ExpressArt® Kits - Publications
Publications (PDF)
New Product Line "ExpressArt RNAready kits" for RNA isolation
The FFPE RNAready kit is based on the technology developed by Prof. Rolf Jaggi,
Bern University, Switzerland. Results with this technology are presented in the 2 following publications. |
Oberli, Popovici et al., 2008 (1.18 MB)
Expression profiling with RNA from formalin-fixed, paraffin-embedded material. BMC Medical Genomics 2008, 1:9.
A simple procedure with a novel demodification/decrosslinking step was established for RNA isolation from FFPE material of diagnostic tumor samples. The RNA is suitable for quantitative gene expression measurements by RT-QPCR, and QPCR data were compared for FFPE RNAs obtained with the novel procedure, or with commercial FFPE RNA isolation kits. The new procedure resulted in consistently lower Ct values, combined with less variability. In comparisons with intact RNAs isolated from the same tumors (from parallel, fresh-frozen samples), correlation coefficients between intact and RNA from FFPE material were 0.83 to 0.97.
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Schobesberger et al., 2008 (0.8 MB)
Gene expression variation between distinct areas of breast cancer measured from paraffin-embedded tissue cores. BMC Cancer 2008, 8:343.
Optimized protocols were developed for RNA isolation from histologically distinct areas. RNA prepared from FFPE tissue cores is suitable for gene expression measurement by quantitative PCR. Distinct molecular scores could be determined from different cores of the same tumor specimen.
Gene expression values were used to calculate scores representing the proliferation status (PRO), the estrogen receptor status and the HER2 status. The PRO scores measured from entire sections were similar to PRO scores determined from invasive ductal carcinoma (IDC) tissue cores. Scores determined from normal tissue cores consistently revealed lower PRO scores than cores derived from IDC or ductal carcinoma in situ (DCIS) of the same block or from different blocks of the same patient.
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Melkus, Rolletschek et al., 2009 (0.86 MB)
The Metabolic Role of the Legume Endosperm: A Non-invasive Imaging Study. Plant Physiol. 151: 1139-1154.
Although essential for normal seed development in the legumes, the metabolic role of the endosperm remains uncertain. We designed non invasive nuclear magnetic resonance (NMR) tools for the in vivo study on key metabolites in the transient liquid endosperm of intact pea seeds. Expression of mRNA sequences encoding amino acid and sucrose transporters was up-regulated earlier in the endosperm than in the embryo, and this activity led to the accumulation of soluble metabolites in the endosperm vacuole. Microdissection and RT-PCR: Frozen seeds were cut into 10 µm sections and mounted onto PET-membrane slides (Carl Zeiss MicroImaging GmbH). Sections were subjected to laser-assisted microdissection using a PALMfi Laser-Microbeam (Bernried, Germany) device. Small tissue samples were collected using laser pressure catapulting and larger ones by a hand-operated micro-needle. Poly(A)-mRNA was extracted directly using the Dynabeads mRNA DIRECT kit (Dynal Biotech), using the manufacturer's protocol. Linear amplification and cDNA synthesis was carried out from ~1ng mRNA using the ExpressArt mRNA amplification Nano kit (AmpTec GmbH), according to the manufacturer's protocol. PCR reactions were based on a template of 20 ng cDNA.
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Sharbel et al., 2009 (1.85 MB)
Molecular signatures of apomictic and sexual ovules in the Boechera holboellii complex. Plant J. 58: 870-882.
The switch to apomixis (the animal analogue is parthenogenesis) is based on de-regulation of developmental pathways originally leading to sexual seed formation.
Individual live ovules were collected, using sterile glass needles. Ten ovules per tube and two samples per accession were collected in this way, and frozen directly in liquid nitrogen and stored at -80°C. RNA from micro-dissected ovule material was isolated using a PicoPure RNA isolation kit with several modifications. The standard lysis buffer was supplemented with 1% v/v NucleoGuard stock solution and 2 µl N-Carrier (AmpTec, http://www.amp-tec.com). Linear mRNA amplification was achieved using an ExpressArt mRNA amplification Pico kit (AmpTec) with several modifications. Approximately 1 ng of total RNA was used per sample. The resulting RNA after the first amplification round was purified using RNeasy MinElute columns (Qiagen), followed by a second and third amplification round. Subsequently, 5 µg of DNA-free amplified mRNA was converted into double-stranded cDNA using a 5'-biotinylated primer, and subsequently used for SuperSAGE generation.
