一种标记cDNA芯片探针的新方法

探讨mRNA长片段反转录PCR技术(RT-LDPCR)在cDNA芯片微量探针标记和信号放大中的应用.首先提取BEP2D细胞的总RNA,然后用两种不同的方法进行标记,一种为RT-LDPCR,用荧光素Cy3-dCTP进行标记;另一种为传统的RNA反转录,用荧光素Cy5-dCTP进行标记.将两种方法标记好的探针等量混合后与含有440个点(44个基因)的cDNA芯片同时杂交,发现二者具有很高的一致性(0.5＜Cy3/Cy5＞2.0).由于RNA反转录法为cDNA芯片探针标记的传统方法,从而验证了RT-LDPCR用于cDNA芯片探针标记的可行性.RT-LDPCR具有对样品总RNA的需要量少和可对样品中信号进行放大的优点,特别适合于对材料来源受到限制的RNA进行标记.     

Linear amplification of catalyzed reporter deposition technology on nylon membrane microarray

The application of microarray analysis to gene expression from limited tissue samples has not been very successful because of the poor signal qualityfrom the genes expressed at low levels. Here we discussed the use of catalyzed reporter deposition (CARD) technology to amplify signals from limited RNA samples on nylon membrane cDNA microarray. When the input RNA level was greater than 10 microg, the genes expressed at high levels did not amplify in proportion to those expressed at low levels. Compared to conventional colorimetric detection, the CARD method requires less than 10% of the total RNA used for amplification of signal displayed onto a nylon membrane cDNA microarray. Total RNA (5-10 microg), as one can extract from a limited amount of specimen, was determined to produce a linear correlation between the colorimetric detection and CARD methods. Beyond this range, it can cause a nonlinear amplification of highly expressed and low-abundance genes. These results suggest that when amplification is needed for any applications using the CARD method, including DNA microarray experiments, precaution has to be taken in the amount of RNA used to avoid skew amplification and thus misleading conclusions.     

CTAB-urea method purifies RNA from melanin for cDNA microarray analysis

Melanin represents a major problem for the study of melanoma by microarrays since it is retained during RNA extraction and inhibits the enzymatic reactions used for probe preparation. Here we report a new method for cleaning RNA from melanin, based on the use of the cationic detergent cetyl-trimethylammonium bromide (CTAB)-urea for RNA precipitation. This method is easy to perform and has a low cost. Purified RNA is recovered with high quality and good yield. CTAB-urea treated RNA from highly pigmented melanoma cells can be successfully reverse transcribed and labeled to obtain probes which can be subsequently used in cDNA microarray experiments, giving consistent and reproducible results.     

CTAB–Urea Method Purifies RNA from Melanin for cDNA Microarray Analysis

Melanin represents a major problem for the study of melanoma by microarrays since it is retained during RNA extraction and inhibits the enzymatic reactions used for probe preparation. Here we report a new method for cleaning RNA from melanin, based on the use of the cationic detergent cetyl‐trimethylammonium bromide (CTAB)–urea for RNA precipitation. This method is easy to perform and has a low cost. Purified RNA is recovered with high quality and good yield. CTAB–urea treated RNA from highly pigmented melanoma cells can be successfully reverse transcribed and labeled to obtain probes which can be subsequently used in cDNA microarray experiments, giving consistent and reproducible results.     

Identification of HCV-1b by Low-Density cDNA Microarray-Based Assay

A rapid and sensitive microarray assay for the detection of HCV-1b was developed in our laboratory and a cDNA fragment library for HCV-1b cDNA microarray probes was constructed. The full-length cDNAs of HCV-1b were digested with restriction endonuclease Sau3A I and the fragments were cloned with the pMD18-T vectors. Positive clones were isolated and identified by sequencing. The cDNA microarray was prepared by spotting the gene fragment on the surface of an amido-modified glass slide using the robotics system and samples were fluorescent labeled by the restriction display PCR (RD-PCR) technique, In the present study, modified protocols were used for probe selection and hybridization temperature. The detection of a microarray was validated by the hybridization and the sequence analysis. A total of 22 different specific gene fragments of HCV-1b ranging from 250 to 750 bp were isolated and sequenced, and these fragments were further used as probes in the microarray preparation. The diagnostic validity of the microarray method was evaluated after the washing and scanning process. The results of hybridization and sequence data analysis showed a significant specificity and sensitivity in the detection of HCV-1b RNA. The method of preparing microarray probes by construction of cDNA fragments library was effective, rapid, and simple; the optimized microarray was sensitive in the clinical detection of HCV-1b. The RD-PCR technique for the sample labeling was useful for significantly increasing the sensitivity of the assay. The cDNA microarray assay can be widely used in the clinical diagnosis of HCV-1b.     

