How to Choose an Extraction Workflow Provider: Tips for CROs
Nucleic acid extraction, the isolation of DNA or RNA from cells and tissues, is a critical preliminary step for many molecular biology assays. Ensuring that these extractions obtain the highest yield and purest DNA and RNA samples is critical for the success of many downstream applications such as PCR, SNP genotyping, and NGS.
Partnering with a nucleic acid extraction service who has instrumentation that delivers consistent, high-quality extractions can provide peace of mind for you and your clients. Your samples are varied and valuable, so choose the best instruments for obtaining the quality your clients need, from an experienced provider you can trust.
In this article, we discuss some points to consider when choosing a provider for nucleic acid extraction equipment.
Nucleic Acid Extraction Methodologies
There are several methods for extracting DNA or RNA. All methods share the basic steps of cell lysis, whereby lipid membranes are disrupted by chemical (eg, detergents, proteases) and/or mechanical means to allow the release of nucleic acids; inactivation of nucleases, to ensure that the extracted nucleic acid is not degraded; and purification, to separate the nucleic acid from cellular debris.
Automated instruments use the following methods for extracting and purifying nucleic acids:
- Precipitation Based Isolation
This method uses high salt and alcohol to precipitate DNA out of a lysate. This technique, which is used for extracting DNA from whole blood, uses a detergent solution to lyse both red and white blood cells at the same time. The released nuclei and mitochondria are collected via centrifugation and then treated with chaotropic salts and proteases. DNA is recovered by alcohol precipitation and then rehydrated with Tris or Tris-EDTA buffer. The isolated long-stranded, high molecular weight genomic DNA can remain stable for 20-25 years at -80°C.
Acquiring the blood and other samples for genomic DNA isolation can be an arduous task, and/or one that cannot be repeated. Thus, it is critical that these samples are processed gently and isolated free of RNA and other contaminants. Precipitation-based extraction is ideal for obtaining high molecular weight DNA required for long-read sequencing. A fully automated precipitation-based workflow is the preferred method for genomic DNA extraction and isolation from large volumes of blood, saliva, buffy coats, and other samples. Precipitation-based extractions produce pure, high molecular weight DNA ready for immediate use and long-term storage. The extracted DNA is pure enough for next gen sequencing.
- Magnetic Bead Based Isolation
This extraction technique utilizes tiny magnetic beads comprised of a paramagnetic core surrounded by a layer of binding matrix (such as silica) that can reversibly bind nucleic acids under certain buffer conditions. After the sample is lysed, it is incubated with magnetic beads. Nucleic acid binds to the beads, which are separated from cellular debris via a strong magnet. The beads are washed with ethanol before an elution buffer is added to release the nucleic acid from the beads.
This extraction protocol can provide fast, high-yield nucleic acid purification from virtually all molecular diagnostic, biological, clinical, and forensic sample types. This technique is extremely flexible, and can isolate DNA and RNA from solid tissues, liquid based samples, or cell line samples. However, the possibility of aspirating beads and contaminating the extracted nucleic acid can cause failure of downstream experiments. Fully automated magnetic bead-based extraction instruments are available that take up little benchtop space while offering speedy protocols for virtually any sample type.
- Membrane Based Isolation
With this method, the cell lysate is transferred to a porous polymer membrane column that will selectively bind nucleic acids, while other cellular components pass through the column upon centrifugation or application of a vacuum. The column is washed and then the nucleic acid is eluted off the membrane.
This method can provide a quick, high-purity, high-yield nucleic acid extraction. Ultra-thin, porous polymer membrane columns, which only require gentle, positive air pressure rather than centrifugation or vacuum, are preferable because the ultra-thin membrane releases DNA/RNA of all fragment sizes with very little to no nucleic acid remaining stuck in the column. In addition, because this protocol does not require centrifugation or powerful vacuum application, the nucleic acid does not get damaged; the resulting nucleic acid typically has fewer cleavages and is of higher molecular weight and higher quality compared with other column protocols. This method is ideal for studies involving many genetic markers, because they require the use of high quality DNA with minimal cleavage.
