How to Buy DNA and RNA Purification Technology



DNA and RNA Purification and Analysis Guide

The purification of DNA and RNA is essential for many downstream applications in life sciences, clinical diagnostics, forensics and drug discovery research.

Many different kits are available for the extraction and isolation of DNA and RNA; these have varying components dependent on the starting material and the application of the purified product. The purified DNA and RNA can be analyzed using many different techniques, including electrophoresis, sequencing, PCR, real-time PCR, northern blotting and cloning.

This Buying Guide provides you with an overview of the key technologies and considerations when purchasing kits and other products for DNA and RNA purification and analysis.

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Sample Origins

Different kits are used for the many varied sources of DNA. These include: human tissues (including those from formalin-fixed paraffin-embedded (FFPE) samples), blood, stool, bodily fluids, hair, rodent, insect, plant, bacteria, yeast, fungi, spores, soil, clinical samples (e.g. biopsy samples, fine needle aspirates), forensic samples (e.g. dried blood spots, buccal swabs), and fingerprints.

Blood Samples

Blood samples are routinely collected for clinical analysis. Nucleic acid isolation from blood requires a method to provide high-quality DNA or RNA without contaminants or enzyme inhibitors. Blood contains a number of enzyme inhibitors that can interfere with downstream analysis. In addition, common anticoagulants, such as heparin and EDTA, can interfere with downstream assays.

Erythrocytes (red blood cells) of human blood do not contain nuclei and are therefore not important for DNA/RNA. The target of isolation from whole blood is leukocytes (white blood cells). Since healthy blood contains approximately 1,000 times more erythrocytes than leukocytes, removing the erythrocytes simplifies the isolation process. This can be accomplished by selective lysis of erythrocytes, which are more susceptible than leukocytes to hypotonic shock and burst rapidly in the presence of a hypotonic buffer.

A common alternative to erythrocyte lysis is Ficoll density-gradient centrifugation. In contrast to erythrocyte-lysis procedures, Ficoll density-gradient centrifugation only recovers mononuclear cells (lymphocytes and monocytes) and removes granulocytes. Mononuclear cells isolated by Ficoll density-gradient centrifugation can then be processed for nucleic acid isolation as with other animal cells.

Plant Samples

Isolation of DNA and RNA from plant material presents a special challenge, with commonly used techniques requiring adaptation before they can be used with plant samples. Several plant metabolites have chemical properties that are similar to nucleic acids, making them difficult to remove from sample preparations. Co-purified metabolites (such as polysaccharides, polyphenolics, and flavones) and contaminants introduced by the purification procedure (such as salts or phenol) can inhibit enzymatic reactions or cause variations in UV spectrophotometric measurements and gel migration. Another consideration in the process of nucleic acid isolation is potential pipetting errors due to increased viscosity.

Heart, Muscle, and Skin Tissue Samples

Isolation from heart, skeletal muscle and skin tissue can be difficult due to the abundance of contractile proteins, connective tissue, and collagen. In order to remove these proteins, which can interfere with DNA/RNA isolation, the sample needs to be treated with a protease or phenol-containing lysis reagents.

FFPE Tissue Samples

FFPE tissue represents a valuable and extensive source of material for biomedical research. With an increasing number of researchers turning toward molecular analysis of FFPE samples, it is becoming increasingly important to develop specific protocols that take into consideration the unique nature of these samples.

Due to fixation and embedding conditions, nucleic acids in FFPE samples are usually heavily fragmented and chemically modified by formaldehyde. The degree of fragmentation depends on the type and age of the sample and on the conditions for fixation, embedding and storage of the sample. Although formaldehyde modification cannot be detected in standard quality control assays, such as gel electrophoresis or lab-on-a-chip analysis, it does strongly interfere with enzymatic analyses.

Steps can be taken to minimize the effects of FFPE storage on the nucleic acids, such as use of thin tissue samples, not over-fixing tissue slices, use of quality paraffin embedding reagents, avoidance of sample staining, and storage at low temperatures.

Viral Samples

Viral analysis of biological and environmental samples requires the use of advanced technologies to assure assay effectiveness. Molecular technologies are essential tools for rapid detection and identification of the most significant viruses. Viruses from clinical samples are often isolated from cell-free body fluids, where their titer can be very low. Virus particles may need to be concentrated before nucleic acid isolation by ultracentrifugation, ultrafiltration, or precipitation.

