNA PCR Array procedure H94 3402398 v Figure 2A 96 Well Pathway Focused and Genome RT miRNA PCR Array Layout 123 4 5 6 7 8 9101112131415 16 17 18 19 2021 22 23 24 A 565 0 00 9 6 7 9 9 9 82 8 4 8 7 89 9 1 2 85 85 87 8 39 31 o 89 84 85 6 v 99 90 er a 8 45 06 86 5 2 8 84 8 86 7 89 89 0 6 e 89 amp 65 e G9 Y 02 D 8 4 5 6 rm n9 v 81 42 amp amp 86 n 09 09 86 08 69 09 E 87 9 0
15. Extract RNA from the tissue using the QlAzol Lysis Reagent included in the miRNeasy Mini Kit Be sure to use a sufficient amount of QIAzol Lysis Reagent During homogenization add a volume of reagent at least ten times greater than the tissue volume b Then further clean up and enrich the small RNA following the protocol provided in the miRNeasy Mini Handbook Whole Blood Samples Remove red blood cells RBC from whole blood samples using a density gradient centrifugation medium for example Lymphoprep Greiner Bio One Catalog 1031966 Isolate small RNA from the white blood cell fraction as described for cultured cells above Previously Isolated Total RNA If you have already prepared total RNA from any biological source material be sure that it used a phenol based method such as TRIzol RNAzol etc Then enrich for small RNA using the miRNeasy Mini Kit cat no 217004 For Other Biological Samples Refer to the existing literature to find isolation protocols for high quality RNA from other biological samples or contact one of our Technical Support representatives For best results from the PCR Array all RNA samples should be suspended in the RNase free water provided with the RNA Isolation kit or alternatively in RNase free 10 mM Tris buffer pH 8 0 DO NOT use DEPC treated water 14 2 RNA Quality Control For best results from the PCR Array all RNA samples should also demonstrate consistent quality according t
16. March 2011 RT miRNA PCR Array Handbook For miRNA expression profiling QIAGEN Sample amp Assay Technologies QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result QIAGEN sets standards in E Purification of DNA RNA and proteins Nucleic acid and protein assays microRNA research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www giagen com Product Use Limitations RT miRNA PCR Arrays are intended for molecular biology applications This product is not intended for the diagnosis prevention or treatment of a disease All due care and attention should be exercised in the handling of the products We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to other applicable guidelines CONTENTS l Background and Introduction Il Materials Provided Ill Additional Materials Required IV Protocol A B RNA Preparation and Quality Control RT miRNA First Strand Kit Performing Real Time PCR Data Analysis Appendix Troubleshooting and Frequently Asked Questions 10 12 14 16
17. NA yield a smaller number of positive calls and increase false negative calls The use of the RT miRNA First Strand Kit maximizes the number of positive calls at low amounts of RNA over other sources of miRNA reverse transcriptase and first strand synthesis kits For successful results and maximum positive call rates we recommend that first time users try starting with 100 ng of small RNA It is also important to use a consistent amount of RNA for each sample to be characterized and compared 15 B RT miRNA First Strand Kit NOTE RNA samples must meet the standards of integrity and purity from protein and organics contamination described on the previous two pages NOTE 5x miRNA RT Buffer 2 on ice briefly vortex the tube to mix the contents well and centrifuge to the bottom of the tube before each use 1 Recommended Amounts of RNA Starting Material a Although the reaction accommodates 50 to 400 ng of small RNA 0 5 to 4 0 ug of total RNA first time users are recommended to start with following amounts of RNA depending on the cataloged product used Arrays or Sets Catalog Formats RNA MAH 001 MAM 001 384 well Pathway Focused MAH 102 MAM 102 E G 50 ng small 0 5 ug total MAH 103 MAM 103 MAH 001 MAM 001 96 well Pathway Focused MAH 102 MAM 102 A C D F 100 ng small 1 0 ug total MAH 103 MAM 103 384 well Genome MAH 3100 MAM 3100 E G 200 ng small 2 0 ug total 96 well Genome MAH 100 MAM
18. X and Fluorescein are used by the real time PCR systems to normalize variation from well to well Therefore these systems tolerate volume variations caused by pipetting error and evaporation 2 How prevent the evaporation of reaction volume from the wells Be sure to carefully and completely seal the PCR Array with the optical thin wall 8 cap strips or the optical adhesive film before placing it into your thermal cycler Also be sure to use a compression pad with the plate formats using the optical film seal Formats C E F and G as directed by the manufacturer of your real time PCR instrument 3 How reliable are the results from the RT miRNA PCR Array Assuming the use of good consistent experimental technique real time PCR methods such as the PCR Array provide highly reproducible results To insure the reliability of your results and to reliably detect smaller fold changes in gene expression from the PCR Array the performance of replicate determinations such as biological triplicates is highly recommended The Data Analysis Template available from our website for the PCR Array uses your replicate PCR Array data to calculate t test p values and to generate a Volcano Plot illustrating the statistically significant fold changes in gene expression If you have additional questions please check our website www SABiosciences com for a more complete listing of Frequently Asked Questions FAQs or call our Technical Support Repr
