A rapid and inexpensive labeling method for microarray gene expression analysis

Background  

Global gene expression profiling by DNA microarrays is an invaluable tool in biological research. However, existing labeling methods are time consuming and costly and therefore often limit the scale of microarray experiments and sample throughput. Here we introduce a new, fast, inexpensive method for direct random-primed fluorescent labeling of eukaryotic cDNA for gene expression analysis and compare the results obtained on the NimbleGen microarray platform with two other widely-used labeling methods, namely the NimbleGen-recommended double-stranded cDNA protocol and the indirect (aminoallyl) method.     

Real-time polymerase chain reaction-based exponential sample amplification for microarray gene expression profiling

Conventional approaches to target labeling for gene expression analysis using microarray technology typically require relatively large amounts of RNA, a serious limitation when the available sample is limited. Here we describe an alternative exponential sample amplification method by using quantitative real-time polymerase chain reaction (QRT-PCR) to follow the amplification and eliminate the overamplified cDNA which could distort the quantitative ratio of the starting mRNA population. Probes generated from nonamplified, PCR-amplified, and real-time-PCR-amplified cDNA samples were generated from lipopolysaccharide-treated and nontreated mouse macrophages and hybridized to mouse cDNA microarrays. Signals obtained from the three protocols were compared. Reproducibility and reliability of the methods were determined. The Pearson correlation coefficients for replica experiments were r=0.927 and r=0.687 for QRT-PCR-amplification and PCR-overamplification protocols, respectively. Chi2 test showed that overamplification resulted in major biases in expression ratios, while these alterations could be eliminated by following the cycling status with QRT-PCR. Our exponential sample amplification protocol preserves the original expression ratios and allows unbiased gene expression analysis from minute amounts of starting material.     

表达谱基因芯片的可靠性验证分析

cDNA芯片是一项新兴的能评估检测全范围mRNA表达水平变化的技术。通过同种组织RNA自身比较实验及不同组织RNA的差异分析实验对cDNA芯片实验的重复性进行检验,利用相关系数(correlation coefficient,R)、变异系数(coefficient of variation,CV)和假阳性率(false positiver ate,FPR)分析eDNA芯片数据的可靠程度,对cDNA芯片实验数据作了整体的评估。结果证实,该芯片系统得到的cDNA表达谱数据相关系数一般大于0．9,平均变异系数15％左右,假阳性率控制在3％以内。还提出一致率(consistence rate,CR)的概念,作为衡量cDNA芯片系统重复性的新参数,同时阐述了该参数优于目前常用的相关系数及变异系数的特点。另外,通过比较芯片制备中点样浓度、mRNA和总RNA以及不同批次芯片和不同标记过程对实验的影响,来分析芯片数据的系统误差来源。并提出重复两次实验,可以克服绝大部分实验系统引入的假阳性。     

A concise guide to cDNA microarray analysis

Microarray expression analysis has become one of the most widely used functional genomics tools. Efficient application of this technique requires the development of robust and reproducible protocols. We have optimized all aspects of the process, including PCR amplification of target cDNA clones, microarray printing, probe labeling and hybridization, and have developed strategies for data normalization and analysis.     

Evaluation of five different cDNA labeling methods for microarrays using spike controls

Background

Several different cDNA labeling methods have been developed for microarray based gene expression analysis. We have examined the accuracy and reproducibility of such five commercially available methods in detection of predetermined ratio values from target spike mRNAs (A. thaliana) in a background of total RNA. The five different labeling methods were: direct labeling (CyScribe), indirect labeling (FairPlay? – aminoallyl), two protocols with dendrimer technology (3DNA^® Array 50? and 3DNA^® submicro?), and hapten-antibody enzymatic labeling (Micromax? TSA?). Ten spike controls were mixed to give expected Cy5/Cy3 ratios in the range 0.125 to 6.0. The amounts of total RNA used in the labeling reactions ranged from 5 – 50 μg.

Results

The 3DNA array 50 and CyScribe labeling methods performed best with respect to relative deviation from the expected values (16% and 17% respectively). These two methods also displayed the best overall accuracy and reproducibility. The FairPlay method had the lowest total experimental variation (22%), but the estimated values were consistently higher than the expected values (36%). TSA had both the largest experimental variation and the largest deviation from the expected values (45% and 48% respectively).

