Microbiome DNA Sampling and Analysis

Microbiome DNA Sampling and Analysis

A microbiome is the collective genetic material of the community of microorganisms living in a specific habitat, which can be a niche related to a living organism, e.g., the skin, mouth, or gut, or in the environment, e.g., in soil or water.

Our understanding of the complex relationships between these different microorganisms and the intricate mechanisms by which they influence health and disease has grown tremendously in recent years. Investigating and evaluating an individual’s microbiome and understanding the impact of altering it are important in many areas of medicine.

Microbiomes comprise bacteria, archaea, viruses, and fungi, and the DNA of these microorganisms is studied to gain insights into their diversity, abundance, and functional potential. However, extracting DNA from complex microbiome samples can be challenging due to low DNA yields and the presence of inhibitors and background DNA from the host organism.

This article discusses how to isolate and analyse DNA from three important human microbiomes; the gut, skin, and oral microbiomes.

Human Microbes

The largest human microbiome by mass is the gut microbiome, which weighs approximately 2kg[i] and performs a wide range of functions critical for maintaining health and wellbeing, e.g., aiding digestion, synthesizing essential nutrients, and metabolizing medications. Imbalances in the gut microbiome, known as dysbiosis, are associated with many health conditions, including chronic diseases[ii], cancers[iii], mental health disorders[iv], and gastrointestinal disorders[v].

The second largest microbial community in humans is the oral microbiome[vi]. Dysbiosis in the oral microbiome can lead to various oral health issues[vii] and systemic diseases.[viii] [ix] However, most oral microbiota are beneficial, contributing to good oral health by protecting against pathogens and maintaining a healthy pH balance. The composition of the oral microbiome may be a proxy for microbiomes in other niches, including that of the gut and lung. 

The skin microbiome has a relatively low biomass, but a healthy skin microbiome is also critical for good health. Interactions between members of the skin microbiota can prevent colonization by pathogenic bacteria[x], and many common skin diseases are associated with dysbiosis of the skin microbiome[xi].

Collecting DNA for Microbiome Analysis

Microbiome composition can vary significantly between individuals and even within the same individual over time. Several factors contribute to success in obtaining a representative sample of DNA for microbiome analysis.

1. Sampling, Shipping, and Storage

Microbiome samples are very sensitive to shipping and storage conditions. Not only does DNA degrade if not stored correctly, but 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. Traditional methods of shipping and storing microbiome samples involved freezing, which presents logistical challenges with maintaining a cold chain during transport, and risks sample loss due to freezer failure.

Isohelix has addressed these issues with their range of microbiome DNA collection kits, which include stabilization reagents that instantly stabilize samples, providing an accurate snapshot of the microbiome. Samples can then be shipped and stored at ambient temperatures.

Gut microbiome sample collection can be achieved at home or in the clinic, using rectal swabs or stool samples. The simplest method, as employed by the Isohelix StoolFix Gut Microbiome stabilisation kit (STF), is to simply brush the outside of a stool sample with a swab, before placing the swab into a tube containing stabilisation solution. This is much easier than the traditional “scoop” method of stool sampling.

For the oral microbiome, saliva is the best sample type. The GeneFix Saliva Microbiome DNA Collector (MFX) is an easy to use oral microbiome DNA collection kit that has been optimised for the collection of oral microbiome samples using saliva.

Finally, Buccal Swabs (SK) can be used to collect samples from surfaces, either in the environment, or for skin microbiome sampling. Various stabilization options are available to preserve swab samples.

StoolFix (STF)
Buccal Swabs (SK)

2. Preventing Sample Contamination

Contamination from external sources such as environmental microbes or human DNA, can confound analysis of the gut microbiome. It is important to control for and minimize such contamination to ensure accurate identification and characterization of microbial species present.

Isohelix collection devices and buffers are free from any traces of human or bacterial DNA, so researchers can be confident in the accuracy of their results. To avoid contamination of oral microbiome samples, patients must avoid eating, drinking, smoking, or brushing their teeth for 30 minutes before sampling.

3. Optimise Extraction

To optimise the extraction of microbial DNA it is important to ensure that collection and stabilization kits are compatible with the microbiome DNA extraction method.

To extract DNA from swabs, for either gut or skin microbiome samples, the BuccalPrep Plus DNA Isolation Kit (BPP) can be used to isolate high quality DNA suitable for all downstream applications. Alternatively, when using Isohelix stabilization buffers such as BuccalFix and StoolFix, BuccalFix Plus DNA Isolation Kit (BFP) can be used to maximize samples.

The Saliva Microbiome DNA collector has been designed for use with the Saliva-Prep2 DNA isolation kit, and produces extremely high yields of microbial DNA (more than twice the yield of other commonly available kits). DNA isolated using Saliva-Prep2 is intact and readily amplifiable, with high purity, and therefore suitable for all downstream analyses.