19. ach corresponding well of the PCR Arrays preferably from a reservoir with an eight channel pipettor or a twelve channel pipettor but only using eight tips NOTE Change pipet tips following each addition to avoid any cross contamination between the wells or reactions 96 Well Pathway Focused Arrays amp 384 well Genome Arrays Add 25 uL of the same cocktail to each well of a 96 well array Add 10 uL of the same cocktail to each well of a 384 well array 96 Well Genome Arrays Add 25 uL of the same cocktail to each well of all four arrays in the set 384 Well Pathway Focused amp 384 well Genome V2 0 Arrays Add 10 uL each sample s cocktail to the appropriate sets of wells of the array NOTE Each 384 well pathway focused plate characterizes four samples in separate sets of 96 wells staggered from one another by only one well The spacing between the tips of standard multi channel pipettors will allow you to properly skip rows or columns when adding each sample Be sure to load each sample into the correct set of wells Use Figure 4 as a guide Sample 1 Sample 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 12 20 21 22 23 24 xlo zlalr Ix x o mlelo w x jn m 9 o 1 2 13 4 5 6 7 8 9 10 1 12 23 14 15 16 17 18 19 20 27 22 23 24 Sample 4 112131415 617 8 9 10 47 12 13 14 15 16 17 18 19 20 21 22 23 2
20. d are shipped in sets of two 2 twelve 12 or twenty four 24 identical arrays C Numbers of Plates and Optical Cap Strips or Optical Adhesive Film Each RT miRNA PCR Array shipment includes the array plates and either twelve 12 optical thin wall 8 cap strips Formats A and D or one 1 optical adhesive film Formats C E F and G per plate D Storage Conditions All components included in this kit are shipped at ambient temperature but must be stored at 20 C upon receipt When stored properly at 20 C their quality is guaranteed for 6 months Ill Additional Materials Required A miRNeasy Mini Kit Cat No 217004 B RT miRNA First Strand Kit Cat No 331401 MANDATORY for a Complete and Successful Experiment The universal primer in each assay is only compatible with the sequence added to the CDNA template by the primer in this kit C RT SYBR Green qPCR Mastermix MANDATORY for a Complete and Successful Experiment Be sure to pick the correct one for the instrumentation in your laboratory 1 96 Well RT miRNA PCR Arrays RT SYBR Green ROX qPCR Mastermix Specifically designed for All ABI and Stratagene Instrumentation Eppendorf Mastercycler ep realplex Instruments with ROX filter set Catalog Number Size 330520 For 2 RT miRNA PCR Arrays 330522 For 12 RT miRNA PCR Arrays 330523 For 24 RT miRNA PCR Arrays RT SYBR Green Fluor qPCR Mastermix Specifically designed for BioRad iCycler MyiQ and iQ5 Ins
21. dent See the Troubleshooting Guide b Positive PCR Control PPC Any impurities in your RNA sample that affect the PCR amplification of the positive control also affect the PCR amplification for your miRNA sequences of interest The average value should be 20 2 on each PCR Array and should not vary by more than two cycles between PCR Arrays being compared Larger differences in average values between samples indicate the presence of different amounts of PCR amplification inhibitors in the different samples and that all of the RNA samples require further purification An average value of CPPS that is consistently greater than 22 for all of your samples may indicate a problem with the cycling conditions or may simply be indicative of the relative sensitivity of your instrument See the Troubleshooting Guide 22 3 Calculate the AC for each pathway focused gene in each plate AC CMO CAS HKG NOTE Choosing the right normalization factor The expression level of the housekeeping genes chosen for normalization in the method must not be influenced by your experimental conditions If one or more such sequences have been previously identified by independent means and if the PCR Array reproduces those results use the average of their values the equation above If an appropriate housekeeping gene has not been previously identified use the average value of all housekeeping genes Or simply use zero 0 in