Downstream Applications

The quality and purity of the extracted nucleic acids provided by an extraction kit should be suitable for the intended downstream application. These applications will be covered in the section Applications of Extracted DNA & RNA, but include next generation sequencing (NGS), PCR, qPCR, Southern blotting, restriction endonuclease digestions and library preparation.

Sample Quantity

The choice of kit to be used depends on the number of cultured mammalian cells and bacterial cells, mg of tissue, volume of blood, mg of soil or mg of plant leaf tissue.

DNA Purification

DNA can be purified using many different methods and the downstream application determines how pure the DNA should be. There are a number of considerations that will determine the kit that is best to use.

In order to extract the DNA from a cell or tissue sample, the cells need to be disrupted by cell lysis. This is usually achieved by chemical and physical methods, such as blending, grinding or sonicating the cell sample. For example, the FastPrep-24 5G benchtop homogenizer, from MP Biomedicals, is designed for high-speed isolation of DNA, RNA and proteins. During the cell lysis process the membrane lipids are removed by adding a detergent or surfactants.

A protease is often used to remove cellular or histone proteins bound to the DNA. RNases are often used to remove unwanted RNA.

This raw DNA product is then purified to remove the detergents, proteins, salts and reagents used during cell lysis step. The most commonly used procedures are:

  1. Ethanol precipitation, usually by ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. Precipitation of DNA is improved by increasing ionic strength, usually by adding sodium acetate.
  2. Phenol-chloroform extraction, in which phenol denatures proteins in the sample. After centrifugation of the sample, denatured proteins stay in organic phase, while the aqueous phase containing nucleic acid is mixed with the chloroform, which removes phenol residues from solution.
  3. Minicolumn purification that relies on the fact that the nucleic acid may bind (adsorption) to the solid phase (silica or other), depending on the pH and the salt content of the buffer.

There are refinements to this technique, which include adding a chelating agent to sequester divalent cations, such as Mg2+ and Ca2+; this prevents enzymes such as DNase from degrading the DNA.

After isolation, the DNA is usually dissolved in slightly alkaline buffer or in ultrapure water.

Tip: Consider your downstream application, both purity and quantity of DNA are important.

Table 1: Common DNA Extraction Kit Components

DNA extraction technologies

Anion-exchange

Silica-membrane technology

Magnetic-particle technology

Technology

Solid-phase, anion-exchange chromatography

Selective adsorption to silica membranes

Binding to magnetic silica particles under controlled ionic conditions

Binding/Elution

Binding: variable salt and pH Elution: variable salt and pH
Requires alcohol precipitation

Binding: high salt
Elution: low salt
Ready-to-use eluate

Binding: high salt

Elution: low salt
Ready-to-use eluate

Application

Delivers ultrapure, transfection-grade DNA for optimal results in sensitive applications

Delivers high-purity nucleic acids for use in most downstream applications

Delivers high-purity nucleic acids for use in most downstream applications

Benefits


- Fast, inexpensive

- No silica-slurry carry over, no alcohol precipitation

- Fast, inexpensive

- Easy to automate, no alcohol precipitation


The most commonly used DNA extraction kits have a variation of three different types of technology, as outlined in Table 1.

Anion-exchange methods yield DNA of a purity and biological activity equivalent to at least two rounds of purification in CsCl gradients, in a fraction of the time. Purified nucleic acids are of the highest possible quality and are ideal for sensitive downstream biological applications, such as transfection, microinjection, sequencing, and gene therapy research.

Silica-membrane technology yields high-purity nucleic acids, suitable for most molecular biology and clinical research applications, such as restriction digestion, ligation, labeling, amplification, and radioactive and fluorescent sequencing.

Magnetic-particle technology yields high-purity nucleic acids, suitable for most molecular biology applications used in clinical research, such as restriction digestion, ligation, labeling, amplification, and radioactive and fluorescent sequencing. Magnetic-particle technology can often be automated to enable fast and economical nucleic acid purification procedures. Magnetic beads are rapidly being adopted in genomics and are replacing filtration kits. Corning® offers a comprehensive line of magnetic bead-based kits for fast, rapid isolation of genomic DNA from various starting samples.

Plasmid Isolation

Plasmid isolation is a basic technique that is performed in most molecular biology laboratories. Multi-sample processing is often required to complete both plasmid isolation and subsequent downstream experimentation.

