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ExpressArt® Kits - Publications

Publications (PDF files are available on request info@amp-tec.com)

Publications [R1] to [R6] (section at the top)
The New Product Line "ExpressArt FFPE RNAready kits" for RNA isolation from FFPE samples is based on the technology developed by Prof. Rolf Jaggi, Bern University, Switzerland. Results with this technology are presented here, now available as FFPE RNAready kit (#9000-A100, AmpTec, Germany); the most advanced version is the FFPE Clear RNAready kit (#9009-A100).

Publications [1] to [53]
Results with ExpressArt mRNA amplification kits and other ExpressArt reagents.

[R6] Cathomas et al., 2012 (0.27 MB)
Efficacy of cetuximab in metastatic castration-resistant prostate cancer might depend on EGFR and PTEN expression: results from a phase II trial (SAKK 08/07). Clin Cancer Res. (2012) 18(21):6049-57.
The EGF receptor (EGFR) is overexpressed in the majority of metastatic castration-resistant prostate cancers (mCRPC) and might represent a valid therapeutic target.
Patients with mCRPC progressing during or within 90 days after at least 12 weeks of docetaxel were included in this phase II trial. The primary endpoint was progression-free survival (PFS) at 12 weeks defined as the absence of prostate-specific antigen (PSA), radiographic, or clinical progression. Evaluation of known biomarkers of response and resistance to cetuximab (EGFR, PTEN, amphiregulin, epiregulin) was conducted. A significantly improved PFS was found in patients with overexpression of EGFR and persistent activity of PTEN.
Molecular analysis. RNA was isolated from paraffin cores (0.6 mm diameter) using a kit for archival material (AmpTec GmbH, Hamburg, Germany; the most advanced version is the FFPE Clear RNAready kit, #9009-A100).
[R5] Jaggi et al., 2011 (0.6 MB)
A Case Study: Molecular Profiling of Breast Cancer from Formalin-Fixed, Archival Material - Gene Expression Profiles from FFPE Samples with Improved RNA Decrosslinking Technology. J Biomol Tech. 2011 October; 22 (Supplement): S45.
Poster presented at the ABRF Conference 2011, San Antonio, TX, USA.
ABRF = The Association of Biomolecular Resource Facilities.
We have developed a novel demodifaction/decrosslinking protocol for RNA recovery from archival (FFPE) material (now available as FFPE RNAready kit from AmpTec GmbH, Germany). The resulting FFPE RNA quality is superior to RNA obtained with other commercial FFPE RNA isolation kits: larger RNAs can be recovered, and RTqPCR data demonstrate less variability and lower Cq values. This FFPE RNA is suitable for differential gene expression measurement by qPCR, high concordance with parallel RNA samples from fresh frozen tissues was observed. Prognosis of breast cancer is determined by clinicopathological and molecular factors. We developed and validated molecular scores reflecting the hormone status (ER, PGR, HER2 scores) and the proliferation status (PRO score) of breast cancer cells. The scores can be combined to an overall RISK score. Molecular scores are independent prognostic parameters, they were validated in postmenopausal patients with estrogen receptor positive breast cancer. Multivariate analysis revealed that PRO and RISK scores outperform conventional parameters (histological grading and Ki-67 labeling index). Molecular scores are based on routine pathological material, testing can be implemented easily into routine diagnosis.
[R4] Wilkens et al., 2011 (0.58 MB)
The homeobox gene HLXB9 is upregulated in a morphological subset of poorly differentiated hepatocellular carcinoma. Virchows Arch (2011) 458:697-708.
The aim of this study was to get more insight into the molecular characteristics of dedifferentiated carcinomas. Microarray-based global gene expression analysis was performed on five poorly differentiated HCC cell lines compared with non-neoplastic hepatic controls and a set of three cholangiolar carcinoma (CC) cell lines. The gene with the highest upregulation was HLXB9. HLXB9 is a gene of the homeobox gene family important for the development of the pancreas. In order to analyse upregulation of HLXB9 in surgical specimens, RNA was prepared from FFPE samples according to the procedure of Oberli et al [see R2, below] (now available as FFPE RNAready kit from AmpTec GmbH, Germany).
[R3] Antonov, Popovici et al., 2010 (0.5 MB)
Molecular risk assessment of BIG 1-98 participants by expression profiling using RNA from archival tissue. BMC Cancer 2010, 10:37.
The purpose of the work reported here is to test reliable molecular profiles using routinely processed formalin-fixed paraffin-embedded (FFPE) tissues from participants of the clinical trial BIG 1-98 with a median follow-up of 60 months.
RNAs from fresh frozen (FF) and FFPE tumor samples were isolated with AmpTec's RNAready and FFPE RNAready kits, respectively. RNAs from FF and FFPE tumor samples of 82 patients were used for quality control, and independent FFPE tissues of 342 postmenopausal participants of BIG 1-98 with ER-positive cancer were analyzed by measuring prospectively selected genes.
Pearson correlation coefficients between FF- and FFPE-derived scores were at least 0.94 and high concordance was observed between molecular scores and immunohistochemical data.
Scores representing the endocrine responsiveness and proliferation status of breast cancers were developed from gene expression analyses based on RNA derived from FFPE tissues. The validation of the molecular scores with tumor samples of participants of the BIG 1-98 trial demonstrates that such scores can serve as independent prognostic factors to estimate disease free survival (DFS) in postmenopausal patients with estrogen receptor positive breast cancer.
[R2] 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.
[R1] 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.
[53] Boytard et al., 2012 (1.11 MB)
Role of Proinflammatory CD68+MR- Macrophages in Peroxiredoxin-1 Expression and in Abdominal Aortic Aneurysms in Humans. ATVBAHA.112.300663 Published online before print December 13, 2012.
