Gènes domestiques

Utilisation pour la normalisation de votre expérience de profilage de l’expression relative des gènes

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RT² Profiler PCR Array

Cat. No. / ID: 330231

RT² Profiler PCR Array

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Les Gènes domestiques sont destinés aux applications de biologie moléculaire. Ces produits ne sont pas conçus pour le diagnostic, la prévention ou le traitement des maladies.

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Trouvez ou créez les dosages et panels spécifiques qu’il vous faut pour identifier vos cibles biologiques d’intérêt.

Caractéristiques

Procédure simple pour identifier rapidement les gènes de référence stables
L’analyse intégrée des données en ligne facilite la sélection
Disponible en plaques de 96 et 384 puits ou en disques de 100 anneaux

Détails produit

Les RT² Profiler Array Housekeeping Genes PCR Arrays sont utilisées pour rechercher des gènes de normalisation potentiels à utiliser dans les analyses Pen real-time PCR. Les gènes domestiques codent pour des protéines qui sont généralement essentielles à l’entretien des fonctions cellulaires et qui restent souvent constantes dans la plupart des conditions expérimentales. Avec cette puce à ADN, vous pouvez facilement établir le profil d’expression de douze (12) gènes domestiques couramment utilisés dans huit (8) échantillons en utilisant la qPCR basée sur le SYBR-green.

Performances

Un système garanti

Les RT² Profiler PCR Arrays sont testés et optimisés en combinaison avec les RT² SYBR^® Green qPCR Mastermixes et le RT² First Strand Kit. Ce test signifie que les performances du RT² Profiler PCR Array sont garanties lorsque ces trois composants sont utilisés conjointement.

Sensibilité

Lorsque vous utilisez le RT² First Strand Kit, commencez par 1 ng ou 5 µg d’ARN total par échantillon.

Reproductibilité

Le système complet de puce à ADN pour PCR présente de fortes corrélations entre les réplicats techniques, les lots et les instruments, avec des coefficients de corrélation moyens > 0,99, ce qui garantit une détection fiable des différences d’expression entre les échantillons biologiques.

Spécificité

Le système de puces à ADN pour PCR, avec un ARN d’entrée de haute qualité, produit des bandes uniques de la taille prédite sans amorce ni autres produits secondaires, ce qui permet d’obtenir des résultats de real-time PCR d’une grande précision.

Efficacité uniforme de l’amplification par PCR

Une efficacité d'amplification pour PCR uniforme est indispensable pour que la technologie des puces à ADN pour PCR permette des comparaisons précises de l'expression génique pour tous les gènes et tous les échantillons. La combinaison unique de notre algorithme propriétaire de conception d’amorces et du test rigoureux de chaque dosage d’amorce garantit la haute performance de chaque dosage d’amorce sur les puces à ADN pour PCR.

Principe

Les RT² Profiler PCR Arrays sont des outils fiables pour l’analyse de l’expression d’un panel ciblé de gènes. Chaque plaque de 96 ou 384 puits, ou puce à ADN pour PCR de disque de 100 puits comprend des dosages d’amorces optimisés par le SYBR Green pour un panel de gènes pertinents, axés sur les voies ou les maladies, ayant fait l’objet de recherches approfondies. Les RT² Profiler PCR Arrays peuvent également être personnalisés pour contenir un panel de gènes adapté à vos intérêts de recherche spécifiques. La conception d’amorces de haute qualité et la formulation du RT² SYBR Green qPCR Mastermix permettent à la puce à ADN pour PCR d'amplifier simultanément 96 ou 384 produits différents spécifiques à un gène dans des conditions de cyclage uniformes.

Cette combinaison confère à la RT² Profiler PCR Array la spécificité et l’efficacité d’amplification élevées requises pour obtenir des résultats précis avec le SYBR Green en temps réel. Les puces à ADN pour PCR sont faciles à utiliser dans tout laboratoire de recherche.

Les RT² Profiler PCR Arrays sont suffisamment sensibles pour être utilisés avec de l’ARN préparé à partir d’échantillons ordinaires (0,1–5 –g d’ARN), d’échantillons FFPE et de petits échantillons (1–100 ng d’ARN).