Bioneer’s AccuPrep® Nano-Plus Plasmid DNA Extraction Kits are designed to accelerate the isolation of highly purified plasmid DNA from cultured bacterial cells. The kits combine alkaline lysis and novel nanotechnology, in which pelleted bacterial cells are resuspended and treated with a proprietary nano-solution.

QIAGEN’s HiSpeed Plasmid Kits allow ultrafast purification of up to 750 µg transfection-grade plasmid or cosmid DNA and offer less than 60 minutes prep time.

Sigma Aldrich's GenElute Five-Minute Plasmid Miniprep Kit features an ultra-streamlined protocol yielding up to 5 µg high-quality plasmid DNA in about five minutes.

The Promega PureYield Plasmid Miniprep System provides a rapid method for purification of up to 15 μg of plasmid DNA from 600 μl to 3 ml of bacteria culture. The system incorporates a unique Endotoxin Removal Wash designed to remove substantial amounts of protein, RNA and endotoxin contaminants from purified plasmid DNA.

Genomic DNA Isolation

Genomic DNA can be extracted from a variety of samples including tissue, cells, blood, serum, plants and forensic samples (see Table 2).

Table 2: Different kits available for extraction of Genomic DNA

Genomic DNA Isolation

Extraction Kits

Technology

Application

FFPE (formalin-fixed, paraffin-embedded tissue samples)

blackPREP FFPE DNA Kit, Analytik Jena Life Science (Figure 1)

Novel chemistry, no toxic solvents

Isolation from tumor samples

QIAamp DNA FFPE Tissue Kit, QIAGEN

QIAamp MinElute Columns

High-quality, ready-to-use DNA. Consistent, high yields. Complete removal of contaminants and inhibitors

EpiTect Plus FFPE Bisulfite Kit, QIAGEN

Unique DNA Protect Technology to prevent DNA degradation

Ready-to-use DNA in small elution volumes

BiOstic® FFPE Tissue DNA Isolation Kit, MO BIO Laboratories Inc.

Novel method without the use of solvents

Pure genomic DNA for use in real-time PCR, SNP genotyping and other genetic analysis methods

Blood

Maxwell® CSC DNA Blood Kit, Promega

VD medical device for automated isolation of human genomic DNA from whole blood

In vitro clinical diagnostic and blood analysis, e.g. in this study for genomic DNA purification from human whole blood

QIAamp DNA Blood Mini QIAcube Kit, QIAGEN (Figure 2)

Fast, automated setup with preloaded rotor adapters

Automated purification of genomic, mitochondrial, or viral DNA

Cells/Tissue

UltraClean® Tissue & Cells DNA Isolation Kit, MO BIO Laboratories Inc.

UltraClean® technology avoids the use of phenols and chloroform

Isolated DNA is high quality and ready for PCR, restriction digests, and other downstream applications.

illustra triplePrep Kit, GE Healthcare

Optimized buffer, columns, and protocol ensure high recovery of gDNA, RNA, and proteins

PCR, restriction digestion, sequencing, array CGH, RT-qPCR, gene expression microarray, SDS-PAGE, Western blotting, 2-D DIGE, and LCMS

MagNA Pure LC DNA Isolation Kit II (Tissue), Roche Applied Science

Isolation procedure based on magnetic-bead technology

High-purity DNA from mammalian tissues for directly in highly sensitive PCR.


blackPrep_ffpe

Figure 1: Analytik Jena's blackPREP FFPE DNA Kit for extraction of DNA from tumor samples

QIAamp_DNA

Figure 2: The QIAGEN QIAamp DNA Blood Mini QIAcube Kit

Plant DNA Purification

There are a number of suitable kits for extraction of DNA from plant matter. Again, consider the sample and the purity of the extracted DNA required for the analysis and downstream application. QIAGEN's DNeasy 96 Plant Kit for isolation of up to 15 µg per well total cellular DNA from plant tissue is a great all-round kit.

RNA Purification

RNA is a biological macromolecule that serves a number of different functions. Messenger RNA (mRNA), transcribed from DNA, serves as a template for synthesis of proteins. Protein synthesis is carried out by ribosomes, which consist of ribosomal RNA (rRNA) and proteins. Amino acids for protein synthesis are delivered to the ribosome on transfer RNA (tRNA) molecules. RNAs are also part of riboproteins involved in RNA processing.

Noncoding RNAs are also important. These are functional RNA molecules that do not translate into proteins. Such RNAs include tRNA and rRNA, as well as small nucleolar RNAs (snoRNA), microRNAs (miRNA), short interfering RNAs (siRNA) and piwi-interacting RNAs (piRNA). They are often involved in the regulation of gene expression.

