Disease profiling arrays: reverse format cDNA arrays complimentary to microarrays.

The identification of differentially expressed genes enables us to understand the molecular mechanisms associated with disease, conditions of stress, drug treatments and developmental processes. Microarrays provide a powerful tool for studying these complex phenomena. Verification of differentially expressed genes and correlation with biological function, which is usually done by northern blot analysis, RNase protection assay or RT-PCR, is the bottleneck in all these protocols. We developed a new type of cDNA array for high-throughput expression profiling of multiple tissues and blood samples (i) for confirmation analysis of statistically significant number of clinical samples (ii) from limited amount of starting material, and (iii) with detailed clinical data from each individual. In contrast to traditional cDNA arrays, these arrays are spotted with a complex cDNA representing the entire mRNA message expressed in a given tissue or blood sample. cDNAs for these arrays were generated using SMART technology and accurately represent the original mRNA population, producing specific and quantitative signals during hybridization. cDNAs on Disease Profiling Arrays were derived from total RNAs of diseased and normal tissues or different blood fractions of patients. These cDNAs were spotted onto nylon membranes along with positive and negative controls. The arrays were then hybridized with gene-specific probes. Hybridization results revealed disease-related as well as patient-specific gene expression patterns between different disease types for these genes. These studies demonstrate that Disease Profiling Arrays are suitable for high-throughput studies comparing the relative abundance of a target gene, for simultaneously detecting differentially expressed genes in a wide variety of tissues and blood samples, and can be used down-stream from cDNA microarrays for confirmation analysis.

Multiple transcription factor profiling by enzyme-linked immunoassay.

Transcription factor-DNA interactions have been widely studied in the regulation of gene expression. We have established an enzyme-linked immunoassay platform to quantify specific transcriptionfactor-DNA interactions. In our assay, dsDNA immobilized on a 96-well plate captures the transcriptionfactor from the nuclear extract of mammalian cells. The DNA-bound transcription factor is detected and quantified by enzyme-linked immunoassay using a transcription factor-specific antibody. We have profiled multiple transcription factors involved in inflammation including NFkappaB p50, NFkappaB p65, c-Rel, c-Fos, CREB-1, and ATF-2. When compared with the traditional electrophoretic mobility shift assay, the enzyme-linked immunoassay shows a 10-fold higher sensitivity, eliminates the use of radioactivity, allows for a high-throughput format, and is faster.

Caveolin-1 is down-regulated in human ovarian carcinoma and acts as a candidate tumor suppressor gene.

To identify novel markers differentially expressed in ovarian cancer versus normal ovary, we hybridized microarrays with cDNAs derived from normal human ovaries and advanced stage ovarian carcinomas. This analysis revealed down-regulation of the caveolin-1 gene (CAV1) in ovarian carcinoma samples. Suppression of CAV1 in ovarian carcinomas was confirmed using a tumor tissue array consisting of 68 cDNA pools from different matched human tumor and normal tissues. Immunohistochemistry demonstrated expression of caveolin-1 in normal and benign ovarian epithelial cells, but loss of expression in serous ovarian carcinomas. In low-grade carcinomas, redistribution of caveolin-1 from a membrane-associated pattern observed in normal epithelium to a cytoplasmic localization pattern was observed. No expression of caveolin-1 was detectable in four of six ovarian carcinoma cell lines investigated. In SKOV-3 and ES-2 carcinoma cells, which express high levels of the caveolin-1 protein, phosphorylation of the 22-kd caveolin-1 isoform was detected. Inhibition of both DNA methylation and histone deacetylation using 5-aza-2'deoxycytidine and Trichostatin A, respectively, relieves down-regulation of caveolin-1 in OAW42 and OVCAR-3 cells which is in part mediated by direct regulation at the mRNA level. Expression of CAV1 in the ovarian carcinoma cell line OVCAR-3, resulted in suppression of tumor cell survival in vitro, suggesting that the CAV1 gene is likely to act as a tumor suppressor gene in human ovarian epithelium.

Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction.

