Preparation Of High-quality RNA Research Articles

Chicken Immunization followed by RNA Extraction and cDNA Synthesis for Antibody Library Preparation.

Effective isolation of specific antibodies from immunological repertoires requires the generation of a diverse library against a specific antigen of interest, as well as efficient selection procedures, such as bio-panning and phage ELISA. Key to this is the generation of a good immune response in the host, followed by preparation of high-quality RNA and cDNA from which a library can be constructed by the amplification and cloning of immunoglobulin heavy and light chain genes. The first step in the construction of such an "immune library" is a successful course of immunization. Detection of a strong serum antibody titer will theoretically then result in a pool of extracted RNA that is enriched for transcripts of genes encoding the antibody of interest. Chicken antibodies have been widely used for research and diagnostic purposes, largely because of both their cross-reactivity to epitopes shared by humans, mice, primates, and other mammals, and their simple characteristics, with chickens featuring single functional copies of V H /J H and V λ /J λ gene pairs. In chickens, antibodies against an antigen of interest can be detected in the serum as soon as 5-7 d after immunization. Once the antibody titer reaches an appropriate level in the serum, the spleen, bursa of Fabricius, and bone marrow are then harvested, and antibody libraries can be prepared from extracted RNA. Here, we describe a protocol for chicken immunization with an antigen of interest, followed by RNA extraction from the relevant tissues and cDNA synthesis, which users can use for antibody library construction.

Quality parameters for RNA preparations from biopsies of ulcerated human skin.

Background: Obtaining high quality RNA from skin biopsies is complex due the physical composition and high content of nucleases of this tissue. This becomes particularly challenging when using compromised skin samples with necrotic, inflammed or damaged areas, such as those from patients suffering skin conditions, which affect more than 900 million people annually. We evaluated the impact of the biopsy size and tissue preservation method on the quality and quantity of RNA extracts. Methods: Skin lesion biopsies were obtained from patients with cutaneous leishmaniasis (CL). Biopsy specimens of 2 mm (n = 10) and 3 mm (n = 59) were preserved in Allprotect® reagent, and 4 mm biopsies in OCT (n = 54). Quality parameters were evaluated using Nanodrop and Bioanalyzer. The informativeness of the extracted samples for downstream analyses was evaluated using RT-qPCR and RNA-Seq. Results: The success rate, based on quality parameters of RNA extraction from tissue biopsies stored in OCT and 2 mm biopsies stored in Allprotect®, was 56% (30/54) and 30% (3/10), respectively. For 3 mm skin biopsies stored in Allprotect® was 93% (55/59). RNA preparations from 3 mm-Allprotect® biopsies had an average RIN of 7.2 ± 0.7, and their integrity was not impacted by sample storage time (up to 200 days at -20°C). RNA products were appropriate for qRT-PCR and RNA-seq. Based on these results, we propose a standardized method for RNA extraction from disrupted skin samples. This protocol was validated with lesion biopsies from CL patients (n = 30), having a success rate of 100%. Conclusions: Our results indicate that a biopsy size of 3 mm in diameter and preservation in Allprotect® for up to 200 days at -20°C, are best to obtain high quality RNA preparations from ulcerated skin lesion biopsy samples.

Quality parameters for RNA preparations from biopsies of ulcerated human skin.

Background: Obtaining high quality RNA from skin biopsies is complex due the physical composition and high content of nucleases of this tissue. This becomes particularly challenging when using compromised skin samples with necrotic, inflammed or damaged areas, such as those from patients suffering skin conditions, which affect more than 900 million people annually. We evaluated the impact of the biopsy size and tissue preservation method on the quality and quantity of RNA extracts. Methods: Skin lesion biopsies were obtained from patients with cutaneous leishmaniasis (CL). Biopsy specimens of 2 mm (n = 10) and 3 mm (n = 59) were preserved in Allprotect® reagent, and 4 mm biopsies in OCT (n = 54). Quality parameters were evaluated using Nanodrop and Bioanalyzer. The informativeness of the extracted samples for downstream analyses was evaluated using RT-qPCR and RNA-Seq. Results: The success rate, based on quality parameters of RNA extraction from tissue biopsies stored in OCT and 2 mm biopsies stored in Allprotect®, was 56% (30/54) and 30% (3/10), respectively. For 3 mm skin biopsies stored in Allprotect® was 93% (55/59). RNA preparations from 3 mm-Allprotect® biopsies had an average RIN of 7.2 ± 0.7, and their integrity was not impacted by sample storage time (up to 200 days at -20°C). RNA products were appropriate for qRT-PCR and RNA-seq. Based on these results, we propose a standardized method for RNA extraction from disrupted skin samples. This protocol was validated with lesion biopsies from CL patients (n = 30), having a success rate of 100%. Conclusions: Our results indicate that a biopsy size of 3 mm in diameter and preservation in Allprotect® for up to 200 days at -20°C, are best to obtain high quality RNA preparations from ulcerated skin lesion biopsy samples.

