Manual: Stratagene QPCR Human Reference Total RNA
Stratagene QPCR Human Reference Total RNAInstruction ManualCatalog #750500Revision BResearch Use Only. Not for Use in Diagnostic Procedures.750500-12
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Stratagene QPCR Human Reference Total RNACONTENTSMaterials Provided. 1Storage Conditions . 1Additional Materials Required . 1Introduction. 2Assay Optimization Applications. 2Assay Standardization Applications. 3Sample Standard Curve Data Generated with the Stratagene QPCR Human ReferenceTotal RNA . 3QRT-PCR Assay Optimization Considerations . 5SYBR Green Assays. 5TaqMan Assays . 5Molecular Beacons Assays. 6Use of a Passive Reference Dye . 7Assay Standardization Considerations . 7QRT-PCR Assay Optimization Protocol. 8Preparing QRT-PCR Reactions. 8RT-PCR Program . 9QRT-PCR Standard Curve Protocol . 10Generating a Standard Curve. 10RT-PCR Program . 12Troubleshooting . 13References . 14Endnotes. 14MSDS Information. 14
Stratagene QPCR Human Reference Total RNAMATERIALS PROVIDEDCatalog #750500Materials providedQuantityStratagene QPCR Human Reference Total RNA25 μg (1 μg/μl in 0.1 mM EDTA/RNase-free H2O)STORAGE CONDITIONSAll Components: –80 CADDITIONAL MATERIALS REQUIREDSpectrofluorometric thermal cyclerQRT-PCR reagents [e.g. Stratagene Brilliant II QRT-PCR Master Mix Kit, 1-Step (Catalog#600809)]Nuclease-free PCR-grade waterRevision BStratagene QPCR Human Reference Total RNA Agilent Technologies, Inc. 2010.1
INTRODUCTIONStratagene QPCR Human Reference Total RNA is a high-quality control forquantitative PCR gene-expression analysis. QPCR Human Reference TotalRNA is composed of total RNA from 10 human cell lines (see the tablebelow), with quantities of RNA from the individual cell lines optimized tomaximize representation of gene transcripts present in low, medium, andhigh abundance. The reference RNA is carefully screened for contaminatinggenomic DNA, the presence of which can complicate interpretation ofQRT-PCR assay data.Stratagene QPCR Human Reference Total RNA Cell Line DerivationsAdenocarcinoma, mammary glandHepatoblastoma, liverAdenocarcinoma, cervixEmbryonal carcinoma, testisGlioblastoma, brainMelanoma, skinLiposarcomaHistiocytic lymphoma; macrophage; histocyteLymphoblastic leukemia, T lymphoblastPlasmacytoma; myeloma; B lymphocyteAssay Optimization ApplicationsThe Stratagene QPCR Human Reference Total RNA is ideally suited foroptimizing QRT-PCR assays. Often only small amounts of experimentalRNA template are available for setting up an expression profiling study.Using the extensive representation of specific mRNA species in the generictemplate, assays may be optimized for a variety of primer/probe systems.This eliminates the use of precious experimental RNA samples for assayoptimization.Protocols for RT-PCR assay optimization using the QPCR HumanReference Total RNA in conjunction with Stratagene’s Brilliant II QRTPCR master mix, 1-step (for TaqMan or molecular beacons probes) areprovided in the Assay Optimization Protocols section. These protocols andguidelines may be adapted for use with other reagent systems and platforms.Guidelines for SYBR Green dye assay optimization are also provided inQRT-PCR Assay Optimization Considerations. The Stratagene QPCRHuman Reference Total RNA is especially useful in optimization of SYBRGreen assays. SYBR Green I dye binds non-selectively to any doublestranded DNA, and thus detects primer-dimers as well as the specificamplicon. This problem is especially pronounced at low targetconcentrations. Very careful primer optimization is required to minimize theformation of non-specific amplification products. Use of the StratageneQPCR Human Reference Total RNA allows extensive experimentationduring assay development without depleting the experimental RNA.2Stratagene QPCR Human Reference Total RNA
Assay Standardization ApplicationsUsing the QPCR Stratagene Human Reference Total RNA as a referencecontrol allows data comparisons from multiple experiments, acrossplatforms, and between laboratories. The reference RNA is produced inextremely large lots and subjected to stringent quality-control measures toensure the availability of consistent reference RNA material over long-termexperimental studies. See Assay Standardization Considerations for moreinformation.Sample Standard Curve Data Generated with the Stratagene QPCRHuman Reference Total RNAResults from an RT-PCR reaction monitored in real-time can be displayedas an amplification plot, which reflects the change in fluorescence duringcycling. This information can be used to quantitate initial copy number1based on the threshold cycle (Ct). Ct is defined as the cycle at whichfluorescence is determined to be statistically significant above background1and is inversely proportional to the log of the