Optimization of liver biopsy RNA sampling and use of reference RNA for cDNA microarray analysis

In this study, we used the rat liver as a model system to optimize the conditions for extracting RNA from liver biopsies for use in cDNA microarrays. We found that a 5-mm biopsy with a 16-gauge needle and storage in RNA later at 4 degrees C were optimal conditions for RNA extraction. The most important factor for the quantity and quality of RNA extraction was the sample diameter. Using the optimized sampling conditions and a cDNA microarray, we compared the expression of genes in the normal and the fibrotic tissues of the LEC rat liver, a model of liver tumorigenesis, with SD rat liver RNA as a reference. We found 29 genes that were up-regulated and 33 genes that were down-regulated in the fibrotic part of the liver. Furthermore, with the help of the reference RNA, we were able to classify the expression profiles into five groups without complex mathematical analyses; without the reference RNA, the genes could be classified into only two groups. Finally, we found that osteopontin was expressed at a very high level in the fibrotic portion of the LEC rat liver. This cDNA microarray result was validated by immunohistochemistry, which showed an elevated expression of osteopontin in the region of cholangiocarcinoma and a lack of expression in normal tissues. With optimized conditions, we should be able to apply the microarray system for routine practice.     

Sequence-matched probes produce increased cross-platform consistency and more reproducible biological results in microarray-based gene expression measurements

Cancer derived microarray data sets are routinely produced by various platforms that are either commercially available or manufactured by academic groups. The fundamental difference in their probe selection strategies holds the promise that identical observations produced by more than one platform prove to be more robust when validated by biology. However, cross-platform comparison requires matching corresponding probe sets. We are introducing here sequence-based matching of probes instead of gene identifier-based matching. We analyzed breast cancer cell line derived RNA aliquots using Agilent cDNA and Affymetrix oligonucleotide microarray platforms to assess the advantage of this method. We show, that at different levels of the analysis, including gene expression ratios and difference calls, cross-platform consistency is significantly improved by sequence- based matching. We also present evidence that sequence-based probe matching produces more consistent results when comparing similar biological data sets obtained by different microarray platforms. This strategy allowed a more efficient transfer of classification of breast cancer samples between data sets produced by cDNA microarray and Affymetrix gene-chip platforms.     

A cut-off based approach for gene expression analysis of formalin-fixed and paraffin-embedded tissue samples

Microarray analysis of formalin-fixed and paraffin-embedded (FFPE) tissue seems to be of importance for the detection of molecular marker sets in prostate cancer (PC). The compromised RNA integrity of FFPE tissue results in a high degree of variability at the probe level of microarray data as shown by degradation plot. We tested methods that reduce the variability by including all probes within 300 nucleotides, within 600 nucleotides, or up to a calculated breakpoint with reference to the 3'-end. Accepted PC pathways such as the Wnt signaling pathway could be observed to be significantly regulated within FFPE microarray datasets. The best representation of PC gene expression, as well as better comparability to meta-analysis and fresh-frozen microarray data, could be obtained with a 600-nucleotide cutoff. Beyond the specific impact for PC microarray data analysis we propose a cutoff of 600 nucleotides for samples for which the integrity of the RNA cannot be guaranteed.     

Array-based nano-amplification technique was applied in detection of hepatitis E virus

A rapid method for the detection of Hepatitis E Virus (HEV) was developed by utilizing nano-gold labeled oligonucleotide probes, silver stain enhancement and the microarray technique. The 5'-end -NH(2) modified oligonucleotide probes were immobilized on the surface of the chip base as the capture probe. The detection probe was made of the 3'-end -SH modified oligonucleotide probe and nano-gold colloid. The optimal concentrations of these two probes were determined. To test the detection sensitivity and specificity of this technique, a conservative fragment of the virus RNA was amplified by the RT-PCR/PCR one step amplification. The cDNA was hybridized with the capture probes and the detection probes on microarray. The detection signal was amplified by silver stain enhancement and could be identified by naked eyes.100 fM of amplicon could be detected out on the microarray. As the results, preparation of nano-gold was improved and faster. Development time also was shortened to 2 min. Thus, considering high efficiency, low cost, good specificity and high sensitivity, this technique is alternative for the detection of HEV.     

Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements

Background  

Comparison of data produced on different microarray platforms often shows surprising discordance. It is not clear whether this discrepancy is caused by noisy data or by improper probe matching between platforms. We investigated whether the significant level of inconsistency between results produced by alternative gene expression microarray platforms could be reduced by stringent sequence matching of microarray probes. We mapped the short oligo probes of the Affymetrix platform onto cDNA clones of the Stanford microarray platform. Affymetrix probes were reassigned to redefined probe sets if they mapped to the same cDNA clone sequence, regardless of the original manufacturer-defined grouping. The NCI-60 gene expression profiles produced by Affymetrix HuFL platform were recalculated using these redefined probe sets and compared to previously published cDNA measurements of the same panel of RNA samples.     

The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements

Over the last decade, the introduction of microarray technology has had a profound impact on gene expression research. The publication of studies with dissimilar or altogether contradictory results, obtained using different microarray platforms to analyze identical RNA samples, has raised concerns about the reliability of this technology. The MicroArray Quality Control (MAQC) project was initiated to address these concerns, as well as other performance and data analysis issues. Expression data on four titration pools from two distinct reference RNA samples were generated at multiple test sites using a variety of microarray-based and alternative technology platforms. Here we describe the experimental design and probe mapping efforts behind the MAQC project. We show intraplatform consistency across test sites as well as a high level of interplatform concordance in terms of genes identified as differentially expressed. This study provides a resource that represents an important first step toward establishing a framework for the use of microarrays in clinical and regulatory settings.