RNA Extraction and Purification
Purified, full-length RNA is required for cDNA cloning. It is also used in some methods for analyzing gene expression and for microarrays utilizing cDNA. RNA is prepared from bacteria, tissue culture cells, tissues and plants. Preparations can be purified messenger (mRNA) or total RNA that includes mRNA, ribosomal (rRNA) and transfer (tRNA) RNA. The scale is from 1 microgram to1 milligram.
RT-PCR, Reverse-Transcriptase Polymerase Chain Reaction
The usual PCR technique copies a piece of DNA and greatly amplifies the copy number for analysis or storage. RT-PCR starts with mRNA or total RNA, makes a cDNA complimentary strand using reverse transcriptase, and then amplifies the product. The product is used to determine the presence of the mRNA and for size determination, sequencing or for quantitation by Quantitative-PCR.
If you already have your gene of interest subcloned we can perform the DNA isolation for you. If not, we are skilled in subcloning and can further perform your gene expression analysis or subclone your gene of interest into a mammalian expression vector. With our experience in cell culture, we can expand your cells and harvest your protein for purification (see Cell Biology and Biochemistry).
DNA Extraction and Purification
Purified DNA is required to prepare DNA libraries or microarrays, to make probes, and to transfect genes. DNA is prepared from bacteria, yeast, tissue culture cells, tissues, or plants. Preparations can be genomic DNA or plasmids. The scale is from 1 microgram to 1 milligram.
Genes can be introduced into cells by suitable molecular biology methods such as electroporation, cationic lipid reagents, or calcium phosphate. A gene can be transfected into cells simply with its regulatory elements or after making a construct to achieve high overexpression levels. Cells can also be transfected with conditionally expressed genes of interest. Transfections in mammalian cells can be transient or permanent. Transient expression lasts only a few days. Permanent expression requires cotransfection with a dominant selectable marker and several rounds of selection for the cell populations that stably integrate the transfected DNA into a cellular gene. This takes approximately three to four weeks.
Cell Cloning and Selection
Cloning is used to obtain a stable culture of homogeneous cells. Over periods of many months in tissue culture, cells can change properties due to somatic gene mutation, and overgrowth of mutated cells. It may be desired to select a rare cell type or the few stably transfected cells in a transfection pool. Cloning is achieved by diluting a culture so that ½ the wells in a 96 well plate contain one cell and ½ contain no cells. At this low cell concentration, conditioned medium (medium from the same cell type harvested at high concentrations) is added to enhance growth. When the clone reaches suitable numbers, aliquots are frozen in order to retrieve cells with the same properties at a future date.
Our scientists can also express your gene of interest. We are able to produce constructs in the system of your choice. From your expression construct we can further express the protein of interest and test it in other cell-based assays. If you are working with siRNA, we have experience in regulating mammalian genes using this technology.
Gene activation or gene expression can be conveniently monitored by a DNA construct that links the regulatory components of the gene to a reporter gene, such as for a fluorescent protein or beta galactosidase.
cDNA Purification for Microarrays
DNA complementary (cDNA) to mRNA is prepared for preparing microarrays for screening in different cell sources. For screening, the total RNA is extracted from tissue or cells and is hybridized to the cDNA immobilized onto a solid support. A detection molecule is applied and is visualized.
DNA Restriction Fragment Mapping
Restriction endonucleases recognize and cut DNA at unique nucleotide sequences. Delineation of these cleavage sites provides a map of the DNA, produces characteristic sizes of the fragments, and allows for the preparation of pieces of DNA to be used for making new vector or plasmid constructs.
Subcloning is used to amplify a desired DNA molecule. After a piece of DNA or a gene is obtained, by synthetic synthesis, restriction digestion, joining to another DNA by a ligase, etc., it is introduced into competent bacteria and transformants expressing the new gene are identified. The bacteria are cloned on selection plates whereby only those cells that contain the desired DNA survive and propagate into colonies. The colonies are “picked” and expanded in liquid cultures.
cDNA and Genomic Libraries
A library is an amplified collection of DNA representing the genes of an organism (genomic library) or of DNA complementary to a preparation of mRNA (cDNA library). Either library can be probed to pull out a specific gene of interest. cDNA libraries are used to clone the expressed version of a gene comprised of the exons. Genomic libraries contain both the exons, introns and regulatory portions of the gene. Bacteriophage lambda and cosmid vectors are used in library construction.
Library Screening and Gene Cloning
A DNA library is screened to determine the presence of a gene and to clone out either a full-length or smaller version of the gene. There are many strategies for cloning from a library. Typically, the library is introduced into bacteria, the bacteria are plated under cloning conditions, replicate plates of the bacterial colonies are made, and the clones are probed for the existence of the desired gene either by hybridization or through the expression of the specific protein. For a positive clone, the DNA is extracted from original plate, recloned and purified.
To unequivocally identify a gene or cDNA, the DNA is submitted for sequencing. Using overlapping portions of the DNA, a contiguous sequence is generated which can be used for data mining.
Transfection, Transient and Stable
Genes can be introduced to cells by suitable molecular biology methods. A gene can be transfected into cells simply with its own regulatory elements, or after making a construct to achieve high, overexpression. Cells can also be transfected to conditionally express a gene of interest. Transfections in mammalian cells can be transient or permanent. Transient expression lasts only a few days. Permanent expression requires cotransfection with a dominent selectable marker and several rounds of selection for the few cells that stably integrate the gene in the cell. This takes approximately three to four weeks .
Metabolic Assays, Labeling and Turnover
Drug effect on metabolism may be measured by radioactive precursor uptake, thymidine, uridine (or uracil for bacteria), and amino acid, into DNA, RNA and proteins. Carbohydrate or lipid synthesis is similarly measured using suitable precursors. Turnover of nucleic acid or protein or the degradation of specific cell components, is measured by prelabeling (or pulse labeling) followed by a purification step and quantitation of remaining label or sometimes by measurement of chemical amounts of the component. Energy source metabolism is also analyzed for optimal cell growth.
Recombinant Protein Expression and Manufacturing
Recombinant proteins are expressed in bacteria, yeast, mammalian or in the baculovirus system. The gene of interest is transfected into the appropriate host together with regulatory elements that allow very high levels of production. Protein production may be intracellular, or may accumulate in the bacterial periplasmic space, or be secreted into the medium of cells.
The scale may be a few mL for analytical purposes, or in batches of up to 20 L in bacterial or yeast fermentation, or up to 50 L in mammalian suspension, hollow fiber or porous bead cultures.
Northern blot analysis involves size separation of the RNA species by electrophoresis followed by identification with labeled gene-specific oligonucleotides or DNA probes. The size and abundance of the desired RNA is determined. The predominant rRNA in a total RNA preparation can also be detected by UV absorbance as a measure of intactness.
Dot Blot and Slot Blot
Specific RNA species in an unfractionated preparation can be measured by immobilizing a sample in a spot (Dot Blot) or in a manifold slot (Slot Blot). Detection is by a labeled DNA probe that hybridizes to the immobilized RNA. For the Dot Blot, quantitation is usually visual whereas the Slot Blot format is more easily quantitated by scanning with a densitometer.
In situ Hybridization
In situ hybridization localizes the expression of a particular mRNA among different cells in a tissue or cell preparation. A cell preparation is fixed on a slide and the RNA contents probed with radiolabeled or fluorescent probe.