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Pietschmann, Beetz et al., 2009 (0.9 MB)
Toll-like receptor expression and function in subsets of human gammadelta T lymphocytes. Scandinavian Journal of Immunology 70: 245-255.
By microarray analysis, we have identified a range of genes differentially regulated in freshly isolated gammadelta T cells by TCR versus TCR plus TLR3 stimulation. RNA was isolated with NucleoSpin RNA-Kit II (Macherey & Nagel) from cell sorter purified peripheral blood gammadelta T cells directly after isolation or after stimulation as described above. For comparison of freshly sorted Vdelta 1 and Vdelta 2 gammadelta T cells, 1 µg total RNA was used for reverse transcription. For comparison of resting and differentially stimulated cells from one donor, 0.5 µg RNA from each sample was amplified using the ExpressArt mRNA Amplification Kit, Micro version (AmpTec, Hamburg, Germany) according to the manufacturer's instructions. About 0.1 µg of the amplified RNA was used for reverse transcription and real time PCR was performed for some of the most interesting differentially expressed genes.
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Nam et al., 2009 (1.0 MB)
Metatranscriptome analysis of lactic acid bacteria during kimchi fermentation with genome-probing microarrays. Int. J. Food Microbiol. 130: 140-146.
We constructed genome probing microarrays (GPM) that are specific to 39 lactic acid bacteria (LAB) in an effort to monitor microbial diversity and biological activity during the fermentation of kimchi, a traditional Korean vegetable product known to contain various health-promoting and immunity-boosting factors.
For RNA extraction, 5 mL aliquots of kimchi soup were pelleted by centrifugation at 5400 g for 10 min. The pellets were then lysed in 1 mL of Trizol reagent (Invitrogen Life Technologies) and 0.4 mL of zirconia-silica beads (0.1 mm diameter; Roth, Karlsruhe, Germany) in a Mini-BeadbeaterTM (Biospec Products, Bartlesville, USA). Labelled cDNAs were prepared from 5 µg RNA samples using the ExpressArt Bacterial mRNA amplification kit.
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Carlucci et al., 2009 (0.3 MB)
A 57-gene expression signature in B-cell chronic lymphocytic leukemia. Biomedicine & Pharmacotherapy 63: 663-671.
The aim of this study is to investigate the gene expression profile, by a specific chip for microarray analysis, in B-CLL lymphocytes with regard to factors involved in apoptosis cascade, signal transduction, purine metabolism enzymes, interleukin expression, enzymes involved in the responses to oxidative stress. We found relevant results in a set of 19 of the 57 genes considered.
Dynabeads were used to enrich for CD19+ cells. About 5 µg total RNA per sample, were amplified and labelled with the ExpressArt Aminoallyl mRNA Amplification Kit, Micro Version. Cy-labelled RNAs were hybridised on a specific custom-chip with 57 gene targets.
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Publications (PDF) |
Knight, Shepherd et al., 2008 (521KB)
TEAD1 and c-Cbl are novel prostate basal cell markers that correlate with poor clinical outcome in prostate cancer. British Journal of Cancer 99: 1849-1858.
Immunohistochemistry and laser microdissection pressure catapulting were combined to isolate 100 to 500 cells from clinical samples. The resulting severely degraded RNAs were amplified by 2-rounds with the ExpressArt TRinucleotide kit. Results of differential expression in microdissected clinical samples were confirmed by RT-PCR, immunofluorescence and furthermore, by effects of knockdown with siRNA in a 3D culture system of prostate cell lines.
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Morris et al., 2008 (760 KB)
Epstein-Barr virus-encoded LMP1 induces a hyperproliferative and inflammatory gene expression programme in cultured keratinocytes. Journal of General Virology 89: 2806-2820.
Comparison of Affymetrix microarray data, obtained with ng amounts of RNA and ExpressArt mRNA amplification versus µg amounts with standard Affymetrix protocols. Differential expression data were generated with three different analysis methods. Microarray data were extensively validated by qRT-PCR, ELISA, immunofluorescence and Western blots. We identified sets of differentially expressed genes that are characteristically expressed in inflammatory and hyperproliferative epidermis, including chemokines, cytokines and their receptors, growth factors involved in promoting epithelial cell motility and proliferation and signalling molecules that regulate actin filament reorganization and cell movement.