Purified RNA is used for many downstream applications (see section below). DNA extraction methods cannot be directly applied to RNA, as RNA is structurally very different from DNA. RNA is single-stranded, while DNA is mostly double-stranded. It is often difficult to isolate intact RNA. RNases, a group of enzymes that degrade RNA molecules, are abundant in the environment, including on hands and on surfaces, and it is difficult to completely remove/destroy RNases. RNA isolation therefore requires cautious handling of samples and good aseptic techniques. It is important to use only RNase-free solutions during the extraction, as well as RNase-free pipette tips and glassware.

Ribonucleases (RNases) are very stable and active enzymes that generally do not require cofactors to function. Since RNases are difficult to inactivate, and even minute amounts are sufficient to destroy RNA, no plastic or glassware should be used without first eliminating possible RNase contamination. Great care should be taken to avoid inadvertently introducing RNases into the RNA sample during or after the purification procedure. In order to create and maintain an RNase-free environment, precautions must be taken during pre-treatment and use of disposable and non-disposable vessels and solutions while working with RNA.

Some sample sources have differences in their RNA or contain substances that can cause problems in RNA isolation and analysis. Special considerations are required when working with these sample sources. In this section, considerations for working with a number of different sources are discussed.

The Bio-Rad Aurum™ Total RNA Mini Kit also produces high-quality DNA-free RNA from a wide range of starting materials, including cultured cells, bacteria and yeast, as well as animal and plant tissues.

This interesting application note from Diagenode describes RNA extraction from tissue using the Bioruptor® (Standard/Plus) and RNA Extraction Kit.

Table 3: Summary of RNA Isolation Kits

Sample Types

RNA Extraction Kits

Technology

Application

FFPE (formalin-fixed, paraffin-embedded tissue samples)

AllPrep DNA/RNA FFPE Kit, QIAGEN

Novel chemistry, no toxic solvents

Isolation from tumor samples

Absolutely RNA FFPE Kit, Agilent Technologies

Specially constructed beads have a large surface area to maximize high yields of polyA-RNA

Variety of downstream applications

High Pure FFPE RNA Micro Kit, Roche Applied Science

Innovative column design

RT-PCR

Blood

innuPREP Blood RNA Kit, Analytik Jena Life Science

Specially optimized lysis buffer

Upon elution in RNase-free water, the final RNA is ready-to-use and can be integrated in subsequent applications immediately

QIAamp RNA Blood Mini Kit, QIAGEN

QIAamp Mini Spin Columns and QIAshredder Spin

Variety of downstream applications

Cells/Tissue

RNeasy 96 Universal Tissue Kit, QIAGEN

Streamlined QIAzol lysis and RNeasy 96 purification

High-performance RNA for all downstream applications

illustra triplePrep Kit, GE Healthcare

Optimized buffer, columns, and protocol ensure high recovery

of gDNA, RNA, and proteins

PCR, restriction digestion, sequencing, array CGH, RT-qPCR, gene expression microarray, SDS-PAGE, Western blotting, 2-D DIGE, and LCMS

Plants

innuPREP Plant RNA Kit, Analytik Jena Life Science

Two Lysis Buffers to process as wide a range as possible of different plant

Variety of downstream applications


RNA Isolation from Bacteria

Bacterial mRNAs differ from eukaryotic mRNAs in a number of essential features. Prokaryotic mRNAs have no 5' cap and only rarely have poly-A tails. The absence of a poly-A tail means that mRNA isolation by hybrid capture is not possible. In addition, oligo-dT primers cannot be used to prime first-strand cDNA synthesis so random primers need to be used instead.

In addition, bacterial mRNAs are highly unstable, with an average half-life of about three minutes for fast-growing bacteria. Sometimes the bacterial mRNA begins to degrade while it is still being translated. This can be a big problem for researchers trying to isolate mRNA from bacteria. Since mRNAs are very rapidly turned over in bacteria, gene expression studies are even more difficult in prokaryotes than in eukaryotes. To accurately preserve gene expression patterns and to maximize the amount of fully intact mRNA isolated, samples need to be stabilized prior to sample harvesting and processing.