An effective and rapid method for RNA preparation from non-conventional yeast species

The increased use of high-throughput RNA-based analysis has spurred the demand for rapid and simple preparation of high quality RNA. RNA preparation from non-conventional yeasts having diverse cell wall and morphological characteristics is often inefficient using current methods adapted for the model yeast, Saccharomyces cerevisiae. We report a simple RNA preparation method based on glass bead-mediated breakage in a formamide/EDTA solution. High quality RNA is generated within 15 min from various non-conventional yeasts species. The obtained RNA can be directly used for experimentation without further RNA purification and buffer exchange.

Single-Step RNA Extraction from Different Hydrogel-Embedded Mesenchymal Stem Cells for Quantitative Reverse Transcription-Polymerase Chain Reaction Analysis.

For many tissue engineering applications, cells such as human mesenchymal stem cells (hMSCs) must be embedded in hydrogels. The analysis of embedded hMSCs requires RNA extraction, but common extraction procedures often produce low yields and/or poor quality RNA. We systematically investigated four homogenization methods combined with eight RNA extraction protocols for hMSCs embedded in three common hydrogel types (alginate, agarose, and gelatin). We found for all three hydrogel types that using liquid nitrogen or a rotor-stator produced low RNA yields, whereas using a microhomogenizer or enzymatic/chemical hydrogel digestion achieved better yields regardless of which extraction protocol was subsequently applied. The hot phenol extraction protocol generally achieved the highest A260 values (representing up to 40.8 μg RNA per 10(6) cells), but the cetyltrimethylammonium bromide (CTAB) method produced RNA of better quality, with A260/A280 and A260/A230 ratios and UV spectra similar to the pure RNA control. The RNA produced by this method was also suitable as a template for endpoint and quantitative reverse transcription-PCR (qRT-PCR), achieving low Ct values of ∼20. The prudent choice of hydrogel homogenization and RNA extraction methods can ensure the preparation of high-quality RNA that generates reliable endpoint and quantitative RT-PCR data. We therefore propose a universal method that is suitable for the extraction of RNA from cells embedded in all three hydrogel types commonly used for tissue engineering.

Partial purification of IBV and subsequent isolation of viral RNA for next-generation sequencing.

RNA viruses are known for a high mutation rate and rapid genomic evolution. As such an RNA virus population does not consist of a single genotype but is rather a collection of individual viruses with closely related genotypes—a quasispecies, which can be analyzed by next-generation sequencing (NGS). This diversity of genotypes provides a mechanism in which a virus population can evolve and adapt to a changing environment. Sample preparation is vital for successful sequencing. The following protocol describes the process of generating a high-quality RNA preparation from IBV grown in embryonated eggs and then partially purified and concentrated through a 30 % sucrose cushion for NGS.

Advances in methods for native expression and purification of RNA for structural studies

Many RNAs present unique challenges in obtaining material suitable for structural or biophysical characterization. These issues include synthesis of chemically and conformationally homogeneous RNAs, refolding RNA purified using denaturing preparation techniques, and avoiding chemical damage. To address these challenges, new methodologies in RNA expression and purification have been developed seeking to emulate those commonly used for proteins. In this review, recent developments in the preparation of high-quality RNA for structural biology and biophysical applications are discussed, with an emphasis on native methods.

