Use of SMART-generated cDNA for gene expression studies in multiple human tumors

We demonstrate here that SMART PCR-amplified cDNAs arrayed on a nylon membrane are suitable for high-throughput tissue expression profiling when starting biological materials are limited. We show that SMART cDNA accurately reflects gene expression patterns found in total RNA by comparing the expression level of several target genes in SMART PCR-amplified cDNAs and their corresponding total RNAs. We also arrayed cDNAs from 68 matched tumor and normal samples on a nylon membrane to determine whether SMART PCR-amplified cDNA could be used for detecting differentially expressed genes in these tissues. These arrays containing normalized tumor and normal cDNAs were hybridized with probes for glutathione peroxidase and gelsolin. The hybridization results revealed cancer-related and patient-specific gene expression differences between tumor and normal tissues for these genes. These studies show that SMART PCR-amplified cDNAs maintain the complexity of the original mRNA population and are thus suitable for high-throughput studies to compare the relative abundance of target genes and to detect differentially expressed genes in a wide variety of tissues simultaneously.     

Postdifferential display: parallel processing of candidates using small amounts of RNA

The necessity of screening differentially expressed candidate genes has imposed a limit on the application of differential display to large-scale analysis of gene expression patterns. Screening candidates has indeed proven a burden because traditional screening methods require the purification of large amounts of RNA. In this article we describe an assay that allows the screening of 240 candidate genes with only 5 microg of total RNA. This assay consists of using cDNA probes synthesized from amplified RNA in differential screening and can be performed in a 96-well plate format.     

Copy number and loss of heterozygosity detected by SNP array of formalin-fixed tissues using whole-genome amplification

The requirement for large amounts of good quality DNA for whole-genome applications prohibits their use for small, laser capture micro-dissected (LCM), and/or rare clinical samples, which are also often formalin-fixed and paraffin-embedded (FFPE). Whole-genome amplification of DNA from these samples could, potentially, overcome these limitations. However, little is known about the artefacts introduced by amplification of FFPE-derived DNA with regard to genotyping, and subsequent copy number and loss of heterozygosity (LOH) analyses. Using a ligation adaptor amplification method, we present data from a total of 22 Affymetrix SNP 6.0 experiments, using matched paired amplified and non-amplified DNA from 10 LCM FFPE normal and dysplastic oral epithelial tissues, and an internal method control. An average of 76.5% of SNPs were called in both matched amplified and non-amplified DNA samples, and concordance was a promising 82.4%. Paired analysis for copy number, LOH, and both combined, showed that copy number changes were reduced in amplified DNA, but were 99.5% concordant when detected, amplifications were the changes most likely to be 'missed', only 30% of non-amplified LOH changes were identified in amplified pairs, and when copy number and LOH are combined ～50% of gene changes detected in the unamplified DNA were also detected in the amplified DNA and within these changes, 86.5% were concordant for both copy number and LOH status. However, there are also changes introduced as ～20% of changes in the amplified DNA are not detected in the non-amplified DNA. An integrative network biology approach revealed that changes in amplified DNA of dysplastic oral epithelium localize to topologically critical regions of the human protein-protein interaction network, suggesting their functional implication in the pathobiology of this disease. Taken together, our results support the use of amplification of FFPE-derived DNA, provided sufficient samples are used to increase power and compensate for increased error rates.     

High-fidelity mRNA amplification for gene profiling

The completion of the Human Genome Project has made possible the comprehensive analysis of gene expression, and cDNA microarrays are now being employed for expression analysis in cancer cell lines or excised surgical specimens. However, broader application of cDNA microarrays is limited by the amount of RNA required: 50-200 microg of total RNA (T-RNA) and 2-5 microg poly(A) RNA. To broaden the use of cDNA microarrays, some methods aiming at intensifying fluorescence signal have resulted in modest improvement. Methods devoted to amplifying starting poly(A) RNA or cDNA show promise, in that detection can be increased by orders of magnitude. However, despite the common use of these amplification procedures, no systematic assessment of their limits and biases has been documented. We devised a procedure that optimizes amplification of low-abundance RNA samples by combining antisense RNA (aRNA) amplification with a template-switching effect (Clonetech, Palo Alto, CA). The fidelity of aRNA amplified from 1:10,000 to 1:100,000 of commonly used input RNA was comparable to expression profiles observed with conventional poly(A) RNA- or T-RNA-based arrays.     

Optimized procedures for microarray analysis of histological specimens processed by laser capture microdissection

Analysis of cell-specific gene expression patterns using microarrays can reveal genes that are differentially expressed in diseased and normal tissue, as well as identify genes associated with specialized cellular functions. However, the cellular heterogeneity of the tissues precludes the resolution of expression profiles of specific cell types. While laser capture microdissection (LCM) can be used to obtain purified cell populations, the limited quantity of RNA isolated makes it necessary to perform an RNA amplification step prior to microarray analysis. The linearity and reproducibility of two RNA amplification protocols--the Baugh protocol (Baugh et al., 2001, Nucleic Acids Res 29:E29) and an in-house protocol have been assessed by conducting microarray analyses. Cy3-labeled total RNA from the colorectal cell line Colo-205 was compared to Cy5-labeled Colo-205 amplified RNA (aRNA) generated with each of the two protocols, using a human 10K cDNA array. The correlation of the gene intensities between amplified and total RNA measured in the two channels of each microarray was 0.72 and 0.61 for the Baugh protocol and the in-house protocol, respectively. The two protocols were further evaluated using aRNA obtained from normal colonic crypt cross-sections isolated via LCM. In both cases a microarray profile representative of colonic mucosa was obtained; statistically, the Baugh protocol was superior. Furthermore, a substantial overlap between highly expressed genes in the Colo-205 cells and colonic crypts underscores the reliability of the microarray analysis of LCM-derived material. Taken together, these results demonstrate that LCM-derived tissue from histological specimens can generate abundant amounts of high-quality aRNA for subsequent microarray analysis.     

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.     

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.     

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.     

A modified cDNA subtraction to identify differentially expressed genes from plants with universal application to other eukaryotes

We have designed a simple and efficient polymerase chain reaction (PCR)-based cDNA subtraction protocol for high-throughput cloning of differentially expressed genes from plants that can be applied to any experimental system and as an alternative to DNA chip technology. Sequence-independent PCR-amplifiable first-strand cDNA population was synthesized by priming oligo-dT primer with a defined 5' heel sequence and ligating another specified single-stranded oligonucleotide primer on the 3' ends of first-strand cDNAs by T4 RNA ligase. A biotin label was introduced into the sense strands of cDNA that must be subtracted by using 5' biotinylated forward primer during PCR amplification to immobilize the sense strand onto the streptavidin-linked paramagnetic beads. The unamplified first strand (antisense) of the interrogating cDNA population was hybridized with a large excess of amplified sense strands of control cDNA. We used magnetic bead technology for the efficient removal of common cDNA population after hybridization to reduce the complexity of the cDNA prior to PCR amplification for the enrichment and sequence abundance normalization of differentially expressed genes. Construction of a subtracted and normalized cDNA library efficiently eliminates common abundant cDNA messages and also increases the probability of identifying clones differentially expressed in low-abundance cDNA messages. We used this method to successfully isolate differentially expressed genes from Pennisetum seedlings in response to salinity stress. Sequence analysis of the selected clones showed homologies to genes that were reported previously and shown to be involved in plant stress adaptation.