ValidPrime™ - TATAA Biocenter
Contents
1. Master mix or master mix components The ValidPrime kit has been validated in a large number of master mixes using the conditions recommended by the manufacturers including Final Annealing concentrations temperature 800 nM primer 200 nM probe 61 C 800 nM primer 200 nM probe 800 nM primer 200 nM probe 59 C 500 nM primer 250 nM probe 60 C 500 nM primer 250 nM probe 60 C 400 nM primer 200 nM probe 60 C 400 nM primer 200 nM probe 60 C 500 nM primer 200 nM probe 60 C Master mix Applied Biosystems TaqMan Gene expression Master Mix Applied Biosystems TaqMan Universal PCR Master Mix Applied Biosystems TaqMan Genotyping Master Mix Finnzymes DyNAmo Flash Probe qPCR Kit Finnzymes DyNAmo ColorFlash Probe qPCR Kit KAPA PROBE FAST qPCR Kit Qiagen QuantiTect Probe PCR Kit Roche LC480 Probes master Concentration per primer and probe in qPCR Table 4 Recommended primer and probe concentrations and annealing temperature in selected commercial master mixes Pipettes and tips available from www tataa com e Vortex and centrifuge Sample RNA DNA e Optionally DNase With ValidPrime measured Can can be corrected for up to 50 gDNA back ground If gDNA background is high it is recommended to reduce it by treating 10 the cDNA with double strand specific DNase that will remove specifically the gDNA and will not degrade2. 1 Laurell et al 2011 lt 5 the measured Cq ie is confounded It can be corrected to GOI I _G Gor C q Ca log 2 RT 9 ART Equation 1 Gol cq RT and Car RT reactions and Cq a is the Cq value that would have been obtained for the RT reaction in absence of gDNA contaminations From Cq ie the correct transcript amount can be calculated are the qPCR Ca values measured for the RT and RT ValidPrime ValidPrime Using ValidPrime the same test for gDNA contamination can be performed and if needed the same correction for background can be made with a much smaller number of reactions The sensitivities of the GOI qPCR assays for gDNA Cq e are tested relative to the ValidPrime assay Cq ge on the DNA DNA provided gDNA standard Well performing GOI assays that have been properly designed to exclusively target mRNA by for example having intron spanning primers shall not amplify the gDNA standard while GOI assays that amplify sequences present in multiple copies in the gDNA will have even lower C values than the ValidPrime assay All samples are then analyzed also with the ValidPrime assay Cq The measurement setup is shown in Table 2 Sample Original GOI 2 c0 ie GOI 4 ValidPrime data Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 gDNA standard Table 2 Experimental setup based on five samples ass
3. Pre processing includes interplate calibra tion efficiency correction various normalization options handling of technical replicates and missing data normalization with paired samples and correction for gDNA contamination using ValidPrime Analyses include absolute quanti fication relative quantification and expression profiling Tutorials are available on www multid se tutorials php and free support is offered on www qpcrfo rum com A6months complimentary license for GenEx Enterprise is included for first time users with the ValidPrime kit To get started send an email to info multid se and state your order number for your ValidPrime kit together with your cus tomer details You will receive a key to activate your free license that can be downloaded from www multid se To purchase additional GenEx licenses or for qPCR data analysis services contact us on order tataa com 11 12 Troubleshooting e do not get any amplification signal The instrument may not have been programmed correctly or there may be a problem with the master mix Establish ifthe problem is in the detection or the amplification by running the samples on a gel Run a new test using the gDNA control with the ValidPrime assay provided e My negative controls are amplified Your reagents are probably contaminated e My samples have same higher C value than my NTC You have used too little cDNA Add more cDNA and try again The cDNA may also be of
4. TATAA Biocenter has great experience and expertise in high resolution gene expression profiling pathogen de tection and small sample single cell analysis TATAA Biocenter AB Odinsgatan 28 411 03 G teborg Tel 46 31 761 57 00 Fax 46 31 15 28 90 E mail info tataa com Website www tataa com
5. the amount of gDNA may vary and RT controls are typically measured on all samples These controls add substantially to the cost of a qPCR study ValidPrime is an assay to test for the presence of gDNA in test samples and when combined with a gDNA control sample replaces all RT controls ValidPrime is highly optimized and specific to a non transcribed locus of gDNA that is present in exactly one copy per haploid normal genome Therefore ValidPrime measures the number of genomic copies present in a sample and can be used for normalization of samples to cell copy number as endogenous control for CNV applications and as control for gDNA background in RTqPCR The ValidPrime kit also contains a gDNA standard that can be used to test the sensitivity of RTqPCR assays for gDNA background ValidPrime In expression profiling experiment the ValidPrime assay is added to the list of assays and the gDNA control is added to the list of samples From the combined measurements with the ValidPrime assay and the gene of interest GOI assays onall samples and on the gDNA control the genomic background contribution to all RTQPCR measurements can be assessed ValidPrime replaces the need to perform RT controls for all reactions and makes RTqPCR profiling easier and substantially cheaper In an expression profiling experiment based on m samples and n assays traditional set up requires m RT reactions plus mxn qPCR controls while u
