This article explains how to optimize and streamline sample collection and processing for nucleic acid analysis, to ensure consistent and reliable results while minimizing sample collection, processing, and shipping costs.

Key things to consider are:

• Sample collection protocols
• Nucleic acid stabilization
• Sample transport
• Streamlined sample processing
• Sample tracking

Use Simple Saliva Sample Collection Protocols

Buccal swabs and saliva samples can be collected non-invasively, which encourages more people to participate in studies. This is especially important for large-scale population studies, where broad participation is crucial for the validity of the results.

Allowing donors to collect samples at home removes the need to travel to a clinic or laboratory, which is particularly beneficial for individuals with mobility issues, busy schedules, or living in remote areas. Collecting samples at home can significantly reduce costs associated with sample collection by eliminating the need for dedicated space and staff to handle sample collection in a clinical setting.

Isohelix sample collection kits include several features that simplify sample collection:

• All kits include clear instructions for use that can be followed by donors at home
• Isohelix GeneFix saliva collection kits have a unique funnel design that prevents spillages or buffer flow back.
• GeneFix™ collection tubes have clear, legible markings to indicate the correct volume of saliva to be collected
• Buccal Swabs are provided in easy-to-handle formats

Nucleic Acid Stabilization

Isohelix offers several options for nucleic acid stabilization that remove the need for a cold chain during sample shipment, significantly reducing the costs and logistical challenges associated with sample storage and transport.

Stabilization Reagents

Including collection tubes prefilled with a nucleic acid stabilization reagent is a simple method of immediately stabilizing samples at the point of collection, with minimal handling steps and cross-contamination risk.

For swabs and saliva collection, Isohelix collection tubes are pre-filled with BuccalFix or GeneFix stabilizing buffers that completely inhibit all enzymatic and microbial activity following sampling. As a result, DNA is fully stabilized, and the structure and integrity of the DNA are maintained. DNA can be stored in BuccalFix at room temperature for over 2 years and in GeneFix for up to 5 years.

Nucleic Acid Stabilization via sample desiccation

For shorter-term storage, RapiDri™ Swab kits (RD-01) include a microporous moisture-wicking pouch that rapidly dries the swab, stabilizing the DNA on the swab matrix, where it is protected from degradation. DNA can be stabilized for 3 months at ambient temperature, allowing safe and easy sample transport and storage.

Alternatively, Isohelix Dri-Capsules are silica gel capsules that offer an easy-to-use alternative to chemical stabilization for the long-term stability of buccal DNA on a swab head before isolation. DNA stabilized using Dri-Capsules is stable for 3 years at ambient temperature. 

Streamline Sample Transport

If collection tubes and sample packaging are sufficiently robust, stabilized samples can be sent to the laboratory for processing using regular mail, a straightforward and cost-effective method of sample transport.

There are a range of products available from Isohelix to simplify shipping:

• Isohelix collection tubes comply with UN3373 on leak-proof sample transport that specify that collection tubes must withstand defined physical pressures of 95KPA. 
• Mailing boxes are suitable for GeneFix Saliva collectors and have space to fit a transport pack underneath. They are designed to fit through most letterboxes and are certified to UN3373. They include a security sealing strip.
• Transport packs are suitable for saliva and swab samples and include an absorbent pad to contain leakages, a separate document pocket, and are tested to resist 95kPa.
• Saliva transport packs are specifically designed for saliva samples, featuring a slimmer profile for easier postage

Medium and High-Throughput Sample Processing

For large numbers of samples, automated systems reduce handling errors, save hands-on time, and increase processing throughput. Genefix High Throughput tubes (GFXH) for saliva collection have a key-fitting cap and locking base designed to fit automated de-capping machines. For buccal swabs, SwabCatcher tubes include a unique cap design that automatically removes swabs and is suitable for automated and high-throughput systems.

Isohelix also offers magnetic bead-based nucleic extraction kits, Buccal-Mag, and Saliva-Mag. These kits can be used manually or with most openly programmable automated DNA isolation and liquid handling systems fitted with magnetic racks and heat blocks.

Alternatively, for saliva collection, Isohelix has adapted the Saliva-Prep2 kit for use with 1.2ml or 2.0ml 96 – Deep Well Plates. This gives a manual method for isolating 96 samples per plate, requiring only low-cost, widely available benchtop laboratory equipment.

Sample Tracking

Easy and accurate sample tracking is a key component of sample collection and isolation. All GeneFix Saliva Collection tubes include a unique 10-digit, alphanumeric 1D barcode on the side of the tube which allows for full traceability of the sample from production to distribution and testing. Tubes can be specified with a 2D barcode on the base of the tube that corresponds with the 1D barcode on the side. Having a 2D barcode available on the base of the tube allows automation systems to scan the barcodes while in racking to maintain traceability when using high-throughput methods. Barcodes printed in triplicate on cryogenic labels are also available for attaching to tubes or paper. All barcodes are scannable on common readers, including those widely available on mobile phones, so that even patients at home can scan their devices to provide additional traceability.