Abdominal aortic aneurysms (AAAs), dilations of the infrarenal aorta, are characterized by inflammation and oxidative stress. The purpose of this study was to determine which subpopulation of macrophages is present in AAAs and is involved in upregulation of PRDX-1 in aneurysmal disease. Immunohistochemistry with antibodies against CD68 and mannose receptor (MR) was used to determine the subtype of macrophages in AAA tissue samples (n=33); laser capture microdissection to isolate each subtype; and quantitative-reverse transcriptase-polymerase chain reaction, Western blot, and ELISA to assess PRDX-1 mRNA and PRDX-1protein levels in both types. Laser capture microdissection-isolated CD68+MR- and CD68+MR+ macrophages, characterized by quantitative-reverse transcriptase-polymerase chain reaction (TNF, IL1B, MRC1, and CCL18) and Western blot (stabilin and hemoglobin), validated the microdissected subtypes. RNA was isolated from LCM-dissected areas and amplified in 2 rounds with the ExpressArt TRinucleotide mRNA amplification Nano kit (AMS-Biotechnology, Abingdon, United Kingdom). PRDX-1 mRNA and PRDX-1 protein were both more abundant in CD68+MR- than CD68+MR+ macrophages in AAA. These findings suggest that the proteins or mRNAs expressed by the proinflammatory CD68+MR- macrophages may contribute to aneurysmal pathology.
[52] Geiger et al., 2012 (1.45 MB)
The Stringent Response of Staphylococcus aureus and Its Impact on Survival after Phagocytosis through the Induction of Intracellular PSMs Expression. PLoS Pathog 8(11): e1003016.
The stringent response is initiated by rapid (p)ppGpp synthesis, which leads to a profound reprogramming of gene expression in most bacteria. In Staphylococcus aureus, (p)ppGpp synthesis upon amino acid deprivation is achieved through the synthase domain of the bifunctional enzyme RSH (RelA/SpoT homolog). Wild-type strain HG001 and rshSyn, codY double mutants were analyzed by transcriptome analysis to delineate different consequences of RSH-dependent (p)ppGpp synthesis. RNA isolation after phagocytosis was performed as described by the manufacturer of the RNA isolation kit (#9007-A100, ExpressArt RNAready Add-On, AmpTec, Hamburg).
[51] Srivastava et al., 2012 (1.56 MB)
Gene expression profiling through microarray analysis in Arabidopsis thaliana colonized by Pseudomonas putida MTCC5279, a plant growth promoting rhizobacterium. Plant Signaling & Behavior 7(2): 235-245.
The present study reports microarray analysis of Arabidopsis thaliana plants inoculated with Pseudomonas putida MTCC5279 (MTCC5279) which resulted in significant increase in growth traits as compared with non-inoculated control. The gene expression changes, represented by oligonucleotide array (24652 genes) have been studied to gain insight into MTCC5279 assisted plant growth promotion: 882 genes were found to be differentially expressed. RNA and microarray analysis. Ten micrograms of the total RNA was converted to cRNA using ExpressArt aminoallyl mRNA amplification Kit (AmpTec GmbH) as per the manufacturer's instruction. Amplified aminoallyl cRNA was purified and 20 micrograms were coupled with fluorescent dyes Cy3 and Cy5 for a dual channel experiment with dye swap. Fifteen micrograms of labeled control and treated samples were mixed and hybridized to Arabidopsis thaliana 25 K OciChip (Ocimum Biosolutions Ltd.).
[50] Ivanidze et al., 2011 (2.60 MB)
Inclusion Body Myositis. Laser Microdissection Reveals Differential Up-Regulation of IFN-gamma Signaling Cascade in Attacked versus Nonattacked Myofibers. AJP 179(3): 1347-1359.
Sporadic inclusion body myositis (IBM) is a muscle disease with two separate pathogenic components, degeneration and inflammation. By using IHC, we classified myofibers from five patients with sporadic IBM as attacked (AIBM) or nonattacked (NIBM) and isolated the intracellular contents of myofibers separately by laser microdissection. For comparison, we isolated myofibers from control persons (HCTRL). The samples were analyzed by microarray hybridization and quantitative PCR. HLA-I up-regulation was observed in AIBM and NIBM, whereas HCTRL were negative for HLA-I. In contrast, the inducible chain of the interferon (IFN) gamma receptor (IFNGR2) and several IFN-gamma-induced genes were up-regulated in AIBM compared with NIBM and HCTRL fibers. Confocal microscopy confirmed segmental IFNGR2 up-regulation on the membranes of AIBM, which positively correlated with the number of adjacent CD8+ T cells. Thus, the differential up-regulation of the IFN-gamma signaling cascade observed in the attacked fibers is related to local inflammation, whereas the ubiquitous HLA-I expression on IBM muscle fibers does not require IFNGR expression.
Laser Microdissection, RNA isolation & RNA amplification. Before the experiments, the RNA quality of each biopsy was assessed by Agilent Bioanalyzer 2100. We used a staining and laser microdissection protocol. Briefly, cryostat sections were double stained for CD8alpha and HLA-ABC. To inhibit RNase activity, all aqueous solutions were pretreated with diethylpyrocarbonate and contained 3 U/μl Protector RNase inhibitor (Roche, Mannheim, Germany) and 2% purified bovine serum albumin (B4287; Sigma). .... This protocol preserves RNA integrity to a large extent (see Supplemental Figure S1 at http://ajp.amjpathol.org). Three rounds of linear T7-basedtranscriptome amplification were performed using the ExpressArt mRNA Amplification Kit, Pico Version (AmpTec, Hamburg, Germany), according to the instructions of the supplier, with the following exceptions: for synthesis of the first cDNA strand, the master mix was prewarmed at 45ºC, and 1 μl of RNase R was added to each reaction for the RNA removal step. The resulting yields of amplified RNA (aRNA) were between 30 and 40 μg, derived from <10 ng of input total RNA.