Cell-free RNA in Plasma or Serum (or Other Body Fluids)

RNA, especially miRNA, associated with proteolipids (vesicles) or proteins can be found in bodily fluids, including plasma, serum, urine, and in cell culture supernatants. The concentration is much lower than that of cellular RNA, but approximately tenfold higher than cell-free DNA in human plasma. The RNA is relatively stable, with a half-life of about two days in human whole blood. Nonetheless, the RNA can be degraded by repeated freeze-thaw cycles. As with viral RNA in cell-free body fluids, addition of carrier RNA may be necessary during RNA isolation of this RNA.

Automated Nucleic Acid Purification

The automation of nucleic acid purification has evolved at unprecedented levels recently, mainly due to the now high throughput nature of downstream clinical and research applications. In this section, the applications of several types of automated systems are described.

This study by Eppendorf describes an automated method for the purification and analysis of DNA from microbial and food cultures purified using the MO BIO Laboratories Inc. PowerMag Microbial DNA Isolation Kit on the epMotion® 5075 TMX. Discover automated Genomic DNA Purification of Marine Organisms on the epMotion® 5075 VAC from Eppendorf.

Read about an optimized method for the automated extraction of high-quality DNA from bovine tissue samples using the LGC's sbeadex tissue kit, incorporating a novel two-step binding mechanism, and the oKtopure high-throughput extraction platform.

The innuPREP DNA Kit I-KFml, from Analytik Jena, has been optimized for automated DNA extraction using the KingFisher® ml processor from Thermo Fisher Scientific. The kit yields highly pure DNA from whole blood samples, as well as from paraffin-embedded tissue samples, mouse tails or buccal swabs. The extraction method is based on the principle of magnetic particle separation.

The Agencourt SPRI magnetic bead based method, from Beckman Coulter, is used for highly efficient miRNA and total RNA purification from FFPE tissues. The RNA is suitable for size selection and automated NGS library construction.

This study demonstrates a flexible automated solution for the isolation of plant genomic DNA without compromising yield or purity using the MACHEREY-NAGEL NucleoSpin Plant II Midi kit on Tecan's Freedom EVO® platform.

Aurora Biomed has combined its expertise in automated liquid handling with Promega’s MagneSil® Blood Genomic Kit to offer an automated and fully validated application for Genomic DNA isolation. Read the application note to find out more about this new method.

AMSBIO's MagSi-DNA cleanFIX is used with PerkinElmer's JANUS® Automated Workstations in this article for high throughput nucleic acid purification. The article describes a 384-well clean-up automated solution, increasing throughput, reliability and data robustness with a reduced amount of reagent consumption.

Tip: Look for automated systems that are suitable for the high throughput requirements of your downstream applications

DNA & RNA Analysis

Nucleic Acid Gel Electrophoresis

Gel imaging and nucleic acid binding dyes are widely used in today’s life science laboratories to visualize DNA fragments in agarose gels. This application note from UVP describes a method for safe gel imaging with blue light excitation using GelGreen™ Dye GelDoc-It® Imaging System and Visi-Blue™ Converter Plate.

Nucleic Acid Quantitation

Quantification of nucleic acid samples prior to analysis is an important QC step in order to verify suitability for downstream molecular applications, such as qPCR, RT-qPCR, STR analysis, SNP genotyping and DNA sequencing. The QIAxpert Instrument, from QIAGEN, can quantify nucleic acids from up to 16 samples in less than two minutes.

The Thermo Scientific NanoDrop One UV-Vis microvolume spectrophotometer, figure 3, is ideal for quantifying and analyzing the quality of extracted DNA, RNA, and protein samples before use in downstream applications.

Jonathan

Figure 3: Hear about the features of the new Thermo Scientific NanoDrop One

The DS-11 FX+ Spectrophotometer / Fluorometer from Denovix provides fluorescence, microvolume UV-Vis and cuvette-based absorbance in a single instrument, ideal for quantitative measurements using volumes as small as 0.5 to 1 μL.

Download this application note about the quantification of mammalian DNA using spectral content profiling on the Xpose system from Trinean to learn how to use the technology and interpret the analysis data.

Once the sample of DNA or RNA has been quantified, it can be amplified for further analysis and applications.

Applications of Extracted DNA & RNA

Extracted and purified DNA is used for many downstream applications in a number of fields, including clinical diagnostics, basic research, life sciences and forensics.

Depending on the purity and quantity of the nucleic acids, applications include PCR and RT-PCR, restriction digestion and cloning, sequencing, array CGH, gene expression microarray, SDS-PAGE, Northern blotting, 2-D DIGE, and LCMS. Some of these are covered here with associated application notes.

