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A new method for amplification and labeling of RNA is assessed that permits gene expression microarray analysis of formalin-fixed paraffin-embedded tissue (FFPET) samples. Valid biological data were obtained using gene expression microarrays of diffuse large B-cell lymphoma (DLBCL) FFPET samples. We examined 59 matched DLBCL patient samples, FFPET, and fresh/frozen. The samples contained both prognostic subgroups of DLBCL: germinal center B-cell (GCB) and activated B-cell (ABC). Fresh/frozen (FF) samples were amplified by both the traditional Eberwine oligo-dT method and a new method described herein. The matching FFPET samples were also amplified using the new method. Here we detail the comparison of results from all three datasets of matched samples. An established classification model built from previous data accurately classified these new samples. This new method provides a useful technology advance for microarray analysis of FFPET archival samples.

Background: Global analysis of the genome, transcriptome, and proteome is facilitated by the recent development of tools for large-scale, highly parallel analysis. We describe a novel nucleic acid amplification system that generates products by several methods. 3′-Ribo-SPIA™ primes cDNA synthesis at the 3′ polyA tail, and whole transcript (WT)-Ribo-SPIA primes cDNA synthesis across the full length of the transcripts and thus provides whole-transcriptome amplification, independent of the 3′ polyA tail. Methods: We developed isothermal linear nucleic acid amplification systems, which use a single chimeric primer, for amplification of DNA (SPIA) and RNA (Ribo-SPIA). The latter allows mRNA amplification from as little as 1 ng of total RNA. Amplification efficiency was calculated based on the delta threshold cycle between nonamplified cDNA targets and amplified cDNA. The amounts and quality of total RNA and amplification products were determined after purification of the amplification products. GeneChip® array gene expression profiling and real-time PCR were used to test the accuracy and reproducibility of the method. Quantification of cDNA products (before and after amplification) at the 2 loci along the transcripts was used to assess product length (for evaluation of the 3′-initiated Ribo-SPIA) and equal representation throughout the length of the transcript (for evaluation of the whole transcript amplification system, WT-Ribo-SPIA™). Results: Ribo-SPIA–based global RNA amplification exhibited linearity over 6 orders of magnitude of transcript abundance and generated microgram amounts of amplified cDNA from as little as 1 ng of total RNA. Conclusions: The described methods enable comprehensive gene expression profiling and analysis from limiting biological samples. The WT-Ribo-SPIA procedure, which enables amplification of non–polyA-tailed RNA, is suitable for amplification and gene expression analysis of both eukaryotic and prokaryotic biological samples.

Gene expression profiling using microarrays requires microgram amounts of RNA, which limits its direct application for the study of nanogram RNA samples obtained using microdissection, laser capture microscopy, or needle biopsy. A novel system based on Ribo-SPIA technology (RS, Ovation-Biotin amplification and labeling system) was recently introduced. The utility of the RS system, an optimized prototype system for picogram RNA samples (pRS), and two T7-based systems involving one or two rounds of amplification (OneRA, Standard Protocol, or TwoRA, Small Sample Prototcol, version II) were evaluated in the present study. Mouse kidney (MK) and mouse universal reference (MUR) RNA samples, 0.3 ng to 10 μg, were analyzed using high-density Affymetrix Mouse Genome 430 2.0 GeneChip arrays. Call concordance between replicates, correlations of signal intensity, signal intensity ratios, and minimal fold increase necessary for significance were determined. All systems amplified partially overlapping sets of genes with similar signal intensity correlations. pRS amplified the highest number of genes from 10-ng RNA samples. We detected 24 of 26 genes verified by RT-PCR in samples prepared using pRS. TwoRA yielded somewhat higher call concordances than did RS and pRS (91.8% vs. 89.3% and 88.1%, respectively). Although all target preparation methods were suitable, pRS amplified the highest number of targets and was found to be suitable for amplification of as little as 0.3 ng of total RNA. In addition, RS and pRS were faster and simpler to use than the T7-based methods and resulted in the generation of cDNA, which is more stable than cRNA.

Tumorigenic Agrobacterium strains incite the formation of crown gall tumors at wound sites on a wide variety of dicotyledonous plants as well as some monocotyledonous species. The second region of the Ti plasmid essential for tumor formation is the virulence (vir) region. The plant signals are recognized and transduced by the products of two vir genes, virA and virG. These two genes are members of the highly conserved class of two-component sensory transduction systems, virA coding for the sensor protein and virG for the response regulator. In addition to the Ti plasmid-encoded virulence genes, several chromosomal loci are important for tumor formation. Thus, genes on both the chromosome and the Ti plasmid are required for tumorigenesis, and two-component regulatory systems that are involved in virulence are located on each replicon. The VirA/VirG system of Agrobacterium is one of the few two-component systems in which the signal compounds are known. For two reasons, this class of constitutively vir-expressing virA mutant may be difficult to isolate. First, according to the model, this mutant VirA would have to harbor mutations that bypass both the Off and the Standby modes to become constitutively activated. Second, a mutant strain that is constitutively expressing its vir genes is likely to be less fit, and therefore revertants would arise at a high frequency. The pleiotropic nature of the phenotype suggests that any relation to virulence may be indirect.