Here, we describe a fast, simple method for constructing full-length cDNA libraries using SMART technology. This novel procedure uses the template-switching activity of Moloney murine leukemia virus (MMLV) reverse transcriptase to synthesize and anchor first-strand cDNA in one step. Following reverse transcription, three cycles of PCR are performed using a modified oligo(dT) primer and an anchor primer to enrich the cDNA population for full-length sequences. Starting with 1 microgram human skeletal muscle poly(A)+ RNA, a cDNA library was constructed that contained 3 x 10(6) independent clones with an average insert size of 2 kb. Sequence analysis of 172 randomly selected clones showed that 77% of cDNA clones corresponding to known genes contained intact open reading frames. The average length of complete open reading frames was 2.4 kb. Furthermore, 86% of the full-length clones retained longer 5' UTR sequences than the longest 5' end deposited in the GenBank database. cDNA libraries generated using this method will be useful for accelerating the collection of mRNA 5' end sequence information, which is currently very limited in GenBank.

Generation of full-length cDNA libraries enriched for differentially expressed genes for functional genomics.

Here, we describe the application of a RecA-based cloning technology to generate full-length cDNA libraries enriched for genes that are differentially expressed between tumor and normal tissue samples. First, we show that the RecA-based method can be used to enrich cDNA libraries for several target genes in a single reaction. Then, we demonstrate that this method can be extended to enrich a cDNA library for many full-length cDNA clones using fragments derived from a subtracted cDNA population. The results of these studies show that this RecA-mediated cloning technology can be used to convert subtracted cDNAs or a mixture of several cDNA fragments corresponding to differentially expressed genes into a full-length library in a single reaction. This procedure yields a population of expression-ready clones that can be used for further high-throughput functional screening.

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.

RecA-mediated affinity capture: a method for full-length cDNA cloning.

We describe an improved method for rapid cloning of full-length cDNA from cDNA libraries. This approach is based on the ability of Escherichia coli RecA protein to form a stable nucleoprotein complex with a linear single-stranded DNA probe and homologous sequences in circular double-stranded DNA. Hybridization of RecA-coated biotinylated DNA probes to homologous plasmid DNA creates triple-stranded complexes, which are then captured on streptavidin-coated magnetic beads. Following magnetic separation of the hybrid molecules, the enriched plasmid population is recovered by alkaline treatment, precipitated, resuspended and used to transform bacteria. Typically, many clones can then be recovered by colony hybridization screening of a single plate of the enriched library. We have used this technology to clone full-length and alternatively spliced forms of the human bcl-xL cDNA from a human liver cDNA library.

Suppression subtractive hybridization: a versatile method for identifying differentially expressed genes.

A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a technique called suppression PCR, and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. As a result only one round of subtractive hybridization is needed and the subtracted library is normalized in terms of abundance of different cDNAs. It dramatically increases the probability of obtaining low-abundance differentially expressed cDNA and simplifies analysis of the subtracted library. The SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes. This chapter provides detailed protocols for the generation of subtracted cDNA and differential screening of subtracted cDNA libraries. As a representative example we demonstrate the usefulness of the method by constructing a testis-specific cDNA library as well as using the subtracted cDNA mixture as a hybridization probe. Finally, we discuss the characteristics of subtracted libraries, the nature and level of background nondifferentially expressed clones in the libraries, as well as a procedure for the rapid identification of truly differentially expressed cDNA clones.

Quantitative rt-PCR.

Although reverse transcriptase polymerase chain reaction (RT-PCR) is an extremely sensitive method of mRNA analysis, obtaining quantitative information with this technique can be difficult. This is caused primarily by the fact that there are two sequential enzymatic steps involved: the synthesis of DNA from the RNA template and PCR. In practice, the exponential nature of PCR and the practical aspects of performing PCR pose the most serious obstacles to obtaining quantitative information. With some adaptations, however, RT-PCR can yield accurate quantitative results.

PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori.

Genes that are characteristic of only certain strains of a bacterial species can be of great biologic interest. Here we describe a PCR-based subtractive hybridization method for efficiently detecting such DNAs and apply it to the gastric pathogen Helicobacter pylori. Eighteen DNAs specific to a monkey-colonizing strain (J166) were obtained by subtractive hybridization against an unrelated strain whose genome has been fully sequenced (26695). Seven J166-specific clones had no DNA sequence match to the 26695 genome, and 11 other clones were mixed, with adjacent patches that did and did not match any sequences in 26695. At the protein level, seven clones had homology to putative DNA restriction-modification enzymes, and two had homology to putative metabolic enzymes. Nine others had no database match with proteins of assigned function. PCR tests of 13 unrelated H. pylori strains by using primers specific for 12 subtracted clones and complementary Southern blot hybridizations indicated that these DNAs are highly polymorphic in the H. pylori population, with each strain yielding a different pattern of gene-specific PCR amplification. The search for polymorphic DNAs, as described here, should help identify previously unknown virulence genes in pathogens and provide new insights into microbial genetic diversity and evolution.