[49] Hu et al., 2012 (0.70 MB)
A Global View of the Oncogenic Landscape in Nasopharyngeal Carcinoma: An Integrated Analysis at the Genetic and Expression Levels. PLoS ONE 7(7): e41055.
Previous studies have reported that the tumour cells of nasopharyngeal carcinoma (NPC) exhibit recurrent chromosome abnormalities. These genetic changes are broadly assumed to lead to changes in gene expression which are important for the pathogenesis of this tumour. However, this assumption has yet to be formally tested at a global level. Very little correlation is observed between the level of TPG and TSG expression and genomic copy number except for loss of expression in homozygous deletions and one highly amplified segment which shows enhanced gene expression. Individual NPC tumours each express a large number of dysregulated, putative, tumour-suppressing and tumour-promoting genes but almost 60% of these genes can be either upregulated or downregulated in different types of tumour. This suggests that the simplistic classification of genes as TSGs or TPGs may not be entirely appropriate and that the concept of oncosuppressors may be more extensive than previously recognised.
Microdissection, Nucleic Acid Extraction and Amplification. 8 micron thick cryosections were transferred onto PALM membrane slides (P.A.L.M. Microlaser Technologies) and air dried on ice for about 1 minute. Slides were immersed for 2 minutes in cold 75% ethanol, tapped dry and stained for 30 seconds in cold haematoxylin and eosin (9:1) containing 1% NucleoGuard (AmpTec, Hamburg). Excess stain was tapped off and slides were washed in cold nuclease-free water for 30 seconds, cold 75% ethanol for 1 minute, cold 100% ethanol for 1 minute then air dried. Cells for analysis were excised by laser microdissection and pressure catapulting using a PALM MicroBeam instrument and caught on PALM Adhesive Caps. A minimum of 200,000 μm2 of tissue was collected for each DNA or RNA extraction. RNA was extracted by adding 100 μl of RLT buffer (Qiagen) supplemented with 1 μl of N-carrier (AmpTec) and 1 μl of NucleoGuard followed by incubation at room temperature for 15 minutes. Extracted RNA was cleaned up using a Qiagen RNeasy mini kit, including the on-column DNase step as per the manufacturer's instructions. The eluted RNA was collected by ethanol precipitation in the presence of 1 μl P-carrier (AmpTec) and washed twice with 80% ethanol. After checking the quality using the pico assay on an Agilent Bioanalyser, RNA was subjected to three rounds of amplification followed by biotin labelling using an ExpressArt TR Nano amplification kit (AmpTec) and an Affymetrix IVT labelling kit as previously described [65].
[48] Kaur et al., 2012 (1.07 MB)
DMA, a Bisbenzimidazole, Offers Radioprotection by Promoting NFkB Transactivation through NIK/IKK in Human Glioma Cells. PLoS ONE 7(6): e39426.
5.0 μg of total RNA was used for amplification using the ExpressArt mRNA amplification kit. Amplified aRNA (aminoallyl antisense RNA) was then labelled with Cy3 post-labelling reactive dye pack (GE Healthcare, United Kingdom) at RT. 20 μg of labelled aRNA in 200 μl of Ocimum's Hyb buffer was denatured at 95ºC for 3 min and 100 μl was used for hybridization performed at 50ºC for 2 h on Tecan HS 4800 automated Hyb station. Human 40 KA and 40 KB OciChip (OcimumBiosolutions, Hyderabad) consisting of 20160 and 19968 spots respectively were printed on Corning epoxy coated glass slides using Omnigrid (Gene Machines). Oligos were printed using spotting buffer A at 50% humidity and 22ºC. RNA samples were then hybridized and processed according to OciChipTM 40K Array manual specifications. Our studies show that NIK/IKK mediated NFkB activation is more intensified in cells over expressing NIK and treated with DMA, alone or in combination with ionizing radiation, indicating that DMA promotes NIK mediated NFkB signaling. This subsequently leads to the radioprotective effect exhibited by DMA.
[47] Bruder et al., 2012 (1.08 MB)
Target specificity of an autoreactive human γδ-T cell receptor in myositis. J. Biol. Chem. 287: 20986-20995
In polymyositis and inclusion body myositis, muscle fibers are surrounded and invaded by CD8-positive cytotoxic T cells expressing the αβ-T cell receptor (αβ-TCR) for antigen. In a rare variant of myositis, muscle fibers are similarly attacked by CD8-negative T cells expressing the γδ -TCR (γδ-T cell-mediated myositis).We investigated the antigen specificity of a human γδ-T cell receptor (TCR) previously identified in an autoimmune tissue lesion of γδ-T cell mediated myositis. We show that this Vγ13Vδ2-TCR, termed M88, recognizes various proteins from different species. Several of them belong to the translational apparatus, including some bacterial and human aminoacyl-tRNAsynthetases (AA-RS). Specifically, M88 recognizes histidyl-tRNA-synthetase, an antigen known to be also targeted by autoantibodies called anti-Jo-1. The M88 target epitope is strictly conformational, independent of post-translational modification, and exposed on the surface of the respective antigenic protein. Extensive mutagenesis of the translation initiation factor-1 from E. coli (EcIF1), which served as a paradigm antigen with known structure, showed that a short α-helical loop around amino acids 39 to 42 of EcIF1 is a major part of the M88 epitope. Mutagenesis of M88 showed that the complementarity determining regions 3 of both γδ-TCR chains contribute to antigen recognition. M88 is the only known example of a molecularly characterized γδ-TCR expressed by autoaggressive T cells in tissue. The observation that AA-RS are targeted by a γδ-T cell and by autoantibodies reveals an unexpected link between T-cell and antibody responses in autoimmune myositis.