PCR & RT-PCR

The polymerase chain reaction (PCR) is a technique used to amplify DNA sequences in vitro, and is widely used for many applications, such as molecular biology, microbiology, genetics, diagnostics, clinical laboratories, forensic science, environmental science, food science, hereditary studies and paternity testing. Download the How to Buy PCR Technology guide for more information about PCR kits and thermal cycling equipment.

Some kits, such as the Agilent Technologies SureDirect Blood PCR Kit, enable direct DNA amplification from many sample types including liquid blood, dried blood on paper, serum, and plasma. Inhibitor resistance and throughput, and common PCR challenges associated with blood samples are averted through the activity of a newly engineered Taq.

Northern Blot Analysis

The Northern blot is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample.

With Northern blotting it is possible to determine gene expression levels during differentiation, morphogenesis, as well as abnormal or diseased conditions. This application note from LI-COR discusses methods for Northern blotting analysis, biotin probe labeling using PCR amplification, Northern blot hybridization and biotin detection for Northern blots, using the Odyssey® Imaging systems.

RNA Sequencing

RNA-seq (RNA Sequencing), also called Whole Transcriptome Shotgun Sequencing (WTSS), is a technology that uses the capabilities of next-generation sequencing to reveal a snapshot of RNA presence and quantity from a genome at a given moment in time. There are a number of technologies available to achieve this, for example the Fluidigm C1 Single-Cell Auto Prep System provides a new high throughput workflow for single-cell mRNA sequencing. The SMARTer Stranded RNA-Seq Kit from Takara Clontech provides a solution for generating libraries for Illumina® sequencing platforms. This application note describes how the SMARTer kit is used to prepare RNA from challenging samples such as small quantities of FFPE tissue.

Microarray Analysis

Microarray-based gene expression hybridization is a powerful and proven technique for studying differential gene expression signatures, and is especially valid for cancer research. RNA extracted from freshly frozen tissues is optimal for microarray analysis; however, in many cases as described above, FFPE tissues are the only samples available. This application note, from Agilent Technologies, describes an optimized microarray method for RNA extracted from FFPE, in this case archived colon cancer samples.

Next Generation Sequencing

Next generation sequencing is a necessary requirement for today’s high throughput research and clinical needs. One of the bottlenecks for NGS is the amount of time and resources required for library preparation; this is true whichever sequencing instrument you choose. For a comprehensive review of NGS sequencers and library construction kits, please see our How to Buy Next Generation Sequencing Technology guide. This application note from UVP, LLC describes a two-dimensional process to separate DNA for NGS library construction.

Forensic Investigation

DNA and RNA extracted from various materials, plants, cells and tissues from a crime scene are used to gain data and evidence about the crime. Technologies for PCR, sequencing, electrophoresis and other analysis tools are used frequently.

For example, the FBI recently granted approval for the Promega PowerPlex Fusion 6C System for use in laboratories that generate DNA records for the National DNA Index System (NDIS).

Cutting-edge DNA coding was the key technology used by leading forensic geneticist, Dr Jari Louhelainen of Liverpool John Moores University, UK, to discover the identity of the notorious London serial killer ‘Jack the Ripper’, almost 126 years after the last murder victim was discovered. In this exclusive interview, Dr Louhelainen spoke to SelectScience about his work to retrieve and analyze the DNA from a shawl taken from one of the murders.

You can also learn more about the forensic study of soil in our interview with Professor Lorna Dawson, Principal Soil Scientist in the Environmental and Biochemical Sciences group and Head of Forensic Soil Science, at the James Hutton Institute, UK,

Molecular Diagnostics

DNA/RNA can be extracted from a wide range of biological patient specimens such as blood, plasma, swabs, saliva, urine, stool and biopsy preparations. The first application of PCR in clinical diagnostics was in testing for genetic disease mutations. Molecular diagnostic laboratories routinely test for a large variety of mutations, such as those found in hematological disorders, for example Factor V Leiden and Prothrombin 20210; cancer mutations such as epidermal growth factor receptor gene (EGFR) in non-small cell lung cancers and KRAS in colorectal cancer; and prenatal testing by amniocentesis, to name a few. Extracted genetic material can also be used for procedures such as tissue typing in transplant patients.