Summary

By implementing simple standardized collection methods, and optimizing processing and shipping, the integrity and reliability of saliva and buccal swab samples can be preserved, ensuring the best possible nucleic acid analysis results whilst minimizing time and cost.

To find out more about how Isohelix products simplify sample collection, CLICK HERE

The quality of DNA samples affects the quality of data produced by genomic analyses, and as analysis techniques become more sophisticated, DNA quality becomes even more important. For example, long-read sequencing enables the detection of variants that are often difficult to resolve with short reads, but it requires high-quality, high-molecular-weight input DNA to be successful.

DNA integrity must be maintained from the moment of sample collection to when the sample is processed in the lab to produce the best results. This can be particularly challenging with large genetic studies where DNA samples are collected from donors at home. Samples may be in transit for several days and exposed to wide temperature fluctuations before they reach the lab. DNA samples are particularly vulnerable to degradation in hotter, humid conditions.

The DNA integrity of a sample can be reduced during shipping and storage via several mechanisms :

• Enzymes (e.g. nucleases) and chemicals present in samples may degrade DNA
• Repeated freeze-thaw cycles, particularly if samples are exposed to low temperatures during shipping, may shear long pieces of DNA into shorter fragments
• Microbes present in samples may grow, adding to the proportion of microbial DNA present, and also producing chemicals and enzymes that may damage target DNA.

Microbial DNA present in samples can be a particular problem for microbiome analysis. During transport and storage, some microorganisms in the sample may continue to grow at the expense of others, meaning that the sample is no longer representative of the original community.

Traditionally, freezing was the standard method for DNA preservation, but freezing presents logistical challenges, especially in field settings or when samples need to be transported over long distances. Freezing requires a lot of energy, the cold chain must be maintained, and the weight of the ice used to keep samples frozen adds to shipping costs. If the cold chain fails during shipment or storage and the sample is subjected to freeze-thaw cycles, then this can cause DNA shearing.

Fortunately, alternative techniques are available that can maintain DNA integrity without the need for freezing. In this article, we’ll explore some of the methods of maintaining DNA integrity within saliva, swabs, and stool samples.

DNA Preservation using Desiccation

Desiccation, or drying, is a simple method for DNA preservation that works well for swab samples. Silica gel or similar desiccants are used to absorb moisture from the environment surrounding the DNA sample. By removing water from the sample, desiccation inhibits the activity of nucleases and other enzymes that could degrade DNA. Desiccation is particularly useful for preserving DNA in field settings where access to freezers may be limited.

Isohelix SGC Dri-Capsules are a fast and simple way of stabilizing buccal swab samples. A silica gel capsule is placed in the collection tube sitting on top of the swab shaft, maintaining DNA integrity for up to three years.[i]

Alternatively, the Rapi-Dri swab kit from Isohelix contains an easy to use buccal swab and a quick-drying pouch with a unique matrix that rapidly stabilizes samples by removing water. The pouch then acts as a sealed shipping unit for sample transport. Sample integrity can be maintained even if samples are subjected to particularly hot and humid environments. [ii]

Stabilizing DNA using Chemical Stabilization Reagents

Stabilization of DNA from Buccal Cells

Swabs are a straightforward method of collecting donor DNA.

For swabs that have been used to collect buccal cells, the collection tubes in the Isohelix BuccalFix DNA Stabilisation and lysis kit contain a buffer that has been specifically formulated to allow long term storage of swab samples at room temperature, enabling the subsequent isolation of high DNA yields and purity from the stabilized swabs.

Stabilization of DNA in Saliva Samples

Saliva samples can also be collected using swabs. To maintain DNA integrity with saliva samples, Isohelix SaliFix™ swab collectors  are recommended. The kits contain SaliFix™ SwabCatcher tubes prefilled with SaliFix™ buffer that is optimized to work with saliva swab samples

For larger volumes of saliva, e.g. 1-3ml,  GeneFix DNA/RNA collector tubes are pre-filled with a stabilization reagent that can stabilize DNA at room temperature for over five years.

If, however, you are interested in microbial DNA rather than human DNA, Isohelix has developed a new chemistry designed to maximize the collection and preservation of microbial DNA from human saliva samples. The reagent is included in the GeneFix Microbiome DNA Collector Kit, and can be used to provide a snapshot of the microbiome at the point of collection, stabilizing samples for over 12 months.

Stabilization of DNA from Stool Samples

Studies investigating individual gut microbiomes require the purification of microbial DNA from faecal samples. It is important that these samples are collected and stabilised correctly to ensure that the microbiome at the point of collection is analysed, and results are not biased by sample handling after collection.

Isohelix swabs, combined with StoolFix stabilization buffer, can be effectively used to collect and preserve microbial DNA from faecal samples. Samples are collected by brushing the stool lightly with each face of the swab and are stable at room temperature for up to two months.

Conclusion

Isohelix offers a range of DNA and RNA stabilization solutions that maintain nucleic acid integrity at room temperature for buccal cells, saliva, and stool samples. All stabilization products are optimised for use with Isohelix DNA extraction kits. 