Generation of an amplified E. coli cDNA library
E. coli mRNA was amplified using the "ExpressArt Bacterial mRNA amplification kit" (AmpTec), which yields amplified cDNA. In the final synthesis step, cDNA was synthesized according to the protocol for second round amplification with the exception that primer C was replaced by a primer that contained a NotI restriction site (5'-ATAGTTTAGCGGCCGCGGGAGATTTTTTTTTTTT-3' (The NotI site is underlined)). This product was finally digested with NotI. Due to the enzymes contained in the amplification kit, the library contains a blunt end on the other side. It was finally inserted into the plasmid pET21c(+) previously digested with SmaI and NotI.
[46] Radchuk et al., 2012 (1.53 MB)
Fertility in barley flowers depends on Jekyll functions in male and female sporophytes. New Phytologist. 194 (1): 142-157.
Sexual reproduction of small grain cereals is poorly investigated, making discovery of novel genes and functions a challenging priority. Barley gene Jekyll appears to be a key player in grain development; however, its role in flowers has remained unknown. Here, we studied RNAi lines of barley, where Jekyll expression was repressed to different extents. The impact of Jekyll on flower development was evaluated based on differential gene expression analysis applied to anthers and gynoecia of wildtype and transgenic plants, as well as using isotope labeling experiments, hormone analysis, immunogold- and TUNEL-assays and in situ hybridization. Jekyll is expressed in nurse tissues mediating gametophyte–sporophyte interaction in anthers and gynoecia, where JEKYLL was found within the intracellular membranes. The repression of Jekyll impaired pollen maturation, anther dehiscence and induced a significant loss of fertility. The presence of JEKYLL on the pollen surface also hints at possible involvement in the fertilization process. We conclude that the role of Jekyll in cereal sexual reproduction is clearly much broader than has been hitherto realized.
[45] Thompson et al., 2012 (2.82 MB)
RNA profiling and ChIP-Sequencing reveal that PTF1a stabilizes pancreas progenitor identity via the control of MNX1/HLXB9 and a network of other transcription factors. Mol Cell Biol. 32(6):1189-1199.
Pancreas development is initiated by the specification and expansion of a small group of endodermal cells. Several transcription factors are crucial for progenitor maintenance and expansion, but their interactions and the downstream targets mediating their activity are poorly understood. Among those factors, PTF1a, a basic helix-loop-helix (bHLH) transcription factor which controls pancreas exocrine cell differentiation, maintenance, and functionality, is also needed for the early specification of pancreas progenitors.
We used RNA profiling and chromatin immunoprecipitation (ChIP) sequencing to identify a set of targets in pancreas progenitors. We demonstrate that Mnx1, a gene that is absolutely required in pancreas progenitors, is a major direct target of PTF1a and is regulated by a distant enhancer element. Pdx1, Nkx6.1, and Onecut1 are also direct PTF1a targets whose expression is promoted by PTF1a. These proteins, most of which were previously shown to be necessary for pancreas bud maintenance or formation, form a transcription factor network that allows the maintenance of pancreas progenitors. In addition, we identify Bmp7, Nr5a2, RhoV, and P2rx1 as new targets of PTF1a in pancreas progenitors.
cDNA profiling. Dorsal pancreatic E10.5 anlagen (500 to 1,000 cells) were homogenized, and RNA was purified using the RNeasy minikit (Qiagen) including on-column DNase treatment (Qiagen). RNA yields were about 5 ng from 3 dorsal buds. For microarray profiling, cDNA with a T7 promoter incorporated was generated and two amplification rounds were performed (AmpTec, ExpressArt mRNA amplification kit) (Fig. 1A).
[44] Mihailovich et al., 2012 (1.32 MB)
Widespread generation of alternative UTRs contributes to sex-specific RNA binding by UNR. RNA 18: 53-64.
To investigate sex-specific functions of UNR, we have identified its RNA targets in adult male and female flies by high-throughput RNA binding and transcriptome analysis. RNA obtained from RIP of cytoplasmic extracts was used to generate probes for microarray hybridization using the pico version of the ExpressArt mRNA Amplification Kit (Artus, comment: now available from AmpTec, Hamburg, Germany). A comparison of the targets identified by RIP-Chip (Microarray-based) and RIP-seq (based on Illumina Solexa sequencing) indicated an overlap of ~15%. Several factors could contribute to this limited overlap, including the use of a different starting material for RIP-Chip and RIP-seq (cytoplasmic vs. total extracts, respectively). A further obvious difference is the absence of non-coding RNAs on the microarrays used in this study: custom-made microarrays printed at the Transcriptome Platform of Barcelona University.
Comment from AmpTec: Notably, both approaches were limited to RNAs with 3'-polyA tails. Whereas the more recent ExpressArt TR RNA amplification kits do not require a 3'-polyA, while maintaining preferential 3'-priming and selection against ribosomal RNAs. This opens new perspectives for microarray experiments (they could cover much broader probe sets) and for combinations with deep sequencing approaches, like Solexa.
[43] Whitaker et al., 2011 (1.0 MB)
Serotonergic modulation of startle-escape plasticity in an African cichlid fish: a single-cell molecular and physiological analysis of a vital neural circuit. J Neurophysiol 106: 127-137.
In the present study, we pharmacologically perturbed the function of the 5-HTR subtype 2 (5-HTR2) in A. burtoni DOMs and SUBs and showed that 5-HT plays an important role in determining the socially regulated plasticity in startleescape performance, as described previously for this species (Neumeister et al. 2010). Using transcriptomics, we then demonstrated that high-quality mRNA can be harvested [and amplified faithfully] from single M-cells, paving the way for single-neuron molecular analyses of the M-cell system.
RNA was isolated from whole brain of A. burtoni individuals and from single M-cells. We had previously shown that the vast majority of RNA species amplify faithfully [see Duftner et al., 2008, available as download number [12] in this publication list]. We subjected the RNA samples obtained from the three whole brains and the three M-cells (2 replicates each) to two rounds of linear amplification. To facilitate the generation of almost full-length molecules we used the picoRNA amplification system by Ocimum Biosolutions.