Rt-PCR methods and applications.

Control of gene transcription, the process in which a gene's DNA sequence serves as a template for mRNA synthesis, plays a critical role in the multistep process that regulates gene expression. Gene transcrtption levels within a cell change in response to a wide variety of signals that occur during cell development, differentiation, and normal physiological function. Changes in transcrip tion levels also occur in response to disease and other factors. In turn, these changes in transcription levels cause variations in the steady-state levels of indivtdual mRNAs. Thus, analysis of specific mRNA levels 1s vital in a broad range of research areas.

Quantitative rt-PCR.

Although reverse transcriptase polymerase chain reaction (RT-PCR) is an extremely sensitive method of mRNA analysis, obtaining quantitative information with this technique can be difficult. This is caused primarily by the fact that there are two sequential enzymatic steps involved: the synthesis of DNA from the RNA template and PCR. In practice, the exponential nature of PCR and the practical aspects of performing PCR pose the most serious obstacles to obtaining quantitative information. With some adaptations, however, RT-PCR can yield accurate quantitative results.

Identification of an alternative form of human lactoferrin mRNA that is expressed differentially in normal tissues and tumor-derived cell lines.

Lactoferrin (LF), traditionally known as an iron-binding protein present in high concentrations in milk and various secretions, has emerged as a multifunctional protein involved in many aspects of the host defense against infection. Recently, LF has been shown to inhibit the growth of solid tumors and reduce experimental metastasis in mice, suggesting that LF also may play a role in the defense against tumorigenesis. Here we provide the sequence of the cDNA and promoter region, the chromosome assignment, and tissue expression pattern of a novel form of LF mRNA (delta LF). The sequence of delta LF mRNA is nearly identical to that of LF mRNA; however, at the 5' end, we find a novel sequence that replaces the N-terminal signal peptide sequence of LF mRNA. We map the delta LF mRNA to human chromosome 3 and find that both delta LF and LF sequences colocalize to the same cloned 90- to 150-kb genomic DNA fragment. We further show that the delta LF mRNA is the product of alternative splicing of the LF gene and likely is specified by use of an alternative promoter. Although we find delta LF mRNA at various levels in 20 of 20 adult and fetal human tissues, we do not find delta LF mRNA in any of 14 diverse tumor-derived cell lines.

Differential screening of a subtracted cDNA library: a method to search for genes preferentially expressed in multiple tissues.

We have developed a new strategy for differential screening of genes that are expressed in two or more tissues, and have used it to identify genes that are preferentially expressed in both testis and ovary. In this approach, testis-specific cDNAs were first generated using the suppression subtractive hybridization technique, cloned and arrayed in microplates. The inserts from the subtracted testis-specific library were amplified by PCR and spotted on filters. The dot blots were screened by hybridization using either testis- or ovary-specific subtracted cDNA mixtures as probes. By screening about 2000 putative clones from the subtracted testis-specific library, we identified 14 candidate genes that hybridized to both cDNA probes. Northern blot analysis showed that 12 clones were preferentially or exclusively expressed in testis and ovary. Nucleotide sequence analysis indicated that three of the identified clones represented cDNAs from novel genes.

Full-length cDNA cloning and determination of mRNA 5' and 3' ends by amplification of adaptor-ligated cDNA.

