Our biobank service includes:
- Cryogenic long-term storage of fresh tissue specimens, in liquid nitrogen tanks
- Repository for long-term storage of serum, plasma and urine in ultra-low freezers.
- Storage of nucleic acids (DNA and RNA) and proteins samples in ultra-low and normal freezers.
Patients written consent is mandatory for all stored samples. All specimens are inspected upon receipt and labeled with specific unique codes, for secured identification. A database is maintained for easy access to samples.
Nucleic acids isolation services
Our infrastructure has separate facilities and spaces for isolation the DNA, RNA and proteins from different specimens, including: tissue (frozen and FFPE), blood, urine, cervical lavage and exosomes.
The isolation of nucleic acids (DNA, RNA, miRNA) is performed using standard validated protocols. Depending on the final applications, upon request, we can perform nucleic acids isolation based on classical phenol:chloroform procedure and/or silica membrane-based protocols, specific for tissue (fresh and FFPE), whole blood, urine, cervical lavage, exosomes (ex Qiagen, Norgen, Invitrogen, Agilent, Zymo, etc). Our specialists will advise the collaborators in choosing the best method related to their research interest. All isolated nucleic acids samples are evaluated for quantity (Qubit3.0 and Nanodrop ND-1000) and quality (Bioanalyzer 2100). Upon request, we can provide long-term storage of the samples in our biobank.
Nanotechnology-based nucleic acids quantity and quality control
Nucleic acids quality and quantity are essentially for downstream molecular analysis. Our lab has the possibility to perform nanotechnology-based evaluation of nucleic acid samples, therefore only small volumes of samples (1ul) are consumed for each analysis. Thus, high accuracy data related to quality and quantity for DNA, RNA, miRNA and complementary synthesis sequences (cDNA, cRNA, microarray probes) as well as proteins can be provided for limited samples (laser capture microdissection (LCM), fine needle aspiration or needle biopsies, or small number of cells such as tumor circulating cells (CTCs) or cancer stem cells (CSCs)).
Our infrastructure includes Bioanalyzer 2100, a Nanodrop ND-1000 spectrophotometer and a Qubit 3.0 fluorimeter.
Real-Time PCR analysis
Gene expression analysis
We can monitor gene expression by two main analysis methods: absolute or relative quantification. Absolute quantification enables the monitoring of a single target sequence based on a standard curve and express the results as an absolute value such as copies/ml (used for experiments that monitor one or two genes of interest, i.e. BCR-ABL evolution during disease progression and treatment). Relative quantification compares the levels of two different target sequences in a single sample and expresses the results as a ratio of these targets (gene of interest reported to gene of reference-experiments that monitor multiple genes, for example pathways).
Pathway analysis using PCR array (96 genes of interest)
We have the possibility to monitor up to 84 genes in a specific pathway using SYBR Green detection and relative quantification analysis (PCRarray Pathway focused plates from Qiagen). Thus, we can determine the molecular signaling of disease-involved pathways, target genes regulation by mRNA, lncRNAs or miRNAs, treatment targets identification, etc.
SNP mutations identification
Genotyping is performed by using Simple Probes for PCR amplicons detection and monitoring. Fluorescently labeled single probes are designed to hybridize specifically to the target sequence that contains the SNP of interest. Based on the melting behavior of the hybridization probe, we can determine if the analyzed samples are wild type or mutant and homo/heterozygous (HPV genotyping).
In vitro studies:
Our lab has a stock of different stabilized cancer cell lines of breast, cervical, colon, ovarian, etc, or normal cells such as fibroblasts, osteoblasts, stem cells, etc which permit pharmacologic studies of different compounds (chemical or natural) on live cells. Our experience in cellular effects monitoring include: proliferation and toxic effects which can be assessed by using colorimetric/fluorimetric assays such as the classical MTT assay, alamar blue, etc; apoptosis evaluation can be measured by using on-chip flow cytometry. This analysis calculates the percent of apoptotic cells in live cell population based on dual staining of cells with a live marker and Annexin V, a specific marker for apoptotic cells. Cell adhesion and attachment can be quantified by culturing the cells on different extracellular matrixes (laminin, collagen, etc) and measure the time necessary for developing focal adhesion complexes. Cell motility can be visually quantified by analyzing the cell morphology when cultured on an ECM compound. Invasiveness potential is assessed by culturing cells in transwell systems, and counting the cells on both sides of the wells. Stemness can be assessed by using sphere and hanging drop assays to determine the cells self-renewal and survival properties. These assays are based on cells ability to survive in serum free and anchorage independent conditions which specifically select the stem cells from a heterogeneous population of cells.
In vivo studies:
Our lab disposed of an animal facility that breads NMRI, BALB/C, C57BL/6 mice that can be used in basic research and nonclinical drug discovery and development. We can perform toxicity, pharmacological, delivery, tumor growth and inhibition studies.
With more than eight years experience in microarray analysis, we can provide comprehensive genome-wide screening services using Agilent microarray platform. Our services range from experimental design, target preparation, quality control, hybridization and imaging to bioinformatic support. Taking advantage of Agilent technology, both one and two-color microarray experiments are available using standard or custom arrays (1x180K, 4x44k, 4x180k, 8x60k) for different species (human, mice, rat, cow, canine, chicken, pig, maize, rice, Arabidopsis, etc) and small quantities of biological material (20-100 ng). Currently, we provide gene expression profilling of mARN, miRNA and lncRNA. At request we perform comparative genomic hybridization (CGH) and methylation.
Available instruments: Agilent DNA Microarray Scanner with SureScan High-Resolution Technology, Agilent 2100 Bioanalyzer, NanoDrop ND-1000 spectrophotometer and Qubit 3.0 fluorometer.
Bioinformatic and statistical analysis
We can assist you with analysis of gene expression microarray experiments including: setting experimental design, processing data from raw intensities (normalization, probeset summarization, filtering), gene lists, sequence annotations, identification of biological significance (GO, GSEA, KEEG, IPA), uploading data to public repositories (e.g. GEO). We also performed analysis of CGH array data and DNA-metylation data. We use custom routine R/Bioconductor, Gene Spring GX, Ingenuity Pathway Analysis.
Our expertise also includes statistical analysis of clinical and other molecular data (qRT-PCR gene expression, ELISA, in vitro and in vivo studies, etc) and their integration into biological context.
We can asses genes functionality by means of silencing or over-expression assays. Genes of interest (mRNAs, lncRNAs) can be cloned into plasmid expression or siRNAs can be designed against these transcripts. These gene modulators are delivered into cells to increase (plasmids) or decrease (siRNAs) gene expression, therefore allowing the study of the biological events triggered by these antagonistic treatments. By measuring the subsequent cellular and molecular effects we can determine the pathways and inherent functions regulated by any gene of interest.
miRNAs functionality can be studied by using miRNA mimics and inhibitors. miRNA mimics and inhibitors are small, chemically modified double and single-stranded RNAs respectively, that mimic or specifically bind to and inhibit endogenous miRNAs and enable miRNA functional analysis by up or down-regulation of miRNA activity. By studying the subsequent effects of miRNA overexpression or inhibition on mRNA expression we can determine the miRNA targets and functionality.
In addition to the standard services we provide assistance:
- in writing grants application
- in identifying appropriate technology for specific research goals
- with experimental design and optimization
- with project management
- with result presentation, writing and reviewing manuscripts
You are encouraged to contact our department to discuss potential collaborations and to ask any question.