initial copy number. The moretemplate that is initially present, the fewer the number of cycles it takes toreach the point where the fluorescence signal is detectable abovebackground. The threshold cycle is based on measurements taken during theexponential phase of PCR amplification when PCR efficiency is not yetinfluenced by limiting reagents, small differences in reaction components, orcycling conditions. Ct values determined for a set of standard wells,containing known amounts of the template RNA, may be plotted to generatea standard curve that can be used to assess the quality of the QRT-PCRassay. Standard curve data may also be used to compare and assessdifferences in results obtained from the same assay system acrossexperiments, platforms, or researchers.Figure 1 shows standard curves for targets of high, medium and lowabundance for serial dilutions of the Stratagene QPCR Human ReferenceTotal RNA. The standard curve data were generated on the Mx4000instrument for QRT-PCR reactions prepared with the Brilliant II QRT-PCRmaster mix, 1-step, according to the protocol provided in this manual. Thetable below shows the R2 values and standard curve slopes calculated by theinstrument for each target. The R2 value (always between 0 and 1) is anindication of the quality of the fit of the standard curve to the standard datapoints plotted, with values closer to 1 indicating a better fit of the data to theline. The slope of the standard curve is directly related to the averageefficiency of amplification throughout the cycling program and may be usedto calculate the PCR efficiency for a given template in a given experiment.A reaction with 100% efficiency will produce a slope of –3.32.TargetTarget AbundanceR2 000-3.3798.0IL-5low0.998-3.29101.3Stratagene QPCR Human Reference Total RNAEfficiency (%)3
50Average Ct4030CyclophilinGUS20IL-5100-10123Log Initial Quantity Reference RNA (ng)FIGURE 1 Standard curves for the high-abundance target cyclophilin (bottom curve; diamonds), the medium-abundancetarget GUS (middle curve; squares) and the low-abundance target IL-5 (top curve; triangles). The Stratagene QPCR HumanReference Total RNA was added as 10-fold serial dilutions from 1000 ng to 0.1 ng. QRT-PCR reactions were prepared usingthe Brilliant QRT-PCR master mix, 1-step, and TaqMan probes. The real-time fluorescence data were analyzed on theMx4000 multiplex quantitative PCR instrument.4Stratagene QPCR Human Reference Total RNA
QRT-PCR ASSAY OPTIMIZATION CONSIDERATIONSSummary of Primer and Probe Optimization RecommendationsPrimer ConcentrationOptimization Range (nM)Detection ChemistrySYBR Green I dye50–300TaqMan probe Molecular beaconProbe ConcentrationOptimization Range (nM)—50–600100–500200–600200–500SYBR Green AssaysPrimer Optimization for SYBR Green AssaysDetection with SYBR Green I dye requires very careful primer optimization.It is critical to minimize the formation of non-specific amplificationproducts. This issue becomes more prominent at low target concentrations.To maximize the sensitivity of the assay, it is necessary to use the lowestconcentration of primers possible without compromising the efficiency ofPCR. The optimal concentration of the upstream and downstream PCRprimers is the lowest concentration that results in the lowest Ct and anadequate fluorescence for a given target concentration, with minimal or noformation of primer-dimers. Titrate the concentration of each primer in therange of 50 to 300 nM. The best concentrations of the upstream anddownstream primers are not always of equal molarity.Use of Dissociation Profiles in Primer OptimizationTarget quantification can be inaccurate due to the contributions of primerdimers to the measured fluorescence intensity. Running a dissociationprofile for determining the amount of primer-dimers is stronglyrecommended. The primer concentrations resulting in the lowest occurrenceof primer-dimers and the lowest Ct should be chosen.PCR ProtocolsFor SYBR Green I dye detection, three-step PCR cycling protocols arepreferred over two-step PCR protocols. Set the QPCR instrument to acquiredata at the annealing and the extension steps.TaqMan AssaysPrimer Optimization for TaqMan AssaysWhen developing a QRT-PCR assay, it is generally advantageous tooptimize the primer concentration first, and then complete the probeoptimization using the optimized primer concentrations. Titrate theconcentration of each PCR primer in the range of 50 to 600 nM. The optimalconcentration is the lowest concentration that results in the lowest Ct and anadequate fluorescence for a given target concentration. The bestconcentrations of the upstream and downstream primers are not always ofequal molarity.Stratagene QPCR Human Reference Total RNA5