Comment from AmpTec: The ExpressArt mRNA amplification kits used in this study were manufactured by AmpTec and obtained via our US Distributor Ocimum Biosolutiuons.
[42] Tochio et al., 2011 (0.12 MB)
Presence of amplifiable mRNA in acellular hair shafts: utilization to analyze gene expression profiles of black and white hairs. International Journal of Dermatology 50: 530-534.
This study aimed to prove the presence of amplifiable mRNAs in the hair shaft and then to utilize them to characterize black and white hairs at the gene expression level. RNAs were extracted from black and white scalp hair shafts. Microarray analysis was performed using T7 RNA polymerase-amplified mRNAs, using an AminoAllyl mRNA amplification kit from Artus, Hamburg, Germany (now these kits are available from AmpTec, Hamburg, Germany). The amplified fluorescence-labeled mRNAs of hair shafts were subjected to gene expression assays using a 35,000 probespotted microarray (Human 35K; Filgen, Inc., Nagoya, Japan).
We showed that hair shaft RNAs contained trace amounts of mRNAs, we termed the mRNA "fossil mRNA" (fmRNA) in this study. Totals of 10 and 2% of the detected genes were expressed at levels more than two-fold higher in black and white hairs compared with white and black hairs, respectively. We selected five genes and examined their expression levels in five donors by qRT-PCR. Among them, only hypothetical protein (L1H3 region) human was found to be expressed at 2.69 ± 1.81 (relative ratio) in black hairs. Consequently, fmRNA is expected to be a useful source of information for the study of phenotypic changes in hairs at the molecular level.
[41] Raj et al., 2011 (1.63 MB)
Changes in the Cytokine and Toll-Like Receptor Gene Expression Following Infection of Indigenous and Commercial Chickens with Infectious bursal disease virus. Indian J. Virol. 22(2):146-151.
Indigenous chickens are considered to be more disease resistant than their commercial counterparts in many parts of the world. RNA was isolated from control and virus infected spleen tissues. For the microarray experiment, RNA was amplified using ExpressArt mRNA amplification kit Micro version (Artus GmbH, Germany; now these kits are available from AmpTec, Hamburg, Germany) using 2.5 μg of total RNA. Amplified RNA was labeled with Cy3 Post-Labelling Reactive Dye Pack (GE Healthcare UK Limited, UK). Five μg of the labeled RNA was used for hybridization with the custom OciChipTM (Ocimum Biosolutions, Hyderabad, India). In summary, we have shown for the first time that some local innate effectors including TLR3 and IL15 are differentially up regulated in the spleen of indigenous chickens 3 days post-infection. The innate antiviral effect induced through TLR3 up regulation and engagement and Th1 type immunity by IL15 in indigenous chickens probably confer innate resistance to IBDV-induced clinical disease in these native birds.
[40] Vogel¹) & Wheat²), 2011 (0.23 MB)
Accessing the transcriptome: how to normalize mRNA pools. Methods Mol Biol. 772: 105-128. From the Department of Entomology, Max-Planck-Institute for Chemical Ecology, Jena, Germany. ¹) hvogel@ice.mpg.de, ²) cww10@psu.edu
Advances in next generation sequencing continue and transcriptome sequencing will increase in importance as a fast and direct means of accessing the genes them-selves. However, constructing a comprehensive cDNA library for deep sequencing is very difficult and cDNA normalization provides an important means of allocating sequencing across a greater fraction of genes. This publication provides a complete work flow: RNA Isolation from few cells where the combination of NucleoGuard (chemical nuclease inhibitor) and N-Carrier (inert carrier RNA) (both from AmpTec, Hamburg, Germany) prevents degradation and loss of small RNA samples. Amplification of small RNA amounts is achieved with ExpressArt mRNA ampli-fication kits (AmpTec, Hamburg, Germany). Following cDNA synthesis, two different normalization methods are outlined, addressing many of the important issues that need consideration when going from RNA isolation to the cDNA material required for sequencing.
[39] Fuentes et al., 2011 (0.30 MB)
Downregulation of the tumour suppressor p16INK4A contributes to the polarisation of human macrophages toward an adipose tissue macrophage (ATM)-like phenotype. Diabetologia 54(12): 3150-3156.
Laser-capture microdissection of human atherosclerotic plaques was followed by RNA isolation and two rounds of RNA amplification using the ExpressArt TRinucleotide mRNA amplification Nano kit (AmpTec).
Gene expression of p16INK4A in ATMs was analysed and compared with that in monocyte-derived macrophages (MDMs) from obese patients or with macrophages from human atherosclerotic plaques (AMs). Additionally, p16INK4A levels were studied during macrophage differentiation and polarisation of monocytes isolated from healthy donors. The role of p16INK4A in MDMs from healthy donors was investigated by small interfering (si) RNA-mediated silencing or adenovirus-mediated overproduction of p16INK4A. The obtained results show that p16INK4A inhibits the acquisition of the ATM phenotype. The accerelated increase in p16INK4A level may thus influence normal ATM function and contribute to type 2 diabetes risk.
[38] Schiebold et al., 2011 (3.4 MB)
A novel procedure for the quantitative analysis of metabolites, storage products and transcripts of laser microdissected seed tissues of Brassica napus. Plant Methods 2011, 7:19.
This publication provides a guideline and detailed procedures towards the quantitative analysis of laser micro-dissected (LM) tissues in oilseed rape (Brassica napus). This includes protocols for laser microdissection of the seed, and the subsequent extraction and quantitative analysis of lipids, starch and metabolites, including a protocol allowing the parallel analysis of the transcriptome using Brassica-specific microarrays. Some data are presented regarding the compartmentation within the oilseed rape embryo.