22. en we test other sources of enzymes with our RT miRNA PCR Arrays we frequently see primer dimers and other non specific products that confound SYBR Green based real time PCR detection Because each instrument uses a different reference dye to normalize their optics be sure that you use the correct master mix for the instrumentation in your laboratory The universal primer in every assay of the RT miRNA PCR Arrays is specific only for the unique and proprietary sequence incorporated into the cDNA by the universal RT primer in the RT miRNA First Strand Kit The RT miRNA PCR Arrays cannot detect cDNA generated from miRNA using other sources of miRNA first strand synthesis or miRNA reverse transcription kits Similarly CDNA generated from the RT miRNA First Strand Kit cannot be used with other sources of real time qPCR assays for miRNA The Reverse Transcription Controls RTC on the RT miRNA PCR Array can only be evaluated using the RT miRNA First Strand Kit and its built in miRNA External RNA Control ERC These RTC assays do not yield results when used with other sources of reverse transcriptases or first strand synthesis kits The RT miRNA First Strand Kit also includes a proprietary buffer to preferentially reverse transcribe miRNA into cDNA The buffer components and the magnesium concentration in our RT miRNA First Strand Kit are also more compatible with our PCR master mixes than other enzymes or kits providing the RT miRNA PCR Array
23. esentatives at 1 888 503 3187 or 301 682 9200 27 Ordering Information Product Contents Cat no RT miRNA PCR Array Array of assays for miRNA Varies quantification For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www qiagen com or can be requested from QIAGEN Technical Services or your local distributor 28 Notes 29 Notes 30 Trademarks QIAGEN QIAzol RNeasy Rotor Gene QIAGEN Group SYBR Molecular Probes Inc Roche LightCycler Roche Group ROX StepOnePlus Applera Corporation or its subsidiaries Eppendorf Mastercycler Eppendorf AG Stratagene Mx3005P 3000 Mx4000 Stratagene Bio Rad iCycler Chromo4 CFX96 DNA Engine Opticon CFX384 iQ 5 MyiQ Bio Rad Laboratories Inc SmartCycler Cepheid Agilent Agilent Technologies Inc LabChip Caliper Technologies Corp TRIzol Molecular Research Center Inc Registered names trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law Limited License Agreement Use of this product signifies the agreement of any purchaser or user of the RT miRNA PCR Array to the following terms The RT miRNA PCR Array may be used solely in accordance with the RT miRNA PCR Array Handbook and for use wit
24. h components contained in the Kit only QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this Kit with any components not included within this Kit except as described in the RT miRNA PCR Array Handbook and additional protocols available at www giagen com Other than expressly stated licenses QIAGEN makes no warranty that this Kit and or its use s do not infringe the rights of third parties This Kit and its components are licensed for one time use and may not be reused refurbished or resold QIAGEN specifically disclaims any other licenses expressed or implied other than those expressly stated Ur 9v The purchaser and user of the Kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above QIAGEN may enforce the prohibitions of this Limited License Agreement any Court and shall recover all its investigative and Court costs including attorney fees in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the Kit and or its components For updated license terms see www giagen com LIMITED PRODUCT WARRANTY This warranty limits our liability to the replacement of this product in the event the product fails to perform due to any manufacturing defect SABiosciences Corporation makes no other warranties of any kind expressed or implied including without limitation
25. he Genome V2 0 RT miRNA PCR Arrays contain 528 Human or 440 Mouse miRNA sequences as annotated by the Sanger miRBase Release 14 See Figure 2 for the layout of a typical PCR Array The specificity and single mismatch discrimination sensitivity and dynamic range and reproducibility of the miRNA expression analysis is guaranteed when using the complete miRNA PCR Array System To complete the miRNA PCR Array procedure reverse transcribe the experimental small RNA samples into first strand cDNA the template for the PCR using the RT miRNA First Strand Kit See Figure 1 for an overview of the miRNA PCR Array procedure Then mix the template with one of our instrument specific and ready to use RT SYBR Green qPCR Mastermixes Aliquot the mixture into each well of the same plate containing the pre dispensed miRNA specific assays Perform PCR and finally determine relative expression with your real time instrument and the AAC method The simplicity of the RT miRNA PCR Arrays makes them accessible for routine use in every research laboratory Benefits of the RT miRNA PCR Arrays e Genome Wide Coverage Arrays represent either a large panel of the most relevant miRNA in the human and mouse genomes as annotated by the Sanger miRBase Release 14 or pathway or disease focused panels Reliable Sensitive Specific Simple real time PCR method with optimized primer design and reaction formulation improves discrimination and sensitivity