TaqMan Probe Design and OptimizationProbes should have a melting temperature that is 7–10 C higher than theannealing temperature of the PCR primers. For additional considerations indesigning TaqMan probes, refer to Primer Express oligo design softwarefrom Applied Biosystems.Resuspend lyophilized custom TaqMan probes in buffer containing 5 mMTris-HCl, pH 8.0, and 0.1 mM EDTA (low TE buffer).The optimal TaqMan probe concentration should be determined empirically.The optimal concentration is the lowest concentration that results in thelowest Ct and an adequate fluorescence for a given target concentration. TheTaqMan probe concentration can be optimized by varying the finalconcentration from 100 to 500 nM in increments of 100 nM.PCR ProtocolsTwo-step PCR cycling protocols (including a denaturation step and anannealing/extension step) are generally recommended for TaqMan assays.Set the QPCR instrument to acquire data at the annealing/extension step.Molecular Beacons AssaysPrimer Optimization for Molecular Beacons AssaysWhen developing a QRT-PCR assay, it is generally advantageous tooptimize the primer concentration first, and then complete the probeoptimization using the optimized primer concentrations. Titrate theconcentration of each PCR primer in the range of 200 to 600 nM. Theoptimal concentration is the lowest concentration that results in the lowestCt and an adequate fluorescence for a given target concentration. The bestconcentrations of the upstream and downstream primers are not always ofequal molarity.Molecular Beacons Probe Design and OptimizationProbes should have a melting temperature that is 7–10 C higher than theannealing temperature of the PCR primers.Resuspend lyophilized custom molecular beacons probes in buffercontaining 5 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA (low TE buffer).The optimal concentration should be determined empirically for eachmolecular beacon. The optimal concentration is the lowest concentrationthat results in the lowest Ct and an adequate fluorescence for a given targetconcentration. The molecular beacon concentration can be optimized byvarying the final concentration from 200 to 500 nM in increments of100 nM.PCR ProtocolsThree-step PCR cycling protocols (including separate denaturation,annealing and extension steps) are generally recommended for molecularbeacons assays. Set the QPCR instrument to acquire data at the annealingand extension steps.6Stratagene QPCR Human Reference Total RNA
Use of a Passive Reference DyeIt is good practice to include a passive reference dye in QRT-PCR reactionsto compensate for non-PCR related variations in fluorescence. Fluorescencefrom the passive reference dye does not change during the course of thePCR reaction but provides a stable baseline to which samples arenormalized. In this way, the reference dye compensates for changes influorescence between wells caused by slight volume differences in reactiontubes. The reference dye is generally diluted before use in a QRT-PCRreaction, with the extent of dilution dependent on the correspondence ofexcitation and emission wavelengths of the reference dye compared to theexcitation and detection properties of the instrument. Consult the referencedye manufacturer’s protocol for dilution recommendations.ASSAY STANDARDIZATION CONSIDERATIONSIntra-laboratory and inter-laboratory standardization of assays is importantto allow data comparisons across experiments, platforms, or researchers.Within a single laboratory, changes in the performance of the real-timereader and in the reagents used cannot be detected unless an independentstandard is included in the runs. Including standard curve determinationsusing the Stratagene QPCR Human Reference Total RNA provides a meansfor comparing the results from different experiments. It is recommended thatreference RNA standard curve reactions be included at regular intervals toconfirm amplification and detection performance.When two or more laboratories need to compare data, the Stratagene QPCRHuman Reference Total RNA can be used as an inter-laboratory standard.The laboratories involved should run a defined set of reactions (generallystandard curves) on their respective platforms. It is important that thelaboratories use the same sets of reagents (shared primers and probes andthe same supplier for kit reagents) and identical protocols. After the standardcurves have been generated on the various platforms in the variouslaboratories statistical analysis will reveal the quality of each standard curve(the R2-value, slope, y-axis intercept), the sensitivity of the assay/instrumentsystem and the absolute quantification. Statistical analysis methods forcomparing data generated in various laboratories is to date not standardized.One way of analyzing these data is utilizing the calculation of theConfidence Interval (CI) for the data points of the standard curve. Reference2 provides a useful discussion of the calculation and use of confidenceinterval values in comparing data sets. With the CI calculated from theStratagene QPCR Human Reference Total RNA standard curve the qualityof the experimental sample data can be predicted and compared toexperimental sample data generated on another platform.Stratagene QPCR Human Reference Total RNA7
