Studying the complex details of the biological world requires the use of tools that allow effective and sensitive isolation of the individual elements that make up these systems. Almost every molecular biology workflow starts with an extraction step to isolate the target molecule(s) (DNA, RNA, protein) of interest for the downstream applications of purification, amplification, or manipulation. Meridian offers a suite of specially formulated nucleic acid purification reagents to provide streamlined, sensitive and robust options for this routine, yet highly important step of your workflow.
Basic Concepts of Nucleic Acid Purification
The quality and integrity of your extracted nucleic acid will directly affect all subsequent experimentation and downstream results. Protocols will vary depending on the starting sample type and the target of interest.
The basis of a successful nucleic acid isolation workflow involves a number of key steps:
- Effective disruption of the biological matrix (cell, tissue, environmental or biological sample) to release the nucleic acids
- Denaturation of structural proteins associated with the nucleic acids (nucleoproteins)
- Inactivation of nucleases that will degrade the isolated product (RNase and/or DNase)
- Removal of contaminants (proteins, carbohydrates, lipids, biological or environmental elements, unwanted nucleic acids, other cellular debris)
Chemical and/or physical methods are used for the initial disruption to homogenize sample matrix and break open cell walls, releasing nucleic acids along with other cellular components. Disruption of DNase activity is relatively easy, making the isolation of DNA a fairly straight forward technique. RNases, on the other hand, are ubiquitous and extremely stable, thus effective and sensitive RNA extraction methods rely on good laboratory techniques using RNase-free equipment and appropriate protocols for effective RNase inactivation.
Traditional nucleic acid extraction techniques use organic solvents such as phenol-chloroform to separate nucleic acids from the proteins, carbohydrates and cellular debris that will dissolve into aqueous phase. Centrifugation then allows the hydrophilic, nucleic acid-containing layer to be collected, followed by alcohol-precipitation for recovery. Manipulating the pH of the solutions and adding appropriate additives such as isoamyl alcohol and guanidinium thiocyanate, will allow for the isolation of RNA whilst inactivated RNases. Multiple alcohol wash steps are always required to remove high levels of salt that affect downstream applications.
Commercial nucleic acid extraction techniques largely forego the often time-consuming precipitation methods in favor of solid-phase extraction commonly in the form of spin-columns. These silica matrices and other anion-exchange materials change affinity to nucleic acids with changing pH and salt contents, combined with centrifugal force, column-based extraction workflows greatly simplify the various adsorption, washing and elution steps required resulting in a high-quality extraction free from contaminants and suitable for the majority of downstream applications.
Sample Types and Specific Considerations
Most molecular biology techniques rely on high-quality nucleic acid starting material that is free from biological and environmental contaminants.
Excess salts, a variety of organic and inorganic substances, as well as carry-over materials from the extraction processing steps can all be present in the extracted end-product and have the potential to interfere with the chemistry and processes of downstream applications. Along with ensuring that the nucleic acid extraction workflow is rigorously followed, different sample types may require special treatments to ensure resulting extractions are free from potentially inhibitory substances.
Cells, Plasmids and Simple Tissues
Extraction from cell culture is one of the most straightforward and popular sources of nucleic acids and many protocols have evolved to achieve usable nucleic acids from single-tube or “crude-lysis” methodology.
Typically nucleic acid concentration is high in cell cultures and simple tissue samples while the biological medium is low in potential inhibitory substances, such that end-point dilution or use of specially formulated enzymes maximizes efficacy of downstream applications without the need for multiple wash steps. The MyTaq™ Extract PCR Kit from Meridian efficiently streamlines routine DNA extraction from cell cultures and mammalian solid tissue samples (such as mouse tail or ear clips) for high-yield, PCR-ready template with a single-step protocol.
Biological Samples and Paraffin-embedded Tissues
For samples that have lower nucleic acid concentrations, more complex matrices and the potential for more inhibitory substances, it is advisable to employ a more sophisticated workflow that will maximize sensitivity and minimize inhibitory carry-over.
The majority of biological samples can be treated in a similar fashion, which will simplify laboratory workflow. The ISOLATE II Genomic DNA Kit from Meridian is one such multi-purpose, column-based approach that will provide rapid and highly efficient extraction of genomic DNA from a variety of biological samples including buccal swabs, biopsy, hair, paraffin embedded tissues, along with virus, yeast and bacterial samples.
Blood and Fecal Samples
Some biological samples contain unique components that may require specialized protocols to ensure the resulting nucleic acids are free from these inhibitory contaminates.
The haemoglobin in blood, for example, is a known PCR inhibitor, as well as other blood-based proteins, immunoglobulins, hormones etc. thus these samples may warrant specialized treatment. The ISOLATE II Blood DNA Kit from Meridian is specially formulated to deal with these contaminants as well as other potential elements from treatment such as EDTA, heparin or citrate. Fecal samples can also pose problems with high levels of lipids, salts and complex polysaccharides. The ISOLATE II Fecal DNA Kit from Meridian is specially formulated to overcome these challenges.
Plants and Soil
Environmental samples come with a unique set of problems, with humic acids, metal ions and other organic and inorganic debris complicating the extraction process.
Extracted nucleic acids from plants needs to be free of high levels of pectin, polysaccharides, xylan and polyphenols that can all have a downstream inhibitory effect. The ISOLATE II Plant DNA Kit is formulated to remove these inhibitors from a broad range of environmental samples from soil, plant tissues, including bark, compost and even dung.
PCR Products and Gels
Some protocols require purification rather than extraction, such as cleaning up of PCR products, or isolation of target nucleic acids from agarose gels.
The ISOLATE II PCR and Gel Kit is a fast and convenient solution offering high recovery rate and high-quality end product suitable for a range of downstream applications such as cloning, sequencing, or restriction analysis.
Since their discovery a little over 20 years ago, miRNAs, previously overlooked within what was thought to be non-functional genome components, are now understood to be crucial regulators of important cellular functions.
Aberrantly expressed miRNAs are a hallmark of many diseases, including cancer. Effective miRNA profiling calls for reproducible, sensitive and specific tools with turn-around times fast enough to support investigations into what can be a rapidly changing disease progression and treatment environment.
While the majority of traditional RNA isolation methods are inefficient at recovering miRNAs, the ISOLATE II kits offer, fast, unbiased, phenol-free recovery of miRNA from a variety of sample types, including mammalian tissue, cultured cells, blood, biofluids, viruses, bacteria, FFPE and plant tissue.