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Schain et al., 2008 (392 KB)
Evidence for a pathophysiological role of cysteinyl leukotrienes in classical Hodgkin lymphoma. Int. J. Cancer 123: 2285-2293.
RNA was isolated from fresh-frozen clinical biopsies, from Hodgkin Lymphoma cell lines, LMP1-transfected GC B-cells and EBV-infected KMH2 cells. In model experiments, two HL cell lines, L1236 and KMH2, were shown to express functional CysLT1 receptors, responding with a robust calcium signal upon leukotriene (LT) D4 challenge. LTD4 stimulated protein release of tumor necrosis factor-a, interleukin-6 and interleukin-8 by L1236 cells and interleukin-8 by KMH2 cells. Importantly, all these LTD4-induced effects were blocked by the CysLT1 receptor-specific antagonist zafirlukast.
Corresponding studies were performed with laser microdissection: ~150-200 H-RS cells and 200-300 nonmalignant cells were picked from each of the HL tumors. ExpressArt mRNA amplification and microarray analysis of microdissected cells revealed that the CysLT1 receptor is expressed also by primary Hodgkin Reed-Sternberg cells.
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Vockerodt et al., 2008 (425 KB)
The Epstein-Barr virus oncoprotein, latent membrane protein-1, reprograms germinal centre B cells towards a Hodgkin's Reed-Sternberg-like phenotype. Journal of Pathology 216: 83-92.
RNA from FACS-sorted transfected GC B cells was amplified with the ExpressArt mRNA Amplification kit protocol and 10 µg of fragmented cRNA was hybridised to Affymetrix HGU133Plus2 microarrays. Microarray data were evaluated by qRT-PCR, immunohistochemistry, immunofluorescence and Western blots. Gene expression profiling revealed that LMP1 induced in GC B cells transcriptional changes characteristic of Hodgkin's lymphoma cell lines. Strikingly, LMP1 down-regulated the expression of B-cell-specific genes but also induced the expression of ID2, a negative regulator of B-cell differentiation. Our data suggest that in EBV-positive cases, LMP1 is likely to be a major contributor to the altered transcriptional pattern characteristic of Hodgkin/Reed-Sternberg cells, including the loss of B-cell identity.
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Baumforth et al., 2008 (1780 KB)
Expression of the Epstein-Barr Virus-Encoded Epstein-Barr Virus Nuclear Antigen 1 in Hodgkin's Lymphoma Cells Mediates Up-Regulation of CCL20 and the Migration of Regulatory T Cells. Am. J. Pathol. 173:195-204.
Microdissections were performed on cryosections using the PALM laser microdissector. For microarray analysis (Affymetrix HG-U133 Plus 2.0 GeneChips), RNA was extracted from 150-200 Hodgkin Reed-Sternberg cells or from GCs. RNA was amplified in two rounds with the ExpressArt TRinucleotide mRNA amplification kit.
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Campean et al., 2008 (570 KB)
Campean et al., 2008
Laser microdissection (MMI AG, Switzerland), and use of ExpressArt N-Carrier ("Pico RNA Care") for RNA isolation, followed by analysis on Affymetrix RAE230A Microarray GeneChip.
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Su et al., 2008 (424 KB)
Lambda light chain revision in the human intestinal IgA response. J Immunol 181:1264-1271.
Laser microdissection (P.A.L.M. Microlaser Technologies), and use of ExpressArt N-Carrier ("Pico RNA Care") for RNA isolation, followed by analysis with RT-PCR.
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Hu et al., 2008 (256 KB)
Exon-level expression profiling: a comprehensive transcriptome analysis for oral fluids. Clin. Chem. 54(5): 824-832.
Conclusion: "It is feasible to use [very small] samples containing fragmented RNAs to conduct high-resolution expression profiling with coverage of the entire transcriptome and to validate multiple targets from limited amounts."
Goal was the analysis of amplified salivary RNA with Affymetrix Exon Arrays with the challenges of small sample size (< 5 ng total human RNA) and severe RNA degradation. The standard Affymetrix protocol with rRNA removal with magnetic beads (requires >100 ng of intact RNA) and subsequent RNA amplification by random priming (no selectivity against rRNAs, and requires >100 ng RNA) was impossible.