Procédure

Il suffit de mélanger l’ADNc matriciel avec le mélange principal PCR prêt à l’emploi approprié, d’aliquoter des volumes égaux dans chaque puits de la même plaque, puis d’exécuter le programme de cyclage de real-time PCR. Les RT² Profiler PCR Arrays sont compatibles avec tous les instruments QIAGEN, ABI, Bio-Rad, Eppendorf, Roche et Stratagene.

Disposition et contrôles flexibles

Les RT² Profiler PCR Arrays sont disponibles en plaques de 96 ou 384 puits et disques de 100 puits, et sont utilisés pour la surveillance de l’expression de 84 ou 370 gènes liés à un état pathologique ou à une voie, plus 5 gènes domestiques. Chaque RT² Profiler PCR Array comprend également des éléments de contrôle pour :

La normalisation des données
La détection de la contamination par l’ADN génomique
La qualité de l’échantillon d’ARN
Les performances générales de la PCR

Une analyse des données facile à utiliser

Il est possible d’analyser les données à l’aide d’un modèle d’analyse de données basé sur Excel facile à utiliser ou d’un logiciel basé sur le Web. L’analyse des données est basée sur la méthode ΔΔC_T avec normalisation des données brutes par rapport à l’un ou l’autre des gènes domestiques.

Applications

Les RT² PCR Profiler Arrays peuvent être utilisés dans toutes les zones de la recherche biologique et médicale, notamment :

Cancer Inflammation et profilage des cytokines
Cellules souches
Neuroscience
Les voies de transduction du signal
L’adhésion cellulaire et la migration cellulaire
Dépistage et validation des biomarqueurs

Ressources

Simultaneously profile mRNA, miRNA and lncRNA using a simple, complete workflow

For analyzing gene expression data from RT2 Profiler PCR Arrays

Data analysis file for RT² ProfilerRT² Profiler™ PCR Array RT2 RNA QC
Catalog number- 330231
Pathway number- PAXX-999

Poster for download

For pathway-focused gene expression profiling using real-time RT-PCR

For gene expression and genomic analysis

For pathway-focused gene expression analysis

FAQ

The best way to set the threshold is to make sure that your PPC values are between 18 and 22. I would look my first PCR Array, set it so that the PPC is at 20, and see if the same threshold fits for the rest of the arrays.

FAQ ID -2705

Each PCR Array has 5 housekeeping genes. You can use one or an average of the most stable ones to do data analysis.

FAQ ID -2704

There are less than a dozen genes encoded by the mitochondrial genome (all other mitochondrial proteins are encoded by nuclear genes), and they are all transcribed as one transcript (just like any prokaryote), so distinguishing the expression of individual genes by real-time RT-PCR is not possible.

FAQ ID -2680

The RT² Profiler PCR Array is a 96-/384-well plate or 100-well disc that contains gene-specific Primer Assays for a thoroughly researched set of relevant, pathway- or disease-focused genes. It simultaneously profiles the expression of 84 pathway-specific genes, and five housekeeping genes. Each RT² Profiler PCR Array also includes a Genomic DNA Control (GDC) assay, triplicate Reverse Transcription Controls (RTC), and triplicate Positive PCR Controls (PPC).

FAQ ID -2718

If you are unsure of the correct housekeeping gene(s), review the literature and technical information in your field to determine which gene(s) other researchers commonly use. It is recommended that multiple housekeeping genes be utilized for each gene expression experiment, to account for any impact that an experimental condition may have on the expression of an individual housekeeping gene. For a systematic assessment of which housekeeping genes are appropriate for your specific experimental conditions, we recommend using the Housekeeping Genes RT2 Profiler PCR Arrays for human (330231 PAHS-000), mouse (330231 PAMM-000), or rat (330231 PARN-000). These arrays consist of 8 sets of 12 common housekeeping genes. They are a valuable tool for easily identifying genes with a constant level of expression among your different experimental conditions.

FAQ ID -2674

Be sure to carefully and completely seal the qPCR assay plate with fresh, optical, thin-wall, 8-cap strips or adhesive optical film before the plate is placed into the real-time cycler. In addition, refer to your instrument's user's manual to determine whether the real-time cycler manufacturer recommends use of a plate compression pad during the run.

FAQ ID -2679

No. Data Analysis can be done with a little as 2 PCR Arrays. Whether or not you run a sample in triplicate is determined by experimental setup and what you are going to use the data for.