[i] Dri-Capsule_3_Year_Study.pdf (isohelix.com)

[ii] RapiDri-Genotyping-Heat-App-Note-ver-4-020721.pdf (isohelix.com)

Introduction

With advanced genomic analysis applications becoming routine in many labs, the need for high purity, high quality DNA and RNA samples collected using non-invasive sampling methods, such as saliva, is becoming increasingly important.

Nucleic acid extraction from saliva can be challenging due to the presence of contaminants and inhibitors  that can interfere with downstream applications. However, in recent years significant improvements have been made with devices to collect, store, and process saliva to extract DNA and RNA. Good yields of high-quality nucleic acids can now be extracted for use in sensitive downstream applications.

Variations in sample collection and handling can dramatically affect data quality. This article explains how to get the best from your saliva samples by maximizing the purity and yields of DNA and RNA with information on:

• Preventing sample contamination
• Saliva collection from donors
• Nucleic acid stabilization
• Safely shipping saliva samples
• Choosing your DNA/RNA isolation kit
• Isolating high molecular weight (HMW) DNA from saliva   
• Cleaning up your DNA after an initial extraction

Preventing sample contamination

The main sources of human DNA in saliva are epithelial cells and leukocytes. Saliva samples may also include bacterial DNA, allowing for DNA extraction and analysis of the oral microbiome. To prevent contamination from external nucleic acids and other factors that may interfere with the purity of samples, it is important to ensure that donors do not eat, drink, smoke, or chew gum for at least 30 minutes before providing a sample.

Saliva collection from donors

For collection by donors at home without medical supervision, kits such as the GeneFix^™ Saliva DNA/RNA range include a simple to attach funnel that makes it easy to collect saliva in a tube prefilled with stabilization reagent for transport and storage of the sample. 

Traditionally, collection devices were designed to collect 1ml or 2ml saliva, but newer devices such as the GFX-03, can collect up to 3ml if higher yields (300μg+) are required, e.g., for biobanking. For maximum yields, donors must ensure that they don’t include bubbles when measuring the liquid level in the tube as this can result in overfilling, or not collecting enough saliva.

Some donors, e.g., children or elderly patients, may find it difficult to produce saliva. If this is the case, saliva production can be stimulated by gently rubbing the cheeks or using a kit such as the SaliFix^™ Saliva Swab DNA Collection kit which contains absorbent swabs for easy collection of smaller volumes of saliva.

Nucleic acid stabilization

One of the key benefits of using saliva as a source of nucleic acids is that samples can be collected remotely and mailed for analysis. Stabilization reagents protect sample integrity by stabilizing DNA and RNA and preventing microbial growth. GeneFix saliva collection kit tubes are prefilled with a non-hazardous guanidium-free stabilization reagent that stabilizes DNA at room temperature for 5 years and RNA for 2 months. Samples can be shipped and stored at room temperature, removing the costs and logistical challenges associated with cold chain storage and transport. For longer term storage GeneFix kits are also suitable for freezing at temperatures down to -80°C.

Safely shipping saliva samples

Shipping packaging and collection tubes must be manufactured from robust materials that can withstand the rigors of the mailing process to ensure your saliva samples arrive at the lab intact. Isohelix sample collection tubes are pressure tested to 95kPa, and Isohelix^™ TPS-50 transport packs are pressure-tested and adhere to UN3373 packaging standards – including secure sealing strips and absorbent material that retains samples leaking from the primary tube within further layers of packaging, preventing sample cross-contamination.

Choosing your DNA/RNA Isolation Kit

Precipitation methods of DNA isolation such as the GeneFix^™ Saliva-Prep Kit, enable the rapid and efficient extraction of high yields of high molecular weight DNA and can be easily scaled to accommodate different sample volumes. As these kits don’t require additional solvents, columns, or filtration, workflows are easy to automate, with fast handling times and reduced sample steps. The unique precipitation chemistry maximizes recovery of high molecular weight DNA, while minimizing co-precipitation of RNA and degraded low-weight DNA.

Alternatively, silica membrane-based spin columns, such as those included in the Xtreme (XME) and Xtreme-RNA (XMR) kits, allow the purification of very high-purity, high molecular weight DNA or RNA for demanding downstream applications such as next generation sequencing or microarrays.

Isolating high molecular weight (HMW) DNA from saliva

Gentle handling is key to preserving the integrity of your DNA samples and isolating HMW DNA for more demanding applications such as whole genome sequencing (WGS) or microarrays. To prevent shearing, use wide bore tips and pipette slowly, avoiding vortexing or shaking tubes.

Cleaning up your DNA after initial extraction

If, despite your best efforts, your extracted DNA is not of a sufficient concentration or purity for your downstream applications, a DNA clean up kit can be used. The Isohelix DNA clean up kit uses a simple 30 minute protocol to reclaim a “failed” sample, preserving your DNA yields and giving High Purity DNA with 260/280 ratios to 1.8.

Nanodrop Scan Before & After DCU CleanUp

Summary

By following the advice given in this article, high yields of high purity DNA can be extracted from saliva for use in a number of sensitive downstream applications.

GeneFix offers superior performance and high yields when compared with alternative collectors :