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments. In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent <10% of the total cell population. Laser capture microdissection (LMD) has been developed to address this problem. Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited. LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity. Standard techniques are used to recover the total RNA and measure its integrity. This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments. In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent <10% of the total cell population. Laser capture microdissection (LMD) has been developed to address this problem. Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited. LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity. Standard techniques are used to recover the total RNA and measure its integrity. This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

A fast and efficient method for preparation of high-quality RNA from fungal mycelia

BackgroundFungal RNA samples are usually isolated from fungal mycelia grown in liquid culture, which relies on prolific growth of the fungus in liquid media. The fungal biomass is then collected by vacuum filtration, which can result in low recovery for samples with reduced biomass due to poor growth in liquid media.FindingsHere we report an alternative culturing method, based on growth on solid media which is independent of the ability of a fungus to grow in liquid culture. We show that growth on solid media overlayed with a nylon membrane is superior to other culturing methods, producing large amounts of biomass and allowing for easy harvesting of fungal mycelia. Furthermore, we show that mycelium harvested with this method yielded high-quality RNA, superior to RNA isolated from liquid grown mycelium. We also show that inclusion of a second chloroform extraction step in the procedure significantly increases RNA yield.ConclusionsThis method is particularly useful for fungal species that show poor or no growth in liquid media, but are easily cultured on solid media. Culturing can be performed on small petri dishes, which significantly reduces handling and therefore allowing growth and isolation of RNA from multiple strains in a high throughput manner. The obtained RNA samples are of high quality in sufficient quantities for several northern blot experiments or quantitative RT-PCR experiments.

How to measure RNA expression in rare senescent cells expressing any specific protein such as p16Ink4a

Here we describe a carefully optimized method for the preparation of high quality RNA by flow sorting of formaldehyde fixed senescent cells immunostained for any intracellular antigen. Replicative cellular senescence is a phenomenon of irreversible growth arrest triggered by the accumulation of a discrete number of cell divisions. The underlying cause of senescence due to replicative exhaustion is telomere shortening. We document here a spontaneous and apparently stochastic process that continuously generates senescent cells in cultures fully immortalized with telomerase. In the course of studying this phenomenon we developed a preparative fluorescence activated flow sorting method based on immunofluorescent staining of intracellular antigens that can also deliver RNA suitable for quantitative analysis of global gene expression. The protocols were developed using normal human diploid fibroblasts (HDF) and up to 5x107 cells could be conveniently processed in a single experiment. The methodology is based on formaldehyde crosslinking of cells, followed by permeabilization, antibody staining, flow sorting, reversal of the crosslinks, and recovery of the RNA. We explored key parameters such as crosslink reversal that affect the fragmentation of RNA. The recovered RNA is of high quality for downstream molecular applications based on short range sequence analysis, such qPCR, hybridization microarrays, and next generation sequencing. The RNA was analyzed by Affymetrix Gene Chip expression profiling and compared to RNA prepared by the direct lysis of cells. The correlation between the data sets was very high, indicating that the procedure does not introduce systematic changes in the mRNA transcriptome. The methods presented in this communication should be of interest to many investigators working in diverse model systems.

Next Generation Transcriptome Sequencing and Quantitative Real-Time PCR Technologies for Characterisation of the Bemisia tabaci Asia 1 mtCOI Phylogenetic Clade

A programme of functional genomics research is underway at the University of Greenwich, UK, to develop and apply genomics technologies to characterise an economically-important but under-researched Bemisia tabaci (Hemiptera: Aleyrodidae), the Asia 1 mtCOI phylogenetic group. A comparison of this putative species from India with other important B. tabaci populations and insect species may provide targets for the development of more effective whitefly control strategies. As a first step, next-generation sequencing (NGS) has been used to survey the transcriptome of adult female whitefly, with high quality RNA preparations being used to generate cDNA libraries for NGS using the Roche 454 Titanium DNA sequencing platform. Contig assemblies constructed from the resultant sequences (301 094 reads) using the software program CLC Genomics Workbench generated 3 821 core contigs. Comparison of a selection of these contigs with related sequences from other B. tabaci genetic groups has revealed good alignment for some genes (e.g., HSP90) but misassemblies in other datasets (e.g., the vitellogenin gene family), highlighting the need for manual curation as well as collaborative international efforts to obtain accurate assemblies from the existing next generation sequence datasets. Nevertheless, data emerging from the NGS has facilitated the development of accurate and reliable methods for analysing gene expression based on quantitative real-time RT-PCR, illustrating the power of this approach to enable rapid expression analyses in an organism for which a complete genome sequence is currently lacking.