Automated membrane-based extraction instruments provide high purity isolates with a quicker processing time compared with manual methods.
Choosing an Extraction Workflow
An ideal provider will offer several workflows for nucleic acid extraction. In-house extraction experts should be able to guide you to the best extraction method or combination of extraction technologies for your specific samples, taking into account the intended downstream applications and levels of purity needed. Obtaining high yields of high quality extracted nucleic acid can help ensure that your clients’ experiments will be successful. To choose the most appropriate workflow, you will need to consider the following factors:
- Sample type, eg, blood, bacterial, viral, tissue, stool, forensic samples
- Volume of sample, ie, mL or uL
- Nucleic acid, ie, DNA or RNA
- Throughput level, ie, number of samples per run and time per run
- Intended downstream applications, eg, next-gen sequencing, PCR
In-House Extraction Experts
Every extraction project is unique. In-house extraction experts will understand the advantages and disadvantages of the different extraction methods. For example, if high molecular weight DNA is desired, or the sample is a large volume, they will likely recommend a precipitation-based extraction workflow rather than a bead-based workflow. This is due to the number of beads that would be necessary, the risk of DNA shearing, and the possibility of carry-over bead contamination into your DNA sample.
The provider you choose should be able to work with you to develop personalized solutions for your difficult extraction projects. Look for a provider who has a team of in-house extraction experts dedicated to your project’s success. Your provider should also have the newest automated extraction equipment, chemistries and protocols that can be configured to your sample type, volume, and requirements. From common starting materials like blood and saliva, to less common sample types, your provider should work with you to find a workflow that provides the extraction quality you need for your clients’ downstream applications.
Sample Tracking and Reporting
A workflow that enables you to track samples throughout the extraction process can offer your clients peace of mind that their samples are being prioritized and nothing is left to chance. Tracking allows them to know where their samples are and what’s being done to them at all times. Automated equipment offering full sample tracking can seamlessly communicate with laboratory information systems (LIS), can track samples through the extraction process (allowing for a restart in the event of an error), and can send emails or text alerts when the run is complete.
Ensuring Your Samples are Safe
Ensuring that your clients’ valuable samples are secure and organized at all times, from processing, to storage, to shipping, is essential. Certain sample tubes are designed with features to protect and track your samples. Look for workflows that use tubes with the following qualities:
- Permanently laser etched barcodes that will aid in sample identification and tracking, and will also allow data sharing between multiple users and locations
- Internal gaskets within the cap to preventing leaks
- External threads to ensure that your sample does not get lost in the threading. This also allows the tubes to be as small as possible while maintaining their full working volume, thus maximizing storage space
- Sturdy construction to withstand temperatures as low as -196C
- Space saving design that fits in Society for Biomolecular Screening (SBS) racks while taking up less freezer space. SBS racks are the standard that many automated systems use, so your samples can be used interchangeably with any equipment and automated instrumentation
Customer Service
Another factor to consider when purchasing extraction instruments is the quality of the provider’s customer service. A person should answer when you call, and they should be knowledgeable about their instruments and equipped to clearly answer your questions. You may want to consider the size and scope of the extraction provider. Will they be able to give you personalized attention? Sometimes a smaller, boutique service with in-house extraction experts may have more personalized service.
Conclusion
Finding the ideal nucleic acid extraction instruments to meet your needs entails understanding the methodologies and automated workflows that are available. An experienced provider can help guide you to the instruments best suited for your needs. In-house extraction experts can explain the advantages and limitations of extractions options and answer any questions you may have.
When you work with AutoGen, you gain access to high-end extraction equipment that helps maximize your sample yield and quality.
Are you looking for a dependable DNA/RNA extraction partner? Get in touch with AutoGen today. Our experts will be happy to talk with you.