The field of infectious disease has been revolutionized by advances in PCR and nucleic extraction methods. PCR tests are capable of detecting extremely low levels of infection over traditional serology or culture techniques. The rapid detection of infectious disease pathogens in blood samples, enabled by RT-PCR, allows for faster, more specific identification, which can aid the therapeutic choices made by the clinician. Better therapeutic choices are beneficial to the patient, and are also desirable in lowering the rates of antibiotic sensitivity. PCR tests have been developed for a wide range of pathogens including Staphylococcus Aureus, Streptococcus Pneumoniae, Listeria Monocytogenes, Neisseria Meningitidis, and Clostridium Difficile. Modern automated molecular diagnostic analyzers reduce the possibility of contamination and maximize throughput.

Current and Future Trends

The use of extracted DNA and RNA in various applications and research fields is widening every year. With the need for high throughput analysis and downstream applications such as NGS, automation of extraction and purification is key to the future and further expansion into new areas. Some examples of these new areas and new technologies in life sciences and clinical diagnostics are discussed here.

Kits and technologies for co-extraction of DNA and RNA from starting materials are now available. These systems alleviate the need for separate kits and save time for downstream processing techniques such as RT-PCR. The AllPrep DNA/RNA Kits for FFPE, and fresh cells and tissues in a 96-well format provide fast purification using a standardized workflow.

In the field of diagnostics, great strides continue to be taken in the field of infectious diseases. Despite the advances of PCR in detecting infectious pathogens, other detection methods are still more commonly used. PCR methods will continue to be developed to create fast, accurate assays that can be used in laboratories around the world, including developing countries, where the need is greatest.

The potential for molecular diagnostics has much more scope than just infectious diseases; advances have been made in developing blood tests for cancer biomarkers (liquid biopsies), micro-fluidics and lab-on-a-chip methodologies and microarrays. NGS is also moving steadily into the diagnostic market in the fields of oncology and gene expression profiling.

In other applications such as food technology, reports on the detection of DNA in bottled wines have been emerging since 2000. New preliminary research has shown that DNA molecules of up to 5,000 bp could be detected in wine nucleic acid extracts using an extraction method developed in a laboratory in Australia. DNA extraction also plays an important role in the analysis of food fraud. Learn how DNA barcoding is used to fight fish fraud in this SelectScience article and watch this SelectScience video interview with Didier Montet, CIRAD, to learn how biological barcoding of yeast and fungi can be used to identify the geographical origin of foodstuffs.

Summary

There are many different types of DNA and RNA purification kits, and automated extraction systems on the market. Finding the correct kit or system for your starting material and downstream application may an enormous task, but by applying a few simple considerations, selecting the correct kit or system can be made easier.

DNA and RNA extraction kits will continue to develop and new technologies will emerge as the demands for pure DNA or RNA for different applications evolves.

Visit the SelectScience product directory for an overview of the latest DNA and RNA Purification and Analysis technology from leading manufacturers and read user reviews. Keep up-to-date with the latest purification methods by visiting the SelectScience application note and video libraries in life sciences, drug discovery and clinical communities.

Editor's Picks

Kerry Parker Kerry Parker
Editor-in-Chief

NanoDrop 2000/c Spectrophotometers (Thermo Fisher Scientific)

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4 out of 5

"Very sensitive and easy to use. Reproducible results. Small amount of samples needed."
Anca Gafencu, Institute of Cell Biology and Pathology

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innuPREP Plant RNA Kit (Analytik Jena Life Science)

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5 out of 5

"This kit provides an easy way to get high yields of RNA in very little time."
Jasmin Goetzenberger, Karlsruhe Institute of Technology

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BiOstic® FFPE Tissue DNA Isolation Kit (MO BIO Laboratories Inc.)

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5 out of 5

"This FFPE DNA isolation kit was extremely easy to use, and it yielded high quality and quantity of DNA."
Kristin Williams, University of Massachusetts

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Gentra Puregene Blood Kit (1000 ml) (QIAGEN)

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5 out of 5

"The kit is based on salting-out chemistry and is very easy to use."
Nicholas Risch, Empire Genomics



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Wizard® Genomic DNA Purification Kit (Promega)

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5 out of 5

"I was able to extract DNA from fish gonads and coral polyps with this product."
Tracy Sherwood, Mote Marine Laboratory



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Precellys®24 (Bertin Technologies)

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5 out of 5

"Precellys is very useful for protein and RNA extraction."
Antonio Peluso, Universidade Federal de Minas Gerais







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