6. color your favourite qPCR mas termix to easily visualize where the reagent is loaded to your plates and tubes VisiBlue is very easy to use by simple addition to your favorite master mix CelluLyser for rapid and easy lysis and cDNA synthesis The CelluLyser Lysis and cDNA Synthesis Kit enables you to generate cDNA from small samples with minimal losses and hands on time It is particularly useful for single cell analysis By using CelluLyser the entire workflow from cell lysis to RT and qPCR can be performed without washing steps thus elimi nating material loss TATAA Interplate Calibrator Variation Compensation For practical reasons many qPCR studies involve the use of samples that are processed in more than a single batch or in which the sample set is extended over time Even over a short time period variation between qPCR processing runs is observed due to different baseline subtractions and threshold settings The TATAA Interplate Calibrator IPC is used to compensate for the variation between qPCR runs Express your genius TATAA Biocenter with offices in Gothenburg San Francisco and Prague is the leading provider of real time PCR services and the prime organizer of real time PCR work shops globally TATAA Biocenter con ducts commissioned research and training within the field of molecu Q tataabiocenter lar diagnostics and gene expression analysis along with developing real time PCR expression panels
7. User Manual ValidPrime Control for Genomic Background Human and Mouse Probe protocol Version 1 1 September 2012 For use in quantitative real time PCR Q tataabiocenter Table of contents Background gies een Traditional approach based on RT controls ValidPrime nn AA nn Storage Additionally required materials AN devices nnn Amplification protocol vee Pipetting protocol GENEX nun Troubleshooting References AA CN License information Other products from TATAA eine HL dsDNase GenEx software Reference Gene Panel Human or Mouse VisiBlue qPCR mix colorant CelluLyser for rapid and easy lysis and cDNA synthesis TATAA Interplate Calibrator Variation Compensation ValidPrime 10 10 11 12 13 13 13 13 14 Background For accurate gene expression analysis the measured Ca shall reflect the amount of gene transcript present in the sample This requires that the assay is specific and selective for the targeted cDNA and contributions to the signal from primer dimers pseudogenes and genomic DNA are negligible To test for primer dimer formation qPCR is performed in absence of template no template control NTC and to test for genomic DNA gDNA background reverse transcription RT is performed in the absence of reverse transcriptase RT The NTC is sample independent and is performed only once to validate assay performance while
8. ayed for four GO s and ValidPrime including also the gDNA standard GOI A col ValidPrime Cq is shown in black Cq cou blue Cq RT Sample in orange ValidPrime in red Cq sii ValidPrii ValidPri GOI From the measured Cq s ee cq and Cq expected C_for RT col Sample A gDNA gDNA q j controls Cq pare calculated with Equation 2 and as before Equation 1 is used to correct for the gDNA background Table 3 GOI _ GOI ValidPrime ValidPrime Cq RTS 7 Cq gDNA CA sample Cq gDNA Equation 2 Gene 1 Gene 2 Gene 3 Gene 4 ValidPrime Gol Gol Gol Gol Gol Gol Gol Gol Gol Gol Gol Gol CO rie Cary Cana Cria Carro Carna Carro Carro Cana Ceri Carro Cana Sample 1 20 1 SESION 31 1 20 5 SOMOS Sample 2 Sample 3 ME 231 330 2310 318 23 5 319 23 50 30 8 341 3213 225 339 2250 323 332 3341 33 0 31 15 228 328 22 80 320 321 35 90 Sample 5 Table 3 Measured Cq oa calculated Cq using Equation 2 and calculated can using Equation 1 Contents A 6 months complimentary license for GenEx Enterprise the easiest way to correct RTqPCR data for gDNA background GenEx is market leading software for qPCR experimental design and data processing Read more on page 11 1 GenEx license per customer for first time users only Reference standard gDNA 50 ul or 100 ul C 200 ng pl We recommend using 10 ng per qPCR Val
9. idPrime assay primers for 250 rxns 250 ul of primer mix C 10 uM per primer or 1000 rxns 1000 ul of primer mix C 10 uM per primer ValidPrime assay probe for 250 rxns 125 ul of solution C 10 uM or 1000 rxns 500 ul of solution C 10 uM rxns qPCR reactions in 25 ul concentration 400 nM per primer 200 nM probe The ValidPrime assay amplifies agDNA sequence that is present in exactly one copy per haploid genome in a normal cell The sequence has no transcriptional activity and is not present in pure cDNA preparations The assay has very high PCR efficiency E gt 90 in tested commercial master mixes and produces neg ligible amount of primer dimer products Limit of detection LOD is estimated to 4 copies of DNA 0 01 ng of DNA limit of quantification LOQ is estimated to 32 copies of DNA 0 08 ng of DNA Probe is using FAM reporter and BHQ1 quencher ValidPrime Storage The ValidPrime kit can be stored for 1 month at 4 C For long term storage 20 C is recommended Repeated freeze thaw cycles should be avoided Vor tex thoroughly and spin down before use Additionally required materials and devices qPCR instrumentation The ValidPrime kit has been validated on Roche LightCycler 480 Biorad CFX Stratagene MxPro Rotorgene ABI 7500 Fast and is expected to perform excellent on related instruments The Val idPrime probe signal shall be measured on the instrument s FAM channel