High-Quality RNA Preparation from Rhodosporidium toruloides and cDNA Library Construction Therewith

Oleaginous yeast Rhodosporidium toruloides is an excellent microbial lipid producer. Therefore, it is important to develop molecular biology tools to understand the basic mechanism for lipid accumulation and further manipulate the microorganism. High-quality RNA extraction from R. toruloides is particularly challenging due to high level of polysaccharides, lipids, and other secondary metabolites. To obtain an optimal protocol for RNA extraction from R. toruloides, four methods were evaluated. Large difference in RNA yield and quality among these protocols was found. The optimum method was modified RNAiso procedure, where RNA was isolated using liquid nitrogen-RNAiso method with salt precipitation and the addition of β-mercaptoethanol. This method consistently recovered RNA in good quality with high yield. Around 297 μg total RNA per gram of cells was obtained with an average purity measured as A₂₆₀/A₂₈₀ of 2.09. A titer of 10⁵ cfu/ml could be harvested to construct a full-length cDNA library with the RNA sample in this quality. Electrophoresis gel analysis indicated the fragments ranged from 200 bp to 4.0 kb, with the average size of 1000 bp. Randomly picked clones showed the recombination efficiency at 80%. These results showed that RNA of R. toruloides was successfully extracted for the first time using the modified RNAiso method, and the cDNA library was appropriate for screening the genes related to lipid accumulation.

Optimisation of gene expression analysis in Atlantic salmon lenses by refining sampling strategy and tissue storage

Analysis of gene expression in the lens is one of the analytical tools employed to investigate cataract formation in Atlantic salmon (Salmo salar L.). High quality RNA preparations are an essential prerequisite for gene expression analysis. The first aim of the present study was to investigate the possible effects of two methods of tissue preservation on the quality of RNA extracted from Atlantic salmon lenses. RNA was extracted from lenses either stored in RNAlater or flash-frozen in liquid nitrogen. Both tissue preservation methods yielded RNA of similarly high quality. The second aim was to examine if stress related to fish handling and the choice of anaesthesia during the sampling procedure affected gene expression in the lens. Six sampling procedures were tested on groups of sea water adapted Atlantic salmon smolt. Fish were either killed instantaneously (control group) or sampled after 30min anaesthetised with isoeugenol, after 30min without anaesthesia, after 120min anaesthetised with isoeugenol, after 120min with 15min anaesthesia with metacaine or after 120min without anaesthesia. The expression levels of specific genes, of special interest in the study of molecular mechanisms of cataractogenesis, were analysed in lenses by real-time RT-PCR. Fish not anaesthetised had significantly increased levels of heat shock protein 70 (HSP70) mRNA after 30min compared to the control group. Glutathione reductase (GR) and manganese superoxide dismutase (Mn-SOD) were expressed at significantly lower levels in groups of Atlantic salmon sampled after 120min anaesthetised with isoeugenol or metacaine, and anaesthetised with isoeugenol, metacaine or without anaesthesia, respectively. The same expression patterns were found in corresponding gill tissues for these two antioxidant genes. In conclusion, preservation in liquid nitrogen instead of RNAlater is recommended due to practical conditions in RNA extraction. A quick sampling protocol with the use of anaesthetics and not exceeding 30min should be preferred to avoid effects of the sampling procedure on lens gene expression in Atlantic salmon.

High-quality RNA preparation for cDNA library construction of the Antarctic sea–ice alga Chlamydomonas sp. ICE-L

To study the molecular mechanism of the Antarctic sea–ice alga in adaptation to polar sea–ice environments, the RNA was prepared for cDNA library construction of Chlamydomonas sp. ICE-L. Three different methods were tested to prepare total RNA from this psychrophilic, unicellular green alga rich in protein and polysaccharide. Lauryl sodium sulfate- based method allowed a most effective extraction of high-quality total RNA compared to the other methods. Total RNA extracted with this protocol was used for cDNA library construction. The recombination rate of constructed cDNA library was 98.60%, the primary titer was 7.15 × 106 pfu, and an average sequence length was 1.2 kb. These results show that with a high-quality RNA preparation, a cDNA library can be constructed successfully for Chlamydomonas sp. ICE-L.

The Isolation of Binucleated Trophectoderm from Mid-Gestation Bovine Placentae Using Fluorescence Activated Cell Sorting.