26. ll block 384 well A 96 Well Formatted Arrays 1 Pathway Focused Arrays Cat Nos MA 001 MA 102 amp MA 103 in formats A C D amp F These RT miRNA PCR Arrays each represent 88 miRNA sequences and are shipped in sets of two 2 twelve 12 or twenty four 24 identical arrays 2 Genome V2 0 Array Set Cat Nos MAH 200 amp MAM 200 in formats A C D amp F These six array Human and five array Mouse sets represent a combined total of 528 Human 440 Mouse miRNA sequences and are shipped as a duplicate set of the six Human or five Mouse different arrays 3 Genome Array Set Cat Nos MAH 100 amp MAM 100 in formats A C D amp F These four array sets represent a combined total of 352 miRNA sequences and are shipped as a duplicate set of the four different arrays B 384 Well Formatted Arrays 1 Pathway Focused Arrays Cat Nos MA 001 MA 102 amp MA 103 in formats E amp G These RT miRNA PCR Arrays each represent four replicate sets of 88 miRNA sequences and are shipped as a set of four 4 identical arrays 2 Genome V2 0 Array Set Cat Nos MAH 200 amp MAM 200 in formats E amp G These RT miRNA PCR Arrays each represent 528 Human or 440 Mouse miRNA sequences and are shipped in sets of six 6 different arrays for use with 4 samples 3 Genome Array Set Cat Nos MAH 3100 amp MAM 3100 in formats E amp G These RT miRNA PCR Arrays each represent 376 miRNA sequences an
27. nd tip boxes exposed to the air for long periods of time 5 Do not open any previously run and stored PCR Array plate Removing the thin wall 8 cap strips or the adhesive film from PCR Arrays releases PCR product DNA into the air where it will contaminate and confound the results of future real time PCR experiments 13 A RNA Preparation and Quality Control High quality RNA is ESSENTIAL for obtaining good real time PCR results The most important prerequisite for any miRNA expression analysis experiment is consistent high quality small RNA from every experimental sample Therefore the sample handling and RNA isolation procedures are critical to the success of the experiment Residual traces of proteins salts or other contaminants will either degrade the RNA or decrease the efficiency of if not block completely the enzyme activities necessary for optimal reverse transcription and real time PCR performance Although other real time PCR based miRNA detection methods call for the use of total RNA that contains miRNA as the starting input material we highly recommend starting with small RNA 200 nucleotides to reduce background noise for optimal sensitivity 1 Recommended RNA Preparation Methods High quality small RNA for your real time PCR experiment must be prepared using one of the following methods each specific for your biological sample Cultured Cells OR Tissue Samples Use the miRNeasy Mini Kit cat no 217004 a
28. o the following criteria a RNA Concentration and Purity by UV Spectrophotometry NOTE Prepare dilutions and measure absorbance in 10 mM Tris pH 8 0 buffer The spectral properties of nucleic acids are highly dependent on pH i A260 A23o ratio should be greater than 1 7 ii A26o A2so ratio should be greater than 2 0 iii Concentration by Azgo should be greater than 10 ug ml small RNA b RNA Quality amp Integrity Characterize 10 ng of the small RNA on an Agilent Bioanalyzer using an RNA 6000 Nano LabChip The RNA should contain a single sharp peak at a low molecular weight with no smearing and no additional peaks at higher molecular weights Figure 3 Good Small RNA Band Integrity Is Important for Best Results from the miRNA qPCR Array Displayed is an Agilent Bioanalyzer electropherogram of a high quality small RNA preparation showing a sharp low molecular weight peak without a shoulder 3 Amount Considerations The miRNA PCR Array System yields relative gene expression profiles with as little as 50 ng or as much as 400 ng of small RNA per array However the optimal amount of starting material depends upon the relative abundance of the sequences of interest Lower abundance sequences require more RNA higher abundance sequences require less RNA Greater amounts of input RNA yield a greater number of positive calls that is sequences expressed in the linear dynamic range of the method Lower amounts of input R
29. oduct is provided under an agreement between Molecular Probes Inc and SABiosciences and the manufacture use sale or import of this product is subject to one or more of U S Patent Nos 5 436 134 5 658 751 and corresponding international equivalents owned by Invitrogen Corp The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer where such research does not include testing analysis or screening services for any third party in return for compensation on a per test basis The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product of its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research For information on purchasing a license to this product for purposes other than research contact Molecular Probes Inc