AmpTec's universal mRNA-specific linear amplification strategy (ExpressArt TRinucleotide mRNA amplification kit) has overcome both limitations. It provides selection against rRNAs, thus rRNA removal is obsolete, and two amplification rounds are possible to provide the necessary amounts of amplified mRNAs for Exon arrays. Array results were validated by qPCR data and an expression phenotype (male/female) was correctly identified.
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Yanai, Baugh et al., 2008 (345 KB)
Pairing of competitive and topologically distinct regulatory modules enhances patterned gene expression. Mol. Syst. Biol. 4:163.
Study of the embryonic development of Caenorhabditis elegans is hampered by the fact that a complete organism provides only ~ 200 picograms of total RNA. Whole-genome microarray analysis necessitates extensive mRNA amplification. Although a sophisticated version of the standard, Eberwine-based technology has been developed [Baugh et al. (2001), Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res. 29:E29.], a minimum of 10 pooled embryos was still required.
A modification of ExpressArt technology [now available as ExpressArt Pico mRNA amplification kit, #8399-A12] has enabled this large-scale study with sub-nanogram RNA amounts. The RNA from 10 embryos could be safely split in half to provide a back-up for further analyses.
"We used RNAi and time series, whole-genome microarray analyses to systematically perturb and characterize components of a Caenorhabditis elegans lineage-specific transcriptional regulatory network. These data are supported by selected reporter gene analyses and comprehensive yeast one-hybrid and promoter sequence analyses."
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Cummings et al., 2008 (286 KB)
Sexual and social stimuli elicit rapid and contrasting genomic responses. Proc. R. Soc. B 275: 393-402.
An initial evaluation of ExpressArt technology was performed [Duftner et al., 2008] and here, ExpressArt is applied in a differential gene expression study.
"Here we examine preference behaviour of 58 female swordtails, Xiphophorus nigrensis, in four different social environments (attractive and unattractive males, females only, non-attractive males only and asocial conditions) followed by neural gene expression profiling. We used a brain-specific cDNA microarray to identify patterns of genomic response and candidate genes, followed by quantitative PCR (qPCR) examination of gene expression with variation in behaviour. Our microarray results revealed patterns of genomic response differing more between classes of social stimuli than between presence versus absence of stimuli. ..... The identification of stimulus- and behaviour-specific responses opens an exciting window into the molecular pathways associated with social behaviour and mechanisms that underlie sexual selection."
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Duftner et al., 2008 (804 KB)
Efficacy of RNA amplification is dependent on sequence characteristics: implications for gene expression profiling using a cDNA microarray. Genomics 91: 108-117.
Detailed study on potential bias introduced by mRNA amplification with the ExpressArt Nano mRNA amplification kit, provides also a brief overview of the ExpressArt technology. ExpressArt technology was subsequently used in a differential gene expression study [Cummings et al., 2008].
Obtained yields: "Assuming that 1-5% of total RNA correspond to poly(A)+ RNA, the first round of amplification yielded an approximately 1000- to 4000-fold increase, while the second round yielded an approximately 200-fold increase of poly(A)+ equivalents. These amplification factors correlate well with the expected values given by the manufacturer of the amplification kit for 500 ng input material in both the first and the second round of amplification."
"We applied a T7 polymerase-based technique [ExpressArt] to amplify RNA from two tissues of a cichlid fish and compared expression levels of unamplified and amplified RNA on a cDNA microarray. Amplification bias was generally minor and comprised features that were lost (1.3%) or gained (2.5%) through amplification and features that were scored as regulated before but unregulated after amplification (4.2%) or vice versa (19.5%). We examined 10 sequence-specific properties and found that GC content, folding energy, hairpin length and number, and lengths of poly(A) and poly(T) stretches significantly affected RNA amplification. We conclude that, if RNA amplification is used in gene expression studies, preceding experiments controlling for amplification bias should be performed."
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Birgersdotter et al., 2007 (725 KB)
Three-dimensional culturing of the Hodgkin lymphoma cell-line L1236 induces a HL tissue-like gene expression pattern. Leukemia and Lymphoma 48: 2042-2053.