An efficient cDNA amplification procedure is described for determining of the 5' and 3' ends of mRNAs and cloning full-length cDNAs. In this approach, a double-stranded (ds) adaptor is ligated to both ends of a library of ds cDNA by T4 DNA ligase. This adaptor-ligated ds cDNA is then used to selectively amplify 5'- or 3'-cDNA fragments by PCR with a combination of gene-specific and adaptor-specific primers. This is a unified method for 5' and 3' rapid amplification of cDNA ends (RACE) from the same adaptor-ligated ds cDNA template. A specially designed adaptor combines features of "vectorette PCR" and "suppression PCR" technologies that significantly reduce background during amplification. The application of "long and accurate PCR" (LA PCR) technology makes possible the amplification of large RACE products and full-length cDNAs with high fidelity to the original mRNA. We investigated efficacy and limitations of this PCR-based approach for cDNA cloning by amplification of 5'- and 3'-RACE fragments and full-length cDNAs of three members of the abundant human actin gene family (1.3-1.9 kb), the medium abundance transferrin receptor mRNA (5.0 kb) and the low-medium abundance insulin-like growth factor II receptor mRNA (9.1 kb).

Equalizing cDNA subtraction based on selective suppression of polymerase chain reaction: cloning of Jurkat cell transcripts induced by phytohemaglutinin and phorbol 12-myristate 13-acetate.

The major drawback of subtractive cDNA libraries is that the original disproportion in concentrations of different types of transcripts is preserved. This usually makes the isolation of specific rare transcripts extremely difficult. To overcome this difficulty, we propose a strategy that introduces the equalization of concentrations (normalization) of specific transcripts during the subtractive process. This makes possible obtaining both rare and highly abundant transcripts in the resulting subtracted library. This technique has been applied for isolation of transcripts activated upon induction of Jurkat cells by phytohemaglutinin and phorbol 12-myristate 13-acetate. Six novel up-regulated sequences belonging to a low-abundance class of transcripts have been obtained.

Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries.

A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model system, the SSH technique enriched for rare sequences over 1,000-fold in one round of subtractive hybridization. We demonstrate its usefulness by generating a testis-specific cDNA library and by using the subtracted cDNA mixture as a hybridization probe to identify homologous sequences in a human Y chromosome cosmid library. The human DNA inserts in the isolated cosmids were further confirmed to be expressed in a testis-specific manner. These results suggest that the SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes.

Combining the technique of RNA fingerprinting and differential display to obtain differentially expressed mRNA.

We have modified recently developed methods of RNA fingerprinting and differential display based on arbitrarily primed PCR which can be used for detection and cloning of differentially expressed mRNAs. Our protocol requires only a single cDNA synthesis for each different RNA sample, in contrast to the multiple cDNA reactions required for differential display method, followed by selective amplification of cDNA sequence fraction by arbitrary and oligo(dT) primers. We have shown that the longer primers (25-29-mers) allow the use of optimal dNTP concentration and higher stringency PCR. Further improvements include using TaqStart antibody for hot start PCR and thermostable enzyme mixes suitable for long-distance PCR. Long-distance PCR enables the method to display bands of up to 2 kb and should result in a higher fidelity of PCR products to the original RNA template. When these improvements are combined the resulting method is highly reproducible, and more than 85% of the differentially expressed bands can be confirmed by Northern blot analysis.

Selection of hybrids by affinity capture (SHAC): a method for the generation of cDNAs enriched in sequences from a specific chromosome region.

We have established a method for preparing cDNA sublibraries enriched in sequences from specific chromosome regions, called selection of hybrids by affinity capture (SHAC). This procedure can be described in two stages. In the first stage, a particular chromosome region, in this study mouse chromosome 11, was microdissected, followed by PCR amplification with a universal degenerate primer. This material is referred to as the "target" DNA. In the second stage, a mouse liver cDNA library with unique linker-adapter ends, referred to as the "source" cDNA, was hybridized to the biotin-labeled target DNA prepared during the first stage. The resulting DNA duplexes were captured by streptavidin-coated magnetic beads. The cDNAs were released from their biotin-labeled target homologs by alkaline denaturation and recovered by PCR amplification. These cDNAs were referred to as the SHACcDNAs. Specificity of the SHACcDNA to chromosome 11 was verified by FISH analysis. To examine representation of the SHACcDNA, we confirmed the presence of seven genes or single-copy DNA segments known to be localized on mouse chromosome 11, using a dot blot assay. In addition, a second round of SHAC was performed to achieve even higher specificity for the resulting chromosome 11 SHACcDNA. The SHAC technology should facilitate construction of cytogenetically defined cDNA libraries and should assist in the fields of gene discovery and genome mapping.