The trophectoderm of ruminant placentae is comprised of two main cell types; mononucleate cells (MNCs) and binucleate cells (BNCs). BNCs comprise approximately 20% of all trophectoderm cells in cattle, sheep, goats, and deer and are characterized as large, hyperploidic, binucleated cells that invade into the endometrium and fuse with maternal cells to form syncytial plaques. BNCs also produce several hormones, including placental lactogen and progesterone. Developing schemes for isolating BNCs from ruminant placentae will enhance our ability to understand BNC development and function. Therefore, the purpose of this study was to use fluorescence activated cell sorting (FACS) for isolating BNCs from mid-gestation bovine placentae. Pregnant bovine uteri (138.64 ± 6.41 days gestation) were collected at a nearby abattoir and transported to the laboratory on ice. Whole cotyledons (n=6/placenta) were dissected away from each placenta, diced into 5-6 mm cubes and incubated in DMEM containing 25 units/ml Dispase, 10% fetal bovine serum (FBS) and 10mM HEPES at 37°C for 1 h. The tissue homogenate was filtered using a 200μm mesh and centrifuged at 1,200 RPM for 10 minutes at room temperature. Cells were resuspended in Dulbecco's PBS containing 5% FBS and 10μM Vybrant DyeCycle Green at 37°C for 30 minutes. FACS was completed on the cells using a FACS-ARIA cell sorting system. Forward scatter (530nm±15nm) was monitored after excitation (488nM). In the first study (n=5 placentae), cells containing excitation intensities two- to four-times that of the peak value (i.e. diploid cells) were collected. Approximately 1 million cells were collected for each placental preparation. Cells were fixed and immuno-stained for placental lactogen (PL) using a polyclonal antibody directed against ovine PL (generously provided by Dr. Russ Anthony). Cells were examined with epifluorescence microscopy to determine the proportion of BNCs (detection of two nuclei within one plasma membrane) and PL-positive cells. Four individual slides were examined for each placental preparation, and four independent fields were counted on each slide (approximately 125-200 cells/ field). The percentage of BNCs were greater (P=0.0001) in the FASC-sorted preparation (73.8±3.3%) than placental cell preparations sampled before sorting (20.1 ±2.2%). Also, FACS-sorted preparations contained a greater proportion (P=.0002) PL-positive cells than the pre-sorted preparations (65 ±4.3% versus 15.9±1.7%). These cells also were viable after FACS. Membrane integrity is maintained in nearly all cells, and high quality RNA preparations were retrieved from sorted cells. In another study, MNCs and BNCs were sorted so that cells falling in the range of the diploid peak were collected as one fraction (putative MNC fraction) and those cells with 2 to 4 times greater fluorescence were collected as the BNC fraction (n=4 placentae). When compared with the pre-sorted preparations, which contained 12.76±1.17% BNCs, the FASC-sorted MNC population consisted of 5.79±0.77% BNCs whereas the BNC sorted preparations contained 83.07±1.05% BNCs (individual means differ; P<0.01). In conclusion, Dispase digestion and FACS is a useful approach for obtaining enriched populations of BNCs and MNCs from midgestation bovine cotyledons. It is anticipated that this technology will enable researchers to study various cell functions of BNCs and begin to understand what controls their formation. (poster)

Development of an improved protocol to analyse gene expression in temporomandibular joint condylar cartilage of rats using DNA microarrays

During recent years, gene expression analyses based on DNA chip technologies have allowed for the genome-wide identification of genes potentially associated with growth processes in a variety of organs. The present study aims to identify genes differentially expressed in the growing temporomandibular joint cartilage by means of transcriptome analyses. In total, the condylar cartilage of 32 rats comprising 4 age groups (newborn, 10 days, 21 days, 8 weeks) were used for analysis. Transcriptome analyses were carried out using Affymetrix Expression Arrays (Rat Genome 230 2.0 Arrays). The availability of high-quality RNA preparations from homogeneous tissue samples is a fundamental precondition of successful transcriptome analyses using DNA arrays. An optimised preparation protocol allowed RNA isolation of sufficient quality which was validated using capillary electrophoresis. RNA collected from 8 test animals of the 4 age groups respectively was mixed in equimolar RNA pools which served for the transcriptome analyses using Affymetrix arrays. Statistical analysis of the gene expression data indicated the existence of genes differentially regulated in the growing temporomandibular cartilage. This evidence, however, requires validation by RT-PCR using individual animals' RNA. Preliminary candidate genes belong, among others, to the groups of matrix-degrading proteases, protease inhibitors and genes involved in cell growth, apoptosis and bone remodelling. These differentially expressed genes in TMJ growth identified using DNA array technology may possibly contribute to a better understanding of growth biology and provide an approach to necessary therapy.