BuccalPrep Plus (BPP)
BuccalFix Plus (BFP)
Saliva-Prep2 (GSPN)

How to analyse the microbiome

When you have isolated high quality DNA, the two most common methods to measure microbiome depth and diversity are:

  • Targeted sequencing of the 16s rRNA gene of bacteria, and internal transcribed spacer (ITS) region sequencing for fungi. These genes are highly conserved, but have diverged over time and can be used to provide a, “barcode” that can be assigned to specific taxonomies, or counted to identify the frequency of each member of the microbial community.
  • Shotgun metagenomics – untargeted sequencing methods capture all microbial genomes present within a sample. Metagenomic shotgun assemblies are either performed de novo, based on reference genomes, or a hybrid of both. All types of microorganisms can be sequenced, not just bacteria and fungi.

Tools to study the microbiome are rapidly advancing, and as more researchers begin to investigate the role of the microbiome in health and disease, more effort is spent developing new robust statistical methods and analytical tools required for data analysis.

Future Perspectives

Technical advances in DNA sequencing and bioinformatics analysis have enabled us to understand the importance of microbiomes. Understanding their role, including the dynamic interactions with hosts and their microbes, enables new strategies in a wide range of fields from health to ecology and agriculture.

The manipulation of human microbiota and host-microbiome interactions is a valuable management tool for many health conditions, and some beneficial microbes are even considered next-generation drugs.

At the heart of any microbiome study is a need for high-quality DNA suitable for next-generation sequencing. The Isohelix range of products addresses the challenges with microbiome DNA collection and extraction, enabling researchers to obtain the high-quality DNA they need, to conduct their work.


[i] Flint, H.J. The impact of nutrition on the human microbiome. Nutr Rev. Aug;70 Suppl 1:S10-3 (2012).

[ii] Vijay, A., Valdes, A.M. Role of the gut microbiome in chronic diseases: a narrative review. Eur J Clin Nutr 76, 489–501 (2022). 

[iii] Sims TT, et. al, Gut microbial diversity and genus-level differences identified in cervical cancer patients versus healthy controls. Gynecol Oncol. Nov;155(2):237-244 (2019)

[iv] Safadi, J.M., Quinton, A.M.G., Lennox, B.R. et al. Gut dysbiosis in severe mental illness and chronic fatigue: a novel trans-diagnostic construct? A systematic review and meta-analysis. Mol Psychiatry 27, 141–153 (2022)

[v] Wei L, Singh R, Ro S, Ghoshal UC. Gut microbiota dysbiosis in functional gastrointestinal disorders: Underpinning the symptoms and pathophysiology. JGH Open Mar 23;5(9):976-987. (2021)

[vi] Dewhirst, F. E. et al. The human oral microbiome. J. Bacteriol. 192, 5002–5017 (2010).

[vii] Highlander, S. K. et al. Deep sequencing of the oral microbiome reveals sig natures of periodontal disease. PLoS ONE 7, https://doi.org/10.1371/journal. pone.0037919 (2012).

[viii] Wilson, B. A. et al. The oral microbiome of early stage Parkinson’s disease and its relationship with functional measures of motor and non-motor function. PLoS ONE 14, https://doi.org/10.1371/journal.pone.0218252 (2019).

[ix] Matsha, T. E. et al. Oral microbiome signatures in diabetes mellitus and periodontal disease. J. Dent. Res. 99, 658–665 (2020).

[x] Buffie, C. G. & Pamer, E. G. Microbiota-mediated colonization resistance against intestinal pathogens. Nat. Rev. Immunol. 13, 790–801 (2013).

[xi] lebba, V. et al. Eubiosis and dysbiosis: the two sides of the microbiota. New Microbiol. 39, 1–12 (2016)

[i] Karpinets, T.V., Wu, X., Solley, T. et al. Metagenomes of rectal swabs in larger, advanced stage cervical cancers have enhanced mucus degrading functionalities and distinct taxonomic structure. BMC Cancer 22, 945 (2022).

[i] GeneFix™ Saliva Collectors & Kits for Human DNA Oral Microbiome Analysis, Application Note: GeneFix™ MFX, December (2018)

If you want to find out more about how Isohelix can help with your microbiome collection, stabilization and isolation, then CONTACT US

The Best DNA Extraction Methods from Saliva or Buccal Swabs

best dna extraction methods

Isolating nucleic acids from saliva and buccal swabs has become increasingly popular in recent years. Sample collection is easy, safe, and non-invasive, and collection kits can be mailed to donors for self-collection at home if required.

The method of DNA extraction is crucial to the yield and quality of the sample, with high purity DNA required for sensitive downstream analyses such as next generation sequencing, but cruder lysate preparations suitable for PCR.

Historically, the problem with isolating DNA from saliva and buccal swabs over other sample such as blood was low yields and microbial contamination, but improvements in sample collection protocols and devices have solved these issues. To accommodate different sampling protocols, equipment availability, downstream analyses, and storage requirements, Isohelix has developed a number of different extraction kits based on alternative chemistries. Users can select the most appropriate kit for their requirements, allowing them to achieve the best results with their particular application.

Isohelix offers four key technologies each offering user advantages for DNA extraction:

In the following article we discuss the different technologies and when to use them.

1. Precipitation-Based Chemistry: Effective & Fully Optimised

The simplest DNA extraction method offered by Isohelix, precipitation-based chemistry is included in the BuccalFix Plus (BFP)BuccalPrep Plus (BPP) and SalivaPrep-2 (GSPN) kits.