"To overcome some limitations of in vitro established cell-line tumor models for Hodgkin lymphoma (HL), we explored whether culturing in a three-dimensional (3D) matrix could improve the quality of the model..... The gene expression profile of the 3D-cultured HL derived cell-line L1236 was compared with that of suspension-cultured (2D) L1236, as well as to the gene expression profile found in HL tumor samples.
To validate our results we also included a gene expression data set of laser captured Hodgkin- Reed-Sternberg (H-RS) cells..... Between 150 and 200 H-RS cells or nonmalignant cells were isolated.... Following RNA extraction, three rounds of linear T7-based mRNA amplification were performed using the ExpressArt system....
The gene expression profiles were analyzed using Affymetrix technology. We found that the 3D culture affected gene expression of an HL derived cell-line, inducing a more tumor-related expression profile."
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Bose et al., 2007 (646 KB)
Down-regulation of ATM protein in HRS cells of nodular sclerosis Hodgkin's lymphoma in children occurs in the absence of ATM gene inactivation. J Pathol 213:329-336.
Microdissections were performed on cryosections using the Leica or PALM laser microdissector. For microarray analysis (Affymetrix HG-U133 Plus 2.0 GeneChips), RNA was extracted from 200-250 Hodgkin Reed-Sternberg cells and amplified in two rounds with the ExpressArt Nano mRNA amplification kit.
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Eickhoff et al., 2007 (548 KB)
Host cell responses to Chlamydia pneumoniae in gamma-interferon-induced persistence overlap those of productive infection and are linked to genes involved in apoptosis, cell cycle and metabolism. Infection and Immunity 75: 2853-2863.
RNA was isolated from cultured HeLa cells, to compare expression profiles of persistently infected cells with those of mock-infected cells. The ExpressArt Micro mRNA amplification kit was used for subsequent analysis with Affymetrix HG-U133A GeneChips. A high level of technical reproducibility was found, and a set of nineteen differentially expressed genes were selected for qPCR analysis. Microarray data were confirmed for seventeen genes.
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Elvidge, 2006 (1.1 MB)
Microarray expression technology: from start to finish. Pharmocogenomics 7: 123-134.
Technology Report: An overview of methods and platforms is presented, including ExpressArt mRNA amplification with its unique ability to perform up to 3 amplification rounds and using sub-nanogram RNA quantities, combined with adaptations for degraded RNAs. |
Wetzel et al., 2006 (325 KB)
Bone morphogenetic protein-7 expression and activity in the human adult normal kidney is predominantly localized to the distal nephron. Kidney Int. 70: 717-723.
Laser microdissection (MMI AG, Switzerland), and use of ExpressArt N-Carrier ("Pico RNA Care") for RNA isolation, followed by qPCR.
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McLucas et al., 2006 (187 KB)
Global gene expression analysis of the effects of Vinblastine on endothelial cells, when eluted from a thermo-responsive polymer. J. Biomed. Mater. Res. A: 246-253.
Use of ExpressArt mRNA amplification kit (Nano version) with subsequent hybridisation to human 40K OciChip.
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Reis et al., 2006 (322 MB)
Isolation of mouse neuritic mRNAs. J Mol Histol. 37: 79-86.
Laser microdissection and use of ExpressArt mRNA amplification kit (Pico version) to visualize isolated RNA.
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Gomez-Mancilla et al., 2005 (0.47 MB)
Central Nervous System Drug Development: An Integrative Biomarker Approach toward Individualized Medicine. NeuroRx. 2: 683-695.
This review represents an effort to illustrate the integration of state of the art and emerging technologies in drug development supporting the path of individualized medicine.
Citation: "Today, ExpressArt mRNA amplification technology based on TRinucleotide primers allows the complete amplification of all mRNA fragments in severely degraded RNA samples and works very well with extremely low limits of input RNA amounts (picogram range)."
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Okuducu et al., 2005 (1.48 MB)
Cellular retinoic acid-binding protein 2 is down-regulated in prostate cancer. Int. J. Oncol. 27: 1273-1282.
Laser microdissection, subsequent use of ExpressArt mRNA amplification kit (2 amplification rounds), followed by cDNA array hybridisation.
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Zeng et al., 2005 (343 KB)
Transcription factor Gfi1 regulates self-renewal and engraftment of hematopoietic stem cells. EMBO J. 23: 4116-4125.
ExpressArt Carrier ("Pico RNA Care") for RNA isolation from FACS-sorted cells.
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