Conclusion

We demonstrate the usefulness of spike controls in validation and comparison of cDNA labeling methods for microarray experiments.

     

Monitoring of cDNA microarray with common primer target and hybridization specificity with selected targets

Academic researchers are increasingly producing and using cDNA microarrays. Their quality and hybridization specificity are crucial in determining whether the generated data are accurate and interpretable. Here, we describe two methods of monitoring microarray production, the sustainability of DNA attachment, and the specificity of hybridization. The first method consists of labeling an oligonucleotide, which is one of the primers used to amplify all cDNA probes on the array (except for beta-actin and GAPDH) with fluorescent dye and hybridize it to the cDNA microarray. Attachment of the cDNAs on the array after the hybridization procedure was monitored by visualizing fluorescent signals from the spots on the array. In the second method, two selected DNA targets, beta-actin and GAPDH, were labeled with fluorescent dye to hybridize to the cDNA array. Hence, hybridization specificity was demonstrated by obtaining fluorescent signals solely from the genes corresponding to the target.     

Effects of mRNA amplification on gene expression ratios in cDNA experiments estimated by analysis of variance

Background  

A limiting factor of cDNA microarray technology is the need for a substantial amount of RNA per labeling reaction. Thus, 20–200 micro-grams total RNA or 0.5–2 micro-grams poly (A) RNA is typically required for monitoring gene expression. In addition, gene expression profiles from large, heterogeneous cell populations provide complex patterns from which biological data for the target cells may be difficult to extract. In this study, we chose to investigate a widely used mRNA amplification protocol that allows gene expression studies to be performed on samples with limited starting material. We present a quantitative study of the variation and noise present in our data set obtained from experiments with either amplified or non-amplified material.     

Comparison of different labeling methods for the production of labeled target DNA for microarray hybridization

Different labeling methods were studied to compare various approaches to the preparation of labeled target DNA for microarray experiments. The methods under investigation included a post-PCR labeling method using the Klenow fragment and a DecaLabel DNA labeling kit, the use of a Cy3-labeled forward primer in the PCR, generating either double-stranded or single-stranded PCR products, and the incorporation of Cy3-labeled dCTPs in the PCR. A microarray that had already been designed and used for the detection of microorganisms in compost was used in the study. PCR products from the organisms Burkholderia cepacia and Staphylococcus aureus were used in the comparison study, and the signals from the probes for these organisms analyzed. The highest signals were obtained when using the post-PCR labeling method, although with this method, more non-specific hybridizations were found. Single-stranded PCR products that had been labeled by the incorporation of a Cy3-labeled forward primer in the PCR were found to give the next highest signals upon hybridization for a majority of the tested probes, with less non-specific hybridizations. Hybridization with double-stranded PCR product labeled with a Cy3-labeled forward primer, or labeled by the incorporation of Cy3-labeled dCTPs resulted in acceptable signal to noise ratios for all probes except the UNIV 1389a and Burkholderia genus probes, both located toward the 3' end of the 16S rRNA gene. The comparison of the different DNA labeling methods revealed that labeling via the Cy3-forward primer approach is the most appropriate of the studied methods for the preparation of labeled target DNA for our purposes.     

Prokaryotic RNA preparation methods useful for high density array analysis: comparison of two approaches

High density oligonucleotide arrays have been used extensively for expression studies of eukaryotic organisms. We have designed a prokaryotic high density oligonucleotide array using the complete Escherichia coli genome sequence to monitor expression levels of all genes and intergenic regions in the genome. Because previously described methods for preparing labeled target nucleic acids are not useful for prokaryotic cell analysis using such arrays, a mRNA enrichment and direct labeling protocol was developed together with a cDNA synthesis protocol. The reproducibility of each labeling method was determined using high density oligonucleotide probe arrays as a read-out methodology and the expression results from direct labeling were compared to the expression results from the cDNA synthesis. About 50% of all annotated E.coli open reading frames are observed to be transcribed, as measured by both protocols, when the cells were grown in rich LB medium. Each labeling method individually showed a high degree of concordance in replica experiments (95 and 99%, respectively), but when each sample preparation method was compared to the other, ~32% of the genes observed to be expressed were discordant. However, both labeling methods can detect the same relative gene expression changes when RNA from IPTG-induced cells was labeled and compared to RNA from uninduced E.coli cells.     

Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation

There are many sources of systematic variation in cDNA microarray experiments which affect the measured gene expression levels (e.g. differences in labeling efficiency between the two fluorescent dyes). The term normalization refers to the process of removing such variation. A constant adjustment is often used to force the distribution of the intensity log ratios to have a median of zero for each slide. However, such global normalization approaches are not adequate in situations where dye biases can depend on spot overall intensity and/or spatial location within the array. This article proposes normalization methods that are based on robust local regression and account for intensity and spatial dependence in dye biases for different types of cDNA microarray experiments. The selection of appropriate controls for normalization is discussed and a novel set of controls (microarray sample pool, MSP) is introduced to aid in intensity-dependent normalization. Lastly, to allow for comparisons of expression levels across slides, a robust method based on maximum likelihood estimation is proposed to adjust for scale differences among slides.     

猪瘟病毒基因表达谱芯片的可靠性研究

通过同种组织RNA自身比较实验及不同组织RNA的差异分析实验对猪瘟病毒(classic swine fever virus, CSFV)cDNA芯片实验的重复性进行检验.利用相关系数(correlationcoefficient, R)、变异系数(coefficientofvariation, CV)和假阳性率(falsepositiverate, FPR)分析猪瘟病毒cDNA芯片数据的可靠程度,对cDNA芯片实验数据作了整体的评估.结果证实,该芯片系统得到的猪瘟病毒cDNA表达谱数据相关系数一般大于0.9,假阳性率控制在2 %以内.另外,通过比较猪瘟病毒芯片制备中点样浓度、mRNA和总RNA以及不同标记过程对实验的影响,来分析猪瘟病毒芯片数据的系统误差来源,得出结果:重复两次实验,可以克服绝大部分实验系统引入的假阳性.     

Ultrasensitive detection of biomolecules with fluorescent dye-doped nanoparticles

Fluorescent-labeled molecules have been used extensively for a wide range of applications in biological detection and diagnosis. A new form of highly luminescent and photostable nanoparticles was generated by doping the fluorescent dye tris(2'2-bipyridyl)dichlororuthenium(II)hexahydrate (Rubpy) inside silica material. Because thousands of fluorescent dye molecules are encapsulated in the silica matrix that also serves to protect Rubpy dye from photodamaging oxidation, the Rubpy-dye-doped nanoparticles are extremely bright and photostable. We have used these nanoparticles successfully in various fluorescence labeling techniques, including fluorescent-linked immunosorbent assay, immunocytochemistry, immunohistochemistry, DNA microarray, and protein microarray. By combining the high-intensity luminescent nanoparticles with the specificity of antibody-mediated recognition, ultrasensitive target detection has been achieved. In all cases, assay results clearly demonstrated the superiority of the nanoparticles over organic fluorescent dye molecules and quantum dots in probe labeling for sensitive target detection. These results demonstrate the potential to apply these newly developed fluorescent nanoparticles in various biodetection systems.     

Combination of PCR subtraction and cDNA microarray for differential gene expression profiling.

PCR subtraction hybridization has been used effectively to enrich and single out differentially expressed genes. However identification of these genes by means of cloning and sequencing individual cDNAs is a tedious and lengthy process. In this report, an attempt has been made to combine the use of PCR select cDNA subtraction hybridization and cDNA microarrays to identify differentially expressed genes using a nonradioactive chemiluminescent detection method. mRNA from human prolactin (hPRL) or human prolactin antagonist (hPRL-G129R) treated and non-treated breast cancer cells was isolated, and cDNAs were synthesized and used for the PCR subtraction to enrich the differentially expressed genes in the treated cells. The PCR-amplified and subtracted cDNA pools were purified and labeled using the digoxigenin method. Labeled cDNAs were hybridized to a human apoptosis cDNA microarray membrane and identified by chemiluminescence. The results suggest that the strategy of combining all three methods will allow for a more efficient, nonradioactive way of identifying differentially expressed genes in target cells.