하우스키핑 유전자

상대 유전자 발현 프로파일링 실험을 정규화하는 데 사용

GeneGlobe에서 구성

관심 있는 생물학적 표적을 연구하는 데 적합한 표적별 분석 및 패널을 찾거나 맞춤 설계를 진행합니다.

RT² Profiler PCR Array

Cat. No. / ID: 330231

RT² Profiler PCR Array

GeneGlobe에서 구성 가격 보기

GeneGlobe에서 구성

하우스키핑 유전자은/는 분자생물학 분야에 사용하기 위한 것입니다. 이 제품들은 질병의 진단, 예방, 또는 치료 목적으로 사용할 수 없습니다.

GeneGlobe에서 구성

관심 있는 생물학적 표적을 연구하는 데 적합한 표적별 분석 및 패널을 찾거나 맞춤 설계를 진행합니다.

특징

안정적인 참조 유전자를 신속하게 식별하는 간단한 절차
통합된 온라인 데이터 분석으로 손쉬운 선택
96-well 및 384-well 플레이트 또는 링 디스크 100개로 제공

제품 세부 정보

RT² Profiler Array Housekeeping Genes PCR Arrays는 real-time PCR 분석에 사용할 잠재적 정규화 유전자를 스크리닝하는 데 사용됩니다. 하우스키핑 유전자는 일반적으로 세포 기능을 유지하는 데 필수적이며 대부분의 실험 조건에서 보통 일정하게 유지되는 단백질을 암호화합니다. 이 어레이가 있으면 SYBR-Green 기반 qPCR을 통해 샘플 8개에서 일반적으로 사용되는 하우스키핑 유전자 12개의 발현을 손쉽게 프로파일링할 수 있습니다.

성능

보장된 시스템

RT² Profiler PCR Arrays는 RT² SYBR^® Green qPCR 마스터 믹스 및 RT² First Strand Kit와 함께 테스트되고 최적화되었습니다. 이 테스트는 이 세 가지 구성 요소를 모두 함께 사용할 때 RT² Profiler PCR Array 성능이 보장됨을 나타냅니다.

민감도

RT² First Strand Kit를 사용하는 경우 샘플당 총RNA를 1ng 이상 5µg 이하로 시작합니다.

재현성

전체 PCR 어레이 시스템은 평균 상관 계수가 0.99를 초과하는 기술적 복제물, 로트 및 장비 전반에 걸쳐 강력한 상관관계를 보여주므로 생물학적 샘플 간의 발현 차이를 안정적으로 검출할 수 있습니다.

특이성

PCR 어레이 시스템은 고품질의 RNA 투입 시 프라이머-다이머(dimer) 또는 기타 2차 산물 없이 예측된 크기의 단일 밴드를 생성하므로 매우 정확한 real-time PCR 결과를 제공합니다.

균일한 PCR 증폭 효율성

PCR 어레이 기술로 모든 유전자 및 모든 샘플에 걸쳐 유전자 발현을 정확하게 비교하기 위해서는 PCR 증폭 효율성이 균일해야 합니다. QIAGEN의 독자적인 프라이머 설계 알고리즘과 모든 프라이머 분석에 대한 엄격한 테스트의 고유한 조합은 PCR 어레이의 모든 프라이머 분석에 대해 높은 성능을 보장합니다.

원리

RT² Profiler PCR Arrays는 집중된 유전자 패널의 발현을 분석하는 신뢰할 수 있는 도구입니다. 각 96-well 플레이트, 384-well 플레이트 또는 100-well 디스크 PCR 어레이에는 철저하게 연구한 관련 유전자, 경로 또는 질병에 중점을 둔 유전자 패널을 위해 SYBR Green 최적화 프라이머 분석이 포함되어 있습니다. RT² Profiler PCR Arrays는 특정 연구 관심 분야에 맞는 유전자 패널을 포함하도록 맞춤화할 수도 있습니다. 고품질 프라이머 설계와 RT² SYBR Green qPCR 마스터 믹스 제제를 사용하면 PCR 어레이가 균일한 사이클링 조건에서 96개 또는 384개의 다양한 유전자 특이적 산물을 동시에 증폭할 수 있습니다.

이러한 조합은 RT² Profiler PCR Array에 정확한 실시간 SYBR Green 결과를 위해 필요한 특이성과 높은 증폭 효율성을 제공합니다. PCR 어레이는 모든 연구실에서 쉽게 사용할 수 있습니다.

RT² Profiler PCR Arrays는 일반 샘플(RNA 0.1–5µg), FFPE 샘플, 소형 샘플(RNA 1–100ng)에서 준비한 RNA와 함께 사용하기에 충분히 민감합니다.

절차

cDNA 템플릿을 즉시 사용 가능한 적절한 PCR 마스터 믹스와 혼합하고 동일한 플레이트의 각 웰에 동일한 양을 분주한 다음 real-time PCR 사이클링 프로그램을 실행하기만 하면 됩니다. RT² Profiler PCR Arrays는 모든 QIAGEN, ABI, Bio-Rad, Eppendorf, Roche 및 Stratagene 기기와 호환됩니다.

유연한 레이아웃 및 제어

RT² Profiler PCR Arrays는 96-well 플레이트, 384-well 플레이트 및 100-well 디스크 형태로 제공되며, 질병 상태나 경로와 관련된 유전자 84개 또는 370개 그리고 하우스키핑 유전자 5개의 발현을 모니터링하는 데 사용됩니다. 각 RT² Profiler PCR Array에는 다음을 위한 제어 요소도 포함되어 있습니다.

데이터 정규화
유전체 DNA 오염 검출
RNA 샘플 품질
일반 PCR 성능

사용하기 쉬운 데이터 분석

사용하기 쉬운 Excel 기반 데이터 분석 템플릿이나 웹 기반 소프트웨어를 사용하여 데이터를 분석할 수 있습니다. 데이터 분석은 두 하우스키핑 유전자 중 하나에 대해 원시 데이터를 정규화하는 ΔΔC_T 방법을 기반으로 합니다.

응용 분야

RT² PCR Profiler Arrays는 다음을 포함하여 모든 영역의 생물학 및 의학 연구에서 사용할 수 있습니다.

암 염증 및 사이토카인 프로파일링
줄기 세포
신경과학
신호 전달 경로
세포 접착 및 세포 이동
바이오마커 스크리닝 및 검증

리소스

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

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

For analyzing gene expression data from RT2 Profiler PCR Arrays

パスウェイ特異的遺伝子の発現をリアルタイムRT-PCR を用いてプロファイリング

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

For pathway-focused gene expression analysis

For gene expression and genomic analysis

Poster for download

FAQ

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

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

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

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

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

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

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

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

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

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

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

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 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

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

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

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

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

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

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

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

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

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

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

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 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