Different RNA isolation methods were used: Dynabeads mRNA, and total RNA obtained with RNeasy spin column procedure or with a conventional phenol/chloroform extraction method. For microarray analyses we performed a two-round linear amplification, using C&E Version ExpressArt mRNA amplification Nano kit (Amptec GmbH, Hamburg, Germany) according to the manufacturer's protocol. The amplification procedure generates long transcript and yields 2,500-fold to 9,500-fold RNA amplification, based on input mRNA amounts. The high degree of correlation between independent replicates (r ~0.98), suggests that the amplification is highly reproducible.
[37] Weber et al., 2011 (4.0 MB)
CCL17-expressing dendritic cells drive atherosclerosis by restraining regulatory T cell homeostasis in mice. J Clin Invest doi:10.1172/JCI44925.
Immune mechanisms are known to control the pathogenesis of atherosclerosis. However, the exact role of DCs, which are essential for priming of immune responses, remains elusive. We have shown here that the DC-derived chemokine CCL17 is present in advanced human and mouse atherosclerosis and that CCL17+ DCs accumulate in atherosclerotic lesions. In atherosclerosis-prone mice, Ccl17 deficiency entailed a reduction of atherosclerosis, which was dependent on Tregs. Expression of CCL17 by DCs limited the expansion of Tregs by restricting their maintenance and precipitated atherosclerosis in a mechanism conferred by T cells. Conversely, a blocking antibody specific for CCL17 expanded Tregs and reduced atheroprogression. Our data identify DC-derived CCL17 as a central regulator of Treg homeostasis, implicate DCs and their effector functions in atherogenesis, and suggest that CCL17 might be a target for vascular therapy.
Quantitative real-time PCR: Very small RNA amounts required a pre-amplification before RT-qPCR analysis. RNA was isolated from aortic sections and amplified using ExpressArt TRinucleotide mRNA amplification Nano kit (amsbio) Reverse transcription of amplified RNA was performed with a TaqMan Reverse Transcription Kit, and real-time PCR analysis was performed using Pre-Developed TaqMan assays.
[36] Vrzalikova et al., 2011 (0.7 MB)
Down-regulation of BLIMP1α by the EBV oncogene, LMP1, disrupts the plasma cell differentiation program and prevents viral replication in B cells: implications for the pathogenesis of EBV-associated B cell lymphomas. Blood 117, 5907-5917.
Because virus replication in B cells is intimately linked to their differentiation towards plasma cells, we asked if the physiological signals which drive normal B cell differentiation are absent in EBV-transformed cells. We focussed on BLIMP1α, a transcription factor that is required for plasma cell differentiation and which is inactivated in diffuse large B cell lymphomas. We show that BLIMP1α expression is down-regulated following EBV infection of primary germinal centre (GC) B cells and that the EBV oncogene, latent membrane protein-1 (LMP1), is alone capable of inducing this down-regulation in these cells. Our results suggest that LMP1 expression in progenitor GC B cells could contribute to the pathogenesis of EBV-associated lymphomas by down-regulating BLIMP1α in turn preventing plasma cell differentiation and induction of the viral lytic cycle.
Microarray analysis: CD10+ GC B cells were isolated from two donors and transfected as described above. RNA isolated from transfected cells was amplified with ExpressArt mRNA Nano version & Nano plus Version Amplification Kits (AmpTec GmbH, Germany)., Fragmented and biotin-labelled cRNA was hybridized to Affymetrix HG-U133Plus2 microarrays.
[35] Anderton et al., 2011 (0.5 MB)
The H3K27me3 demethylase, KDM6B, is induced by Epstein–Barr virus and over-expressed in Hodgkin’s Lymphoma. Oncogene 30, 2037-2043.
There is now evidence for both increased and decreased activity of the enzymes controlling the methylation of lysine 27 on histone 3 (H3K27) in cancer. One of these enzymes, KDM6B, a histone demethylase, which removes the trimethyl mark from H3K27, is required for the lineage commitment and terminal differentiation of neural stem cells and of keratinocytes. Presented results suggest that KDM6B may also have a role in antigen-driven B-cell differentiation.
Gene expression profiling shows that KDM6B transcriptional targets in germinal centre B (GC B) cells are significantly enriched for those differentially expressed during memory and plasma cell differentiation. Presented results also suggest that aberrant expression of KDM6B may contribute to the pathogenesis of Hodgkin’s Lymphoma (HL), an Epstein–Barr virus (EBV) associated malignancy. KDM6B is over-expressed in primary HL and induced by the EBV oncogene, latent membrane protein (LMP1) in GC B cells, the presumptive progenitors of HL.
Purification and gene expression profiling of transfected of GC B cells: Transfected tonsillar CD10+ germinal centre B (GC B) cells were isolated by staining with anti-LNFGR-FITC and propidium iodide, following which FITC-labelled propidium iodide-negative cells were collected on a MoFlo fluorescence cell sorter (Dako, Glostrup, Denmark). Total RNA was extracted from fluorescence-activated cell-sorted, transfected GC B cells, and then amplified with the ExpressArt mRNA Amplification kit (AmpTec, Hamburg, Germany). Fragmented and labelled cRNA (10 µg) was hybridised to Affymetrix HG-U133Plus2 microarrays.
[34] Beckervordersandforth et al., 2010 (2.5 MB)
In Vivo Fate Mapping and Expression AnalysisReveals Molecular Hallmarks of Prospectively Isolated Adult Neural Stem Cells. Cell Stem Cell 7, 744–758.
Until now, limitations in the ability to enrich adult NSCs (aNSCs) have hampered meaningful analysis of these cells at the transcriptome level. Via a split-Cre technology it was shown that coincident activity of the hGFAP and prominin1 promoters is a hallmark of aNSCs in vivo. Sorting of cells from the adult mouse subependymal zone (SEZ) based on their expression of GFAP and prominin1 isolates all self-renewing, multipotent stem cells at high purity. RNA Isolation: After sorting, the cells were centrifuged for 20 min at 10,000 rpm and lysed in 100 µl of lysis buffer containing N & P carriers (ExpressArt, AmpTec).