FAQ ID -2703

With the additional RT2 PreAMP methodology, only 1 ng of RNA is now needed for PCR Array analysis. Pooling RNA from different sources should only be done when there is not enough sample. We recommend running biological replicates.

FAQ ID -2663

Any DNA contamination will artificially inflate the SYBR Green signal, yielding skewed gene expression profiles and false-positive signals. The most common source of DNA contamination is from PCR products generated during previous experiments. Such contamination is most often due to the improper disposal of tubes, tips, and gels that previously came into contact with PCR products. Additionally, PCR products may also contaminate pipettors, racks, work pads, and commonly used reagents such as water and buffers. To minimize the risk of contaminating your experiment with extraneous DNA, the following steps should be taken:

Remove a single aliquot of water from your PCR-grade stock, sufficient to complete the experiment. This minimizes the number of times that the stock container is opened, thereby minimizing contamination risks.
Use only fresh PCR-grade reagents and disposable labware.
Treat any labware (tubes, tips, and tip boxes) used in PCR with 10% bleach, before discarding.
Maintain a dedicated workspace for PCR setup (perhaps a PCR-only hood), away from areas of the lab where post-PCR work is done, such as running gels, enzyme digestions, and cloning.
Change the lab bench pads/papers often and decontaminate lab benches and labware (racks, pipettors, etc.) before each use by washing with 10% bleach, and/or exposing to UV light for at least 10 minutes. This serves to degrade and/or inactivate contaminating DNA.
Before, during, and after the experiment, minimize the opening and closing of any tubes or plates used during the experiment.

FAQ ID -2654

For real-time detection, the RT² Profiler PCR Array is currently available for most QIAGEN, ABI, BioRad, Eppendorf, Stratagene, TaKaRa, Fluidigm, Cepheid, and Roche real-time instruments. Please refer to the link below, to determine which RT² Profiler PCR Array plate format is compatible with your instrument.

http://www.sabiosciences.com/manuals/PCRArrayGuide.pdf

FAQ ID -2719

The 3 most common negative controls included in a qPCR and/or qRT-PCR experiment are as follows:

1. A no template control (NTC) omits any DNA or RNA template from a reaction, and serves as a general control for extraneous nucleic acid contamination. When using SYBR Green chemistry, this also serves as an important control for primer dimer formation. Within the RT2 Profiler PCR Arrays, the GDC well also serves as a no template control, as this assay is designed to detect Genomic DNA.

2. A no reverse transcriptase control (NRT) or minus reverse transcriptase control (MRT) involves carrying out the reverse transcription step of a qRT-PCR experiment in the absence of reverse transcriptase. This control assesses the amount of DNA contamination present in an RNA preparation.

3. A no amplification control (NAC) omits the DNA polymerase from the PCR reaction. This is a control for background fluorescence that is not a function of the PCR. Such fluorescence is typically attributable to the use of a degraded, dual-labeled probe. This control is unnecessary when utilizing SYBR-Green probe chemistries.

FAQ ID -2672

There is no need to run a standard curve before doing the RT2 PCR Array experiment. Usually we recommend starting with 1000 ng total RNA for a 96-well PCR array.

FAQ ID -2664

We recommend the use of RNeasy Mini Kits. Cultured cells, and freshly isolated white blood cells, may be harvested by centrifugation, and used directly with this kit. For the isolation of high-quality RNA from animal tissues, we recommend RNeasy Plus Universal Kit.

FAQ ID -2657

The qBiomarker PCR Arrays contain gene lists that have been biologically validated and selected to measure the expression of a limited number of genes that are highly predictive for a biological process. Each qBiomarker PCR Array is designed to analyze multiple samples on the same 96-well or 384-well PCR plate. These arrays are best suited for screening and validation applications for a specific biological process. In contrast, the RT2 Profiler PCR Arrays typically have 84 pathway focused genes which are selected based on a different bioinformatic process and are best suited for gene expression profiling applications where a relative fold change result, and not a predictive answer, is necessary.