30. ponding 96 well PCR Array packs 2 x 330500 330510 or 330520 2 x 330502 330512 or 330522 2 x 330503 330513 or 330523 D Equipment NOTE RT miRNA PCR Arrays are NOT recommended for the Cepheid Sma rtCycler the Roche LightCycler 2 0 or the Corbett Research QIAGEN Rotor Gene 1 2 3 For recommendations on specific real time instrumentation thermal cyclers with fluorescent detection see the list of master mixes and plate formats above Calibrated Multi Channel Pipettor RNase DNase free pipette tips and tubes 11 IV Protocol Please read through this entire protocol before beginning your experiment RNA samples are very sensitive to RNase digestion therefore wear gloves and maintain an RNase free work area while performing this protocol NOTE Master Mix and Reverse Transcription Kit Considerations The performance of our RT miRNA PCR Arrays is only guaranteed with our RT SYBR Green qPCR Mastermixes and the RT miRNA First Strand Kit Therefore the use of the complete miRNA qPCR Array System is absolutely essential for obtaining any real time PCR profiling results The chemically modified and tightly controlled HotStart enzyme and other proprietary chemical components in our RT SYBR Green qPCR Mastermixes provide more accurate SYBR Green results by preventing the amplification of primer dimers and other non specific products They also help insure high amplification efficiencies Wh
31. s with maximum levels of sensitivity with ng amounts of small RNA 12 NOTE Preparing a Workspace Free of DNA Contamination For accurate and reproducible PCR Array results it is very important to avoid contamination of the assay with foreign DNA Any DNA contamination will artificially inflate the SYBR Green signal yielding skewed gene expression profiles and false positive signals The most common sources of DNA contamination are the products of previous experiments spread into the air of your working environment Please follow the recommendations below on how to set up and maintain a working environment free of DNA contamination 1 Wear gloves throughout the procedure Use only fresh PCR grade reagents H20 and lab ware tips and tubes 2 Physically separate the workspaces used for PCR setup and post PCR processing or non PCR operations Decontaminate your PCR workspace and lab ware pipettor barrels tube racks etc before each new use with UV light to render any contaminating DNA ineffective in PCR through the formation of thymidine dimers or with 1096 bleach to chemically inactivate and degrade any DNA 3 Close all tubes containing PCR products once you are finished adding or removing volumes Before discarding any lab ware tips or tubes containing PCR products or other DNA treat with 1096 bleach 4 Do not remove the PCR Array plate from its protective sealed bag until immediately ready to use Do not leave lab ware tubes a
32. termine fold change in gene expression the normalized expression of the GOI in the experimental sample is divided by the normalized expression of the same GOI in the control sample 9 5exp 727 Where AAC is equal to AC expt AC control The complete calculation is as follows 990 9 ACUHKG expt E 2104801 expt 9 46 expt 7401801 control 9104801 G HKG control al control 9 56 HKG control 24 E Appendix This additional protocol provides instructions for people who have run MAH 100 or MAM 100 in the past and wanted to extend their study to new MAH 005 amp MAH 006 or MAM 005 arrays 1 Recommended Amounts of RNA Starting Material Arrays or Sets Catalog Numbers Formats RNA MAH 005 MAH 006 96 well Array MAM 105 A C D F 100 ng small 1 0 ug total a Use the same amount of RNA in this reaction for every sample b Lower amounts of small RNA than 50 ng will increase the false negative rate of the miRNA Array method c Do not use more than 400 ng of small RNA 4 ug of total RNA per reaction to avoid expending reagents and inhibiting the reaction 2 Continue with Steps B 2 to B 7 3 Experimental Cocktail Preparation for each sample 2x RT SYBR Green qPCR Mastermix Diluted first strand reaction ddH O Total volume 4 Continue with array loading and real time PCR detection in C 2 and C 3 25 1275 100 117