Development of an Efficient Protocol of RNA Isolation from Recalcitrant Tree Tissues

Isolation of RNA from recalcitrant tree tissues has been problematic due to large amounts of secondary metabolites and interfering compounds in their cells. We have developed an efficient RNA extraction method, which yielded high-quality RNA preparations from tissues of the lychee tree. The method reported here utilized EDTA, LSS, and CTAB to successfully inhibit RNase activities. It was found that a high ionic strength brought about by 2 M NaCl was necessary. In addition, secondary metabolites and other interfering compounds were effectively removed using sodium borate and PVPP under a deoxidized condition. The quality of purified RNA was tested by both RACE and Northern blotting analysis, ensuring that the RNA could be used for subsequent gene expression analysis. This method has been successfully applied to purify RNA from 15 other plant species. In conclusion, the protocol reported here is expected to have excellent applications for RNA isolation from recalcitrant plant tissues.

Lack of Noggin Expression by Cancer Cells Is a Determinant of the Osteoblast Response in Bone Metastases

Prostate and mammary cancer bone metastases can be osteoblastic or osteolytic, but the mechanisms determining these features are unclear. Bone morphogenetic and Wnt proteins are osteoinductive molecules. Their activity is modulated by antagonists such as noggin and dickkopf-1. Differential expression analysis of bone morphogenetic and Wnt protein antagonists in human prostate and mammary cancer cell lines showed that osteolytic cell lines constitutively express in vitro noggin and dickkopf-1 and at least one of the osteolytic cytokines parathyroid hormone-related protein, colony-stimulating factor-1, and interleukin-8. In contrast, osteoinductive cell lines express neither noggin nor dickkopf-1 nor osteolytic cytokines in vitro. The noggin differential expression profile observed in vitro was confirmed in vivo in prostate cancer cell lines xenografted into bone and in clinical samples of bone metastasis. Forced noggin expression in an osteoinductive prostate cancer cell line abolished the osteoblast response induced in vivo by its intraosseous xenografts. Basal bone resorption and tumor growth kinetics were marginally affected. Lack of noggin and possibly dickkopf-1 expression by cancer cells may be a relevant mechanism contributing to the osteoblast response in bone metastases. Concomitant lack of osteolytic cytokines may be permissive of this effect. Noggin is a candidate drug for the adjuvant therapy of bone metastasis.

Analysis of environmental transcriptomes by DNA microarrays

In this work we investigated the correlations between global gene expression patterns and environmental parameters in natural ecosystems. We studied the preferential gene expression of the iron oxidizer bacterium Leptospirillum ferrooxidans to adapt its physiology to changes in the physicochemical parameters in its natural medium. Transcriptome analysis by DNA microarrays can proportionate an instant picture about the preferential gene expression between two different environmental samples. However, this type of analysis is very difficult and complex in natural ecosystems, mainly because of the broad biodiversity and multiple environmental parameters that may affect gene expression. The necessity of high-quality RNA preparations as well as complicated data analysis are also technological limitations. The low prokaryotic diversity of the extremely acidic and iron-rich waters of the Tinto River (Spain) ecosystem, where L. ferrooxidans is abundant, allows the opportunity to achieve global gene expression studies and to associate gene function with environmental parameters. We applied a total RNA amplification protocol validated previously for the amplification of the environmental transcriptome (meta-transcriptome). The meta-transcriptome of two sites from the Tinto River mainly differing in the salt and oxygen contents were amplified and analysed by a L. ferrooxidans DNA microarray. The results showed a clear preferential induction of genes involved in certain physicochemical parameters like: high salinity (ectAB, otsAB), low oxygen concentration (cydAB), iron uptake (fecA-exbBD-tonB), oxidative stress (carotenoid synthesis, oxyR, recG), potassium (kdpBAC) or phosphate concentrations (pstSCAB), etc. We conclude that specific gene expression patterns can be useful indicators for the physiological conditions in a defined ecosystem. Also, the upregulation of certain genes and operons reveals information about the environmental conditions (nutrient limitations, stresses, etc.).