The ability of Agrobacterium strains to infect transformation-recalcitrant maize plants has been shown to be determined mainly by the virA locus, implicating vir gene induction as the major factor influencing maize infection. In this report, we further explore the roles of vir induction-associated bacterial factors in maize infection using the technique of agroinfection. The Ti plasmid and virA source are shown to be important in determining the ability of a strain to infect maize, and the monosaccharide binding protein ChvE is absolutely required for maize agroinfection. The linker domain of VirAC58 from an agroinfection-competent strain, C58, is sufficient to convert VirAA6 of a nonagroinfecting strain, A348, to agroinfection competence. The periplasmic domain of VirAC58 is also able to confer a moderate level of agroinfection competence to VirAA6. In addition, the VirAA6 protein from A348 is agroinfection competent when removed from its cognate Ti plasmid background and placed in a pTiC58 background. The presence of a pTiA6-encoded, VirAA6-specific inhibitor is hypothesized and examined.

The transmembrane sensor protein VirA activates VirG in response to high levels of acetosyringone (AS). In order to respond to low levels of AS, VirA requires the periplasmic sugar-binding protein ChvE and monosaccharides released from plant wound sites. To better understand how VirA senses these inducers, the C58 virA gene was randomly mutagenized, and 14 mutants defective in vir gene induction and containing mutations which mapped to the input domain of VirA were isolated. Six mutants had single missense mutatiions in three widely separated areas of the periplasmic domain. Eight mutants had mutations in or near an amphipathic helix, TM1, or TM2. Four of the mutations in the periplasmic domain, when introduced into the corresponding A6 virA sequence, caused a specific defect in the vir gene response to glucose. This suggests that most of the periplasmic domain is required for the interaction with, or response to, ChvE. Three of the mutations from outside the periplasmic domain, one from each transmembrane domain and one from the amphiphathic helix, were made in A6 virA. These mutants were defective in the vir gene response to AS. These mutations did not affect the stability or topology of VirA or prevent dimerization; therefore, they may interfere with detection of AS or transmission of the signals to the kinase domain. Characterization of C58 chvE mutants revealed that, unlike A6 VirA, C58 VirA requires ChvE for activation of the vir genes.

Gene expression profiling of RNA isolated from formalin fixed, paraffin-embedded (FFPE) tissue samples has been historically challenging. Yet FFPE samples are sought-after because of the in-depth retrospective records typically associated with them rendering these samples a valuable resource for translational medicine studies. Extensive degradation, chemical modifications, and cross-linking have made it difficult to isolate RNA of sufficient quality required for large-scale gene expression profiling studies. NuGEN Technologies' WT-Ovation™ FFPE System linearly amplifies RNA from FFPE samples through a robust and simple whole-transcriptome approach using as little as 50 ng total RNA isolated from FFPE samples. The amplified material may be labeled with validated kits and/or protocols from NuGEN for analysis on any of the major gene expression microarray platforms, including: Affymetrix, Agilent, and Illumina gene expression arrays. Results compare well with those obtained using RNA from fresh-frozen samples. RNA quality from FFPE samples varies significantly and neither sample age nor sample size analysis via gel electrophoresis or the Agilent Bioanalyzer system accurately predict materials suitable for amplification. Therefore, NuGEN has validated a correlative qPCR-based analytical method for the RNA derived from FFPE samples which effectively predicts array results. The NuGEN approach enables fast and successful analysis of samples previously thought to be too degraded for gene expression analysis.

Agrobacterium tumefaciens transfers a piece of its Ti plasmid DNA (transferred DNA or T-DNA) into plant cells during crown gall tumorigenesis. A. tumefaciens can transfer its T-DNA to a wide variety of hosts, including both dicotyledonous and monocotyledonous plants. We show that the host range of A. tumefaciens can be extended to include Saccharomyces cerevisiae. Additionally, we demonstrate that while T-DNA transfer into S. cerevisiae is very similar to T-DNA transfer into plants, the requirements are not entirely conserved. The Ti plasmid-encoded vir genes of A. tumefaciens that are required for T-DNA transfer into plants are also required for T-DNA transfer into S. cerevisiae, as is vir gene induction. However, mutations in the chromosomal virulence genes of A. tumefaciens involved in attachment to plant cells have no effect on the efficiency of T-DNA transfer into S. cerevisiae. We also demonstrate that transformation efficiency is improved 500-fold by the addition of yeast telomeric sequences within the T-DNA sequence.