Comparison of the transcriptome of these purified aNSCs to parenchymal non-neurogenic astrocytes and other SEZ cells reveals aNSC hallmarks, including neuronal lineage priming and the importance of cilia- and Ca-dependent signaling pathways.
[33] Weyrich et al., 2010 (0.6 MB)
Seasonal changes of gene expression in roe deer (Capreolus capreolus) testis measured by expression microarray analysis. Trends Anim Vet Sci J 2010 1(2):5-20.
Gene expression changes in roe deer testis during the transition between the two extreme phases of the circannual testicular development were investigated: arrest of spermatogenesis (December) on one hand and fully activated spermatogenesis (June) on the other.
RNA was isolated from ~30 mg of roe deer testis. Reverse transcription of mRNA, cDNA synthesis and amino-allyl antisense RNA amplification (AA-aRNA) were performed using ExpressArt®-Kits (AmpTec, Hamburg, Germany). Due to the lack of a fully sequenced roe deer genome, we used an 8K expression microarray from cattle as its evolutionary closest and sequenced relative. During the transition from December to June, 622 genes were significantly differentially expressed (p<0.05). Besides the confirmation of data from previous studies, we were able to implicate new genes and pathways such as the Wnt signalling pathway and the Ca2+-ion transport in the interplay between developmental stages of roe deer testis.
[32] Sharma et al., 2009 (0.25 MB)
Role of specific signalling molecules during proliferation of CD4+ T cells in response to different stimuli. BARC Newsletter 309: 293-301.
CD4+ helper T cells, are known to divide in response to diverse stimuli, including mitogens and lymphopenia. Our earlier studies had shown that PI3kinase and mTOR were redundant for Homeostasis Driven Proliferation (HDP), but not for Mitogen Driven Proliferation (MDP). Experiments were carried out to study the gene expression pattern in CD4+ T cells, after Mitogen Driven Proliferation (MDP) and HDP. These cells were used for gene expression analysis, using Ocichip (Ocimum Biosolutions, Hyderabad). Cytokine production was also studied by ELISA. The results indicated, that certain common pathways were involved in HDP and MDP of CD4+ T cells. However, a few pathways were specific to either MDP or HDP. Studies using signaling inhibitors confirmed, that NF-kappaB-dependent pathways were involved in HDP as well as in MDP. However, protein kinase C, PI3Kinase, mTOR and MEK dependent pathways were specifically required for MDP and not for HDP. Further, CD4+ T cells produced different levels of Th1, Th2 or Th17 cytokines, following MDP and HDP
[31] Englert et al., 2009 (0.5 MB)
Amplification of mRNA without 3’ bias from formalin-fixed paraffin-embedded (FFPE) breast cancer tissues and multiplex expression analysis on flow-through microarrays. Cancer Res 69(24 Suppl):Abstract nr 3052. Poster presented at the Thirty-Second Annual CTRC-AACR San Antonio Breast Cancer Symposium. Dec 10-13, 2009.
Total RNA extracted from FFPE samples was subjected to two rounds of amplification with the ExpressArt TRinucleotide kit (AmpTec), which preferentially primes cDNA synthesis near the 3'-ends of mRNA fragments (rather than only at the poly-A sites of intact mRNAs) and selects for mRNAs against ribosomal RNAs. Biotin-labeled anti-sense RNA (aRNA) was hybridized to flow-through microarrays on the Ziplex Automated Workstation (Xceed Molecular). Performance was assessed by the analysis of defined titration mixtures prepared from mRNA from breast cancer and colon cancer FFPE tissue blocks.
Similar signal intensities were observed for 3'-biased probes and probes several hundred bases from the 3'end, confirming that there is no 3' bias in the amplification. This demonstrated the feasibility of amplifying and quantifying sequences at any position within transcripts in degraded mRNA from FFPE samples. Expression differences of less than two-fold may be conveniently analyzed with hundreds of probes on the Ziplex Workstation.
[30] Sharbel et al., 2010 (1.5 MB)
Apomictic and Sexual Ovules of Boechera Display Heterochronic Global Gene Expression Patterns. Plant Cell 22: 655–671.
We have compared the transcriptomic profiles of microdissected live ovules at four developmental stages between a diploid sexual and diploid apomictic Boechera. For each accession, 20 ovules per developmental stage were collected in one tube (with two technical replicates for each stage), frozen directly in liquid nitrogen, and stored at -80°C. RNA was isolated using the PicoPure RNA isolation kit (Arcturus Bioscience).
To prevent degradation of the minute amounts of RNA during the isolation procedure and to enhance the binding efficiency of the RNA on the purification columns, the lysis buffer was supplemented with 1% (v/v) of NucleoGuard stock solution and 2 µl of N-Carrier (AmpTec). For the required amounts of dscDNA for the SuperSAGE method, the RNA had to be amplified. Linear mRNA amplification was achieved using the ExpressArt mRNA amplification kit (AmpTec) with several important modifications. As starting material ~4 ng of total RNA was used in two independent reactions per sample to level out any random methodological effects.
We sequenced >2 million SuperSAGE tags and identified (1) heterochronic tags (n=595) that demonstrated significantly different patterns of expression between sexual and apomictic ovules across all developmental stages, (2) stage-specific tags (n=577) that were found in a single developmental stage and differentially expressed between the sexual and apomictic ovules, and (3) sex-specific (n=237) and apomixis-specific (n=1106) tags that were found in all four developmental stages, but in only one reproductive mode.
[29] Murphy, Buckley et al., 2009 (0.5 MB)
Global MYCN Transcription Factor Binding Analysis in Neuroblastoma Reveals Association with Distinct E-Box Motifs and Regions of DNA Hypermethylation. PLoS ONE 4(12): e8154.