QRT-PCR ASSAY OPTIMIZATION PROTOCOLPreparing QRT-PCR ReactionsNoteThe following assay optimization protocol uses the StratageneBrilliant II QRT-PCR master mix kit, 1-step (catalog #600809),for TaqMan or molecular beacons assays analyzed on aStratagene Mx3000P, Mx3005P, or Mx4000 instrument or the ABIPRISM 7900HT instrument. Other single-tube or two-tubeQRT-PCR reagents may be used (please follow the reagentmanufacturer’s instructions for preparing the reaction mixture).Other QPCR platforms may also be used (please consult theinstrument manufacturer’s instructions for reference dyepreparation and RT-PCR program recommendations).Use the following reaction conditions to optimize the primerconcentrations first, then the probe concentrations. See QRT-PCRAssay Optimization Considerations for the concentration rangesrecommended for each probe system.1.If the reference dye will be included in the reaction, (optional), dilutethe dye solution provided 1:500 (for the Mx3000P, Mx3005P, andMx4000 instruments) or 1:50 (for the ABI Prism 7900HTinstrument) using nuclease-free PCR-grade H2O. When usedaccording to the protocol below, this will result in a final reference dyeconcentration of 30 nM for the Mx3000P, Mx3005P, and Mx4000instruments and 300 nM for the ABI PRISM 7900HT instrument. Keepall solutions containing the reference dye protected from light.NoteIf using a system other than the Mx4000, Mx3000P orMx3005P instruments, the use of the reference dye may berequired for optimal results.2.Thaw the 2 QRT-PCR master mix and store the so
2 Stratagene QPCR Human Reference Total RNA INTRODUCTION Stratagene QPCR Human Reference Total RNA is a high-quality control for quantitative PCR gene-expression analysis.
Introduction to Quantitative PCR Whether you are a novice or experienced user, our goal is to ensure that you are running quantitative PCR (QPCR) experiments quickly, efficiently, and affordably. Our Mx family of QPCR Systems, MxPro QPCR Software, premiere QPCR Systems Service Program, complete line of QPCR and QRT-PCR reagents, and Fast
The TaqPath 1‐Step RT‐qPCR Master Mix User Guide provides a general protocol for performing 1‐step real‐time reverse transcription quantitative PCR (RT‐qPCR) using TaqPath 1‐Step RT‐qPCR Master Mix. Revision Date Description 1.0 10 September 2013 User Guide to accompany new product introduction.
time quantitative PCR (qPCR). As miRNA analysis is a fast changing research field, we have introduced novel technological approaches and compared them to existing qPCR profiling methodologies. qPCR also remains the method of choice for validating results obtained from whole-genome screening (e.g. with microarray).
(iQ SYBR Green supermix) directly from cell lysate preparations. RFU, relative fluorescence units. iScript RT-qPCR sample preparation reagent method: Collect for 10 min Standard method: 30 min DNase treatment Lysis Spin Bind to column matrix RT-qPCR Elute for RT-qPCR 10 min 30 m
device were captured, eluted, and detected by both qPCR and IMS/IFA assays. Three independent experiments were performed for a total of 9 data points for qPCR and 5 replicates were performed for IMS/IFA. Similarly poliovirus bottom filters were analyzed for a total of 6 data points for qPCR and 3 for plaque assay.
7000/7700/7900HT Sequence Detection Systems and the Applied Biosystems 7300 Real Time PCR System, the use of ROX Reference Dye LSR (50X) is recommended. For the Applied Biosystems 7500 Real Time PCR System and the Stratagene Mx3000P, the use of ROX Reference Dye LMP is recommended. The use of ROX Reference Dye LSR or LMP is optional. It
Research Article Selection and Validation of Reference Genes for RT-qPCR Analysis in Spinacia oleracea under Abiotic Stress Hao Xie ,1,2 Bo Li,1 Yu Chang,1 Xiaoyan Hou,2 Yue Zhang,1 Siyi Guo,3 Yuchen Miao,3 Quanhua Wang,2 Sixue Chen,4 Yinghua Su,5 Ying Li ,1 and Shaojun Dai 2 1Key Laboratory of Saline-Alkali Vegetation Ecology Restoration (Northeast Forestry University), Ministry of Education .
Tulang-tulang pembentuk rangka tubuh . 12 3. Tulang-tulang di regio manus tampak . Anatomi hewan ini yang dipelajari adalah anatomi tubuh hewan piara. Pelaksanaan perkuliahan dan praktikum anatomi hewan dilakukan setiap minggu sesuai jadwal dengan beban 3 sks (1-2) pada mahasiswa semester 1. Pelaksanaan meliputi tutorial, pretest, praktikum di laboratorium, pembuatan laporan, dan ujian .