FAQ ID -2438

Assuming 100% amplification efficiency, each step increase in Ct value represents a doubling in the amount of qPCR template. Therefore, evaluating the difference in Ct values between the qPCR assay, and its matching NRT control, leads to the following predictions:

CtNRT - Ct+RT	Fraction of gene expression signal due to contaminating DNA	Percentage of gene expression signal due to contaminating DNA
1	(1/21) = 1/2	50%
2	(1/22) = 1/4	25%
3	(1/23) = 1/8	13%
4	(1/24) = 1/16	6%
5	(1/25) = 1/32	3%

FAQ ID -2688

The most rigorous method to detect genomic DNA contamination, particularly with the RT² qPCR Primer Assays, is to perform a No Reverse Transcriptase (NRT) control. The PCR will have no cDNA template derived from mRNA, and any detectable product could only have been derived from genomic DNA contamination.

FAQ ID -2687

You may be using too much template. Use less input total RNA for reverse transcription, or use template at a greater dilution factor (lower concentration). Do not pipet a volume of less than 1 μl.

FAQ ID -2684

Dissociation curves are carried out at the end of a PCR experiment by following a 3-step procedure.

First, all the components are denatured at 95°C, followed by complete annealing at a set temperature (based on the primer Tm values), followed by a gradual increase in temperature up to 95°C. Fluorescence intensity is monitored during this final temperature increase, resulting in the generation of a melting curve or dissociation curve.

By analyzing the first derivative of such a curve, you can readily assess the homogeneity of the PCR products, including the presence of primer–dimers, thereby determining the specificity of the PCR reaction. It is important to carry out such post-PCR analyses when using SYBR Green probe chemistry due to this reagent's lack of sequence specificity.

FAQ ID -2678

It is critical to include appropriate positive controls in a qPCR experiment to determine if false negatives are being detected in the experiment. Positive controls fall into one of 2 classes.

1. Exogenous positive controls refer to the use of external DNA or RNA carrying a target of interest. If these positive controls are assayed in separate wells/tubes from the experimental sample, they serve as a control to determine whether or not the reverse transcription and/or PCR reaction conditions are optimal. Additionally, exogenous DNA or RNA positive controls may be spiked into the experimental sample(s), and assayed in parallel or in a multiplex format with, the target of interest. These control reactions assess whether the samples contain any components that inhibit reverse transcription and/or PCR.

2. Endogenous positive controls refer to the use of a native target that is present in the experimental sample(s) of interest, but is different from the target under study. These types of controls are often referred to as normalizers, and are typically used to correct for quantity and quality differences between samples.

Within the RT2 Profiler PCR Arrays, the Positive PCR Control (PPC) wells contain a plasmid with a primer assay that detects a sequence it produces. This allows for quick confirmation of the performance of the PCR steps.

The RTC wells include assays that detect the artificial RNA that is spiked in to each sample during the cDNA synthesis step. This ensures the Reverse Transcription step proceeded as needed.

FAQ ID -2673

The Reverse transcription control requires that the reverse transcription is done with the RT² first strand kit. No other cDNA synthesis method can use this control.

FAQ ID - 3534

18S ribosomal RNA is a widely used control for qRT-PCR analyses because of its invariant expression across tissues, cells, and experimental treatments. However, due to its extremely high expression in most cell types, it can sometimes be challenging to use 18S rRNA as an endogenous normalizer for several gene expression assays in the same reaction.

FAQ ID -2675

You can manually set the threshold line. If you are using a catalogued PCR Array, the PPC values should be 20 +/- 2 Cts. Use the same threshold on all of your PCR Arrays.

FAQ ID -2702

The Rn value, or normalized reporter value, is the fluorescent signal from SYBR Green normalized to (divided by) the signal of the passive reference dye for a given reaction. The delta Rn value is the Rn value of an experimental reaction minus the Rn value of the baseline signal generated by the instrument. This parameter reliably calculates the magnitude of the specific signal generated from a given set of PCR conditions. For more information, please refer to your cycler's user manual.

FAQ ID -2681

We determine the amplification efficiency during wet bench testing of our assays using standard curve dilutions, or by single curve analysis. If you would like to calculate the efficiency of each curve using single curve analysis, then you can try Real-Time PCR Miner, LinReg or Dart PCR. Each of these can be found using a GOOGLE search.

FAQ ID -2701

Yes, all you need to do is to organize your data into a “custom PCR Array” file. When you upload it to the website, use the custom PCR array name CUSTOM. The locations of the blank excel spreadsheet is: http://www.sabiosciences.com/pcrarraydataanalysis.php#custom

FAQ ID -2698