10. poor quality Check the quality of the RNA before performing cDNA synthesis e My replicates are not tight With good quality cDNA and good pipetting technique very high reproduc ibility is expected Low amounts of cDNA can lead to higher variation Also low quality cDNA can lead to differences between replicates Check the accuracy and reproducibility of your pipettes It is also possible the qPCR instrument is malperforming get positive ValidPrime signal even after DNAse treatment Often DNAse treatment does not remove all DNA and qPCR will amplify a sin gle molecule Usually solution based DNase treatment is more efficient than column based DNase treatment You may also try the HL dsDNase from TATAA www tataa com which has superior performance to competing dsDNases Usually DNase treatment reduces the gDNA background enough to be ac counted for by ValidPrime and GenEx correction ValidPrime References Henrik Laurell Jason lacovoni David Svec Anne Abot Jean Jos Maoret Jean Francois Arnal Mikael Kubista Correcting RTqPCR data for genomic DNA background Nucleic Acids Res 2012 Mikael Kubista Vendula Rusnakova David Svec Bj rn Sj green and Ales Tichopad GenEx Data Analysis Software In qPCR in Applied Microbiology Editor Martin Filion Horizon Press 2012 Reorder information The ValidPrime kit can be ordered from TATAA by mail at order tataa com from our TATAA webshop on www tataa com o
11. r from the local TATAA distributor in your country Contact For more information about ValidPrime contact us at info tataa com License information PCR is covered by several patents owned by Hoffman La Roche Inc and Hoffman LaRoche Ltd Purchase of the ValidPrimeTM kit does not include or provide a license with respect to any PCR related patents owned by Hoffman La Roche or others TATAA Biocenter does not encourage or support the unauthorised or unlicensed use of the PCR process 13 14 Other products from TATAA HL dsDNase New generation DNase that is specific to double stranded DNA and can be ef ficiently inactivated by heating at 55 C It can be added to your RT reaction to efficiently remove any gDNA without degrading single stranded cDNA It is completely inactivated by the PCR and does not degrade the double stranded PCR product GenEx software Market leading software for qPCR analysis GenEx provides the appropriate tools to analyze qPCR gene expression data and to extract biologically relevant information from the measurements Reference Gene Panel Human or Mouse The panel contains primer sets for 12 commonly used human or mouse refer ence genes A perfect product for finding the most optimal reference gene for your samples A one year license of GenEx Standard software with GeNorm and Normfinder is also included in the kit VisiBlue qPCR mix colorant The VisiBlue mastermix colorant enables you to
12. sing ValidPrime only m n 1 controls are needed Table 1 No of controls 1 20 26 49 10 110 35 490 24 264 49 1176 48 528 73 2352 96 1056 107 121 4704 Traditional RT strategy ValidPrime Table 1 Total number of RT and qPCR controls needed to check for gDNA background using traditional RT approach and ValidPrime In an expres sion profiling experiment based on m samples and n assays traditional set up requires m RT reactions plus mxn qPCR controls while using ValidPrime only m n 1 controls are needed Traditional approach based on RT controls Presence of genomic background in RTqPCR expression profiling is conventionally assessed by running an RT control for each sample that is analyzed by qPCR for all the GOl s Any signal observed in these RT qPCR s is due to presence of contaminating DNA that is amplified by the qPCR assay designed for GOI A common criterion to accept the measured C as not being confounded by gDNA contamination is Can Can gt 5 The estimated GOI concentration is then accurate to at least 96 9 Figure 1 RT controls 100 90 80 70 60 50 40 30 20 10 0 of DNA 0 1 2 3 4 5 6 7 8 C RTO C RT Figure 1 Correlation of AC RT RT with percentage of gDNA in unknown sample Gol Gol If Cq RT Cq RT reflect the RNA concentration using eq
13. the cDNA We recommend heat labile dsDNase HL dsDNase which is efficiently heat inactivated and does not digest the PCR product either HL dsDNase is available from www tataa com Optionally reference cDNA Newly designed assays can be validated on cDNA libraries Several cDNA librar ies are available from www tataa com Amplification protocol Use the recommended amplification protocol for your master mix Optimal annealing temperature of the ValidPrime assay is about 60 C in most master mixes see table 4 for the optimum conditions in selected master mixes Either 2 or 3 step amplification protocol can be used Pipetting protocol Prepare a master mix for each assay using the protocol from the manufacturer including the recommended concentrations of primers and probe Prepare for at least one extra reaction so you do not run out of master mix during the pipetting An NTC is recommended to test for contamination of reagents We recommend 10 ng of gDNA per 10 ul qPCR which on most qPCR instruments should produce a C for the ValidPrime assay in the range 25 30 cycles ValidPrime GenEx Easiest is to use GenEx for correction of RTqPCR data for DNA background and for general qPCR data processing GenEx is market leading software for qPCR experimental design and data processing and is supported by all leading qPCR instrument manufacturers It offers user friendly optimized workflow for qPCR data pre processing and analysis
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