Combination of ExpressArt mRNA amplification and Nimblegen microarrays: The ExpressArt TR Micro Kit (Cat. No. 6199-A30, AmpTec) was used to synthesise double-stranded cDNA from 3 µg total RNA, which was subsequently used to generate amplified amino-allyl antisense RNA (aRNA). The aRNA was coupled to Cy3 reactive dye and 4 µg of Cy3-aRNA was hybridised to the Homo sapiens 4x72K Gene Expression Array from Roche NimbleGen.
Neuroblastoma, a cancer derived from precursor cells of the sympathetic nervous system, is a major cause of childhood cancer related deaths. The single most important prognostic indicator of poor clinical outcome in this disease is genomic amplification of MYCN, a member of a family of oncogenic transcription factors.
We applied MYCN chromatin immunoprecipitation to microarrays (ChIP-chip) using MYCN amplified/nonamplified cell lines as well as a conditional knockdown cell line to determine the distribution of MYCN binding sites within all annotated promoter regions.
Resulting effects on DNA methylation status were correlated with gene expression levels.
[28] 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.
[27] 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.
[26] 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.
[25] 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.
[24] 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.
[23] Thoudam et al., 2008 (40 KB)
Differential gene expression profile of stomach and oral cancer in high risk region of India. Genomic Med. (2008) 2:376
RNA extracted from ten tumor tissues (five with oral squamous cell carcinoma and five with gastric adenocarcinoma) was amplified using ExpressArt AminoAllyl mRNA amplification Kit and labelled with NHS-activated Cy-3 dye. Normal tissue obtained from a site distant to tumour was used as control. The labelled probes were hybridized with Human 40K OciChip Array containing 20,106-probes.
Gene Ontology revealed that genes involved in regulation of actin cytoskeleton, MAPK and Wnt signalling pathways were differentially expressed in both cancers. Genes involved in Neuroactive ligand-receptor interaction and leukocyte transendothelial migration were differentially expressed in gastric adenocarcinoma exclusively. Genes of TGF beta signalling pathway were significantly downregulated in gastric adenocarcinoma while in oral cancer genes of T-cell-receptor and Toll like receptor pathway were significantly upregulated.
[22] Knight et al., 2008 (425 KB)
TEAD1 and c-Cbl are novel prostate basal cell markers that correlate with poor clinical outcome in prostate cancer. Br. J. Cancer 99:1849-1858
Immunohistochemistry of clinical prostate cancer samples was combined with laser microdissection to isolate 200 to 1,000 luminal and basal cells. Isolated RNAs were amplified with the ExpressArt TR Nano kit, followed by hybridisation to genome-wide cDNA arrays. Differential expression data were confirmed by RT-PCR and immunostaining.
[21] Morris et al., 2008 (521 KB)
Epstein-Barr virus-encoded LMP1 induces a hyperproliferative and inflammatory gene expression programme in cultured keratinocytes. J. Gen. Virol. 89: 2806-2820
Affymetrix microarray data were obtained with microgram quantities of RNA by using standard Affymetrix technology, or with nanogram amounts of RNA and amplification with the ExpressArt Nano kit. Differential expression data were generated with three different analysis methods. Microarray data were extensively validated by qRT-PCR, ELISA, immunofluorescence and Western blots.
[20] Schain et al., 2008 (392 KB)
Evidence for a pathophysiological role of cysteinyl leukotrienes in classical Hodgkin lymphoma. Int. J. Cancer: 12: 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. 150–200 H-RS cells and 200–300 nonmalignant cells were isolated by laser microdissection from Hodgkin Lymphoma tumors and amplified with the ExpressArt Nano kit. Microarray data were confirmed by qPCR and immunohistochemistry.
[19] 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. J. Pathol. 216: 83-92.
RNA from FACS-sorted transfected GC B cells was amplified with the ExpressArt Nano mRNA Amplification kit. Microarray data were evaluated by qRT-PCR, immunohistochemistry, immunofluorescence and Western blots.
[18] Baumforth et al., 2008 (1.78 MB)
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 Nano TRinucleotide mRNA amplification kit.
[17] Campean et al., 2008 (570 KB)
Angiopoietin 1 and 2 gene and protein expression is differentially regulated in acute anti-thy 1.1. Am J Physiol Renal Physiol 294: F1174-F1184.
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.
[16] 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.
[15] 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 Nano 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.
[14] 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."
[13] 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."
[12] 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."
[11] Zhang et al., 2008 (0.97 MB)
We describe a simple and highly effective means for global identification of genes that are expressed within specific cell types within complex tissues. It involves transgenic expression of nuclear-targeted green fluorescent protein in a cell-type-specific manner. The fluorescent nuclei are then purified from homogenates by fluorescence-activated sorting, and the RNAs employed as targets for microarray hybridization. We demonstrate the validity of the approach through the identification of 12 genes that are selectively expressed in phloem.
Two consecutive rounds of amplification were done on 5 µl samples (approximately 2–20 ng RNA), using ExpressArt mRNA amplification kits (Nano plus Version; Artus GmbH) according to the manufacturer’s instructions. Aminoallyl-modified UTP was incorporated into the amplified antisense RNA produced during the second round of in vitro transcription.
[10] 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."
[9] 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.
[8] 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.
[7] 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.
[6] 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.
[5] 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.
[4] 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.
[3] 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)."
[2] 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 (two amplification rounds), followed by hybridisation to cDNA microarrays.
[1] Zeng et al., 2005 (343 KB)
Transcription factor Gfi1 regulates self-renewal and engraftment of hematopoietic stem cells. EMBO J. 23: 4116-4125.
ExpressArt N-Carrier and P-Carrier ("Pico RNA Care") for RNA isolation from FACS-sorted cells.
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