33. the place of the average of HK genes C for each group to be compared and rely on the consistency in the quantity and quality of your original input small RNA across your groups to effectively normalize your results 4 When biological and or technical replicates are performed calculate the average value of each gene each well across those replicate arrays for each treatment group 5 Calculate the AAC for each gene across two PCR Arrays or groups AAC AC group 2 AC group 1 Where group 1 is the control and group 2 is the experimental 6 Calculate the fold change for each gene from group 1 to group 2 as 2 AAC OPTIONAL f the fold change is greater than 1 then the result may be reported as a fold up regulation If the fold change is less than 1 then the negative inverse of the result may be reported as a fold down regulation The fold change ratios may also be reported as is 23 NOTE Detailed Mathematical Explanation of AAC Data Analysis Method Due to the inverse proportional relationship between the threshold cycle C and the original gene expression level and the doubling of the amount of product with every cycle the original expression level L for each gene of interest is expressed as 22 To normalize the expression level of a gene of interest GOI to housekeeping gene HKG the expression levels of the two genes are divided 954801 92104801 9 AHR To de
34. tocol See Pages 10 and 11 NOTE An incorrectly chosen PCR Array plate format will not properly fit into your real time PCR instrument and its use will damage the instrument Be sure you have the correct PCR Array format for your instrument before continuing with this protocol See Page 8 NOTE accuracy and precision of your pipetting determines the consistency of your results Be sure that all of your micro pipettors are calibrated before beginning this procedure Also make sure to not introduce any bubbles into the wells of the PCR Array NOTE f unsure of your RNA quality or isolation technique examine the quality of your RNA before this step 1 Experimental Cocktail Preparation For 96 well Arrays use the following table Mix the following components in a 15 ml tube or a multi channel reservoir Qin 2 69 a el da E su Dow 0 0 w g e Sal pos I m 2n g 350 239 3 6 4 6 ou LUN 0 0 oso r i x EN 1 1 294 554 5 lt lt 594 con ov gt zE ors O25 22 e sSzg Org 9 g O pen amp z q lt 2 00 oso o X z2u su u i e e o o o oo 2X RT SYBR Green qPCR Mastermix 1275 uL 5 0 mL 7 5mL 6 25 mL Diluted first strand reaction 100 uL 0 1 mL 0 1mL 0 1 mL ddH20 1175 uL 4 9 mL T AmL 6 15 mL Total volume
35. trumentation Catalog Number Size 330510 For 2 RT miRNA PCR Arrays 330512 For 12 RT miRNA PCR Arrays 330513 For 24 RT miRNA PCR Arrays RT SYBR Green qPCR Mastermix Specifically designed for instrumentation that does not require a reference dye BioRad CFX96 BioRad MJ Research Opticon Opticon 2 and Chromo 4 Roche LightCycler 480 System Eppendorf Mastercycler ep realplex Instruments without ROX filter set Catalog Number Size 330500 For 2 RT miRNA PCR Arrays 330502 For 12 RT miRNA PCR Arrays 330503 For 24 RT miRNA PCR Arrays 10 2 3 The 384 Well PCR Arrays 96 x 4 Format RT SYBR Green ROX qPCR Mastermix Specifically designed for All ABI and Stratagene Instrumentation Catalog Number Size 330521 For 4 384 well RT miRNA PCR Arrays RT SYBR Green Fluor qPCR Mastermix Specifically designed for instrumentation that requires the Fluorescein Reference Dye Catalog Number Size 330511 For 4 384 well RT miRNA PCR Arrays RT SYBR Green qPCR Mastermix Specifically designed for instrumentation that does not require a reference dye BioRad CFX384 Roche LightCycler 480 System Catalog Number Size 330501 For 4 384 well RT miRNA PCR Arrays RT miRNA PCR Arrays MAH 3100 MAM 3100 RT miRNA PCR Arrays MAH 3100 MAM 3100 available in two 2 twelve 12 and twenty four 24 packs Formats E amp G require a quantity of two 2 of the correct master mixes for your instrument of the size for the corres
36. ute Quantitation to begin NOTE For additional help with instrument setup see our Instrument Specific Setup Instructions and Protocol Files at http www sabiosciences com mirnapcrarrayprotocolfiles php Cycles Duration Temperature 1 10 minutes 95 C 15 seconds 95 C 40 30 to 40 seconds 60 C 30 seconds 72 C 1 The 10 minute step at 95 C is required to activate the HotStart DNA polymerase Detect and record SYBR Green fluorescence from every well during the annealing step of each cycle Different instruments need different lengths of time to detect the fluorescent signal Choose the appropriate time for the annealing step 60 C for your instrument 20 e Calculate the threshold cycle Ci for each well using the instrument s software i We highly recommend letting your instrument automatically define the Baseline values but manually setting the Threshold values ii To define the Baseline use the Linear View of the amplification plots and set the instrument to use the readings from cycle number two 2 through two 2 cycle values before the earliest visible amplification usually around cycle number ten 10 but no more than 15 To define the Threshold Value use the Log View of the amplification plots and place it above the background signal but within the lower half to one third of the linear phase of the amplification plot iv IMPORTANT Ensure that the Thresholds are the same across all PCR
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