Investigators are provided with or trained in the following services for CTSC approved protocols: DNA, RNA and protein extraction, genotyping, oligonucleotide synthesis, DNA sequencing, quantitative PCR, RT-PCR, digital PCR, gene expression profiling, next-generation sequencing (NGS) and Western blot analysis.
Oligonucleotide Synthesis & DNA Sequencing: These basic methods of recombinant DNA are arranged by the Molecular Core by sub-contracts. The core will design primers for PCR amplification, genotyping and DNA-sequencing, acquire the primers, genotype and/or sequence samples from clinical studies.
Nucleic Acid and Protein Isolation. The Molecular Core will extract nucleic acids (DNA or RNA) and proteins from clinical samples. The concentration and quality of nucleic acids and proteins will be determined. These nucleic acids or proteins can be used by the PI of the CTSC project or further processed by the Molecular Core.
Genotyping. A number of methods including complete DNA sequencing for polymorphic genes or allele-specific detection methods for known single nucleotide polymorphisms can be implemented by the Molecular Core.
Quantitative PCR and RT-PCR. To assess levels of nucleic acids, quantitative PCR with fluorescent TaqMan or SYBR Green chemistry is used. There are numerous applications, for example, assessment of the levels of virus in samples or comparing the expression levels of one or a few genes and the effects of treatments on those gene expression levels.
Digital PCR: Digital PCR brings PCR analysis to a new level and makes new applications possible by enabling sensitive and precise absolute target quantification without the use of a reference or standard curve. Such applications include precise copy number variation (CNV); rare mutation detection; absolute quantitation of pathogens, nucleic acid standards and next-generation sequencing libraries; low level pathogen detection; and low-level fold change of gene expression.
Gene expression profiling. When pure cell types or well defined tissues are available, gene expression profiling using the Affytemrix GeneChips can be performed. This is a comparative method allowing the effect of treatment on the expression level of several thousand genes to be determined simultaneously. The Molecular Core will assist in experimental design, execution and data interpretation.
Next-Generation Sequencing (NGS): With its unprecedented throughput, scalability, and speed, NGS enables researchers to study biological systems at a level never before possible. The Molecular Core through sub-contracts will arranged the following services to all CTSC investigators: Whole genome sequencing; All exome sequencing; Protein-DNA/RNA interactions (ChIP-Seq, CLIP, etc.); RNA sequencing for SNP detection, expression analysis and small RNA detection; Targeted resequencing; and Whole-genome methylation analysis.
Western blot. The Core will perform Western blot for investigators using the CTSC. Western blot is a widely accepted analytical technique to detect specific proteins in protein samples. After separating protein samples by denaturing polyacrylamide gel electrophoresis, the proteins is transferred to a solid membrane and stained with antibodies specific to a target protein. The signal is visualized by chemoluminescence or flourescence. Multiplex flourescence detection is also available. The immunoblot images and quantitation data will be provided to investigators.