In the first DNA extraction step, buffers in the kits lyse cells in the samples, and then nucleic acids are purified via precipitation, leaving contaminants behind. Proteinase K is added during the protocol to increase purity by digesting contaminating proteins.

Precipitation methods 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 maximises recovery of high molecular weight DNA, while minimising co-precipitation of RNA and degraded low-weight DNA.  

  • Benefits: User-friendly, fast protocol, easy to automate, high molecular weight DNA, scalable, no additional solvents needed.
  • Applications: PCR/qPCR, Microarray, Methylation Array, Sanger Sequencing, Next-Generation Sequencing (NGS).
BuccalFix Plus (BFP)
BuccalPrep Plus (BPP)
SalivaPrep-2 (GSPN)
Xtreme (XME)
Xtreme RNA (XMR)

2. Silica Spin-Columns: Familiar method, reliable results

Silica membrane-based spin columns, such as those included in the Xtreme (XME) and Xtreme-RNA (XMR) kits, are a tried and tested extraction method, allowing the purification of very high-purity, high molecular weight DNA or RNA that can be used in demanding downstream applications such as next generation sequencing or microarrays.

Cells are lysed to release nucleic acids, and then samples are centrifuged to filter them through a silica membrane inside the column. Under the right ionic conditions, nucleic acids selectively bind to the silica membrane, and unwanted proteins and inhibitors are then washed away. Finally, the ionic conditions are altered to elute nucleic acids from the column in an aqueous solution.

Using a silica column maintains the integrity of your nucleic acids while ensuring the removal of traces of PCR inhibitors. Isohelix spin column-based kits can be used to process large numbers of samples and are available in manual or high-throughput formats.

  • Benefits: Very high purity DNA or RNA
  • Applications: PCR/qPCR, Microarray, Methylation Array, Sanger Sequencing, NGS.

3. Magnetic Beads: High-Throughput Solutions

Magnetic-bead-based methods such as Buccal-Mag (BMG) and Saliva-Mag (GSM) can be used to produce high yields and purities, are fast and reliable for medium to high throughput applications, and extraction is performed in the original sample collection tube, reducing waste and cost. Buccal-Mag kits include magnetic beads with high nucleic acid binding capacities with a large surface area for fast attachment, enabling rapid extraction of intact genomic DNA using a bind/wash/elute workflow.

Magnetic separator racks are available for medium-throughput sample processing, (MR-24) or for high-throughput automated processing mag-bead-based kits can be used with most openly programmable automated DNA extraction and liquid handling systems fitted with magnetic racks and heat blocks.            

  • Benefits: Designed for high throughput & automated applications.
  • Applications: PCR/qPCR, Microarray, Methylation Array, Sanger Sequencing, NGS.
Buccal-Mag (BMG)
Mag-Rack MR-24
Salivalyse (SEK)
Buccalyse (BEK)

4. Direct to PCR: Fast PCR-Ready DNA

Direct-to-PCR kits such as the Salivalyse (SEK) and Buccalyse (BEK) allow users to rapidly prepare DNA from saliva and buccal swabs ready for amplification-based analysis methods. This method bypasses the need for time-consuming DNA extraction and purification steps, thus reducing the overall time and effort required for DNA analysis. These kits are ideal for high throughput and automated workflows, and also reduce consumable waste.

  • Benefits: Rapid workflow, easy to automate, reduced waste
  • Applications: PCR, qPCR, Loop-Mediated Isothermal Amplification (LAMP)


Isohelix specialises in providing nucleic acid collection, stabilisation and extraction kits designed and optimised for saliva and buccal swab samples. DNA and RNA isolated using Isohelix products can be used for genotyping, diagnostics, paternity and heredity analysis, forensics, and population studies. Kits avoid the use of hazardous chemicals, and minimise plastic waste, reusing tubes where possible.

As well as providing products, Isohelix experts are always on hand to offer advice and technical support.

Any enquiries or questions can be emailed to us at info@isohelix.com.

PrecipitationSpin ColumnMagbeadsDirect to PCR
Kit NameBuccalFix PlusBuccalPrep PlusSaliva-Prep 2XtremeXtremeRNABuccal-MagSaliva-MagBuccalyseSalivalyse
Sample TypeBuccal SwabBuccal SwabStabilized SalivaBuccal Swab & SalivaBuccal SwabSalivaBuccal SwabSaliva
Time60 mins60 mins60 mins90 mins90 mins60 mins< 15 mins< 20 mins
Automation RequirementHeating block, CentrifugeMagnetic Rack / PlateHeating Block
Expected PurityA260/280 > 1.7 A260/230 > 1.5A260/280 > 1.7 A260/230 > 1.6A260/280 > 1.75 A260/230 > 1.6A260/280 > 1.8 A260/230 > 2.0A260/280 > 1.9 A260/230 > 1.7A260/280 > 1.8 A260/230 > 1.5PCR-Ready DNA
Expected Yield1 – 5μg DNA>100μg DNA1 – 5μg DNA10 – 50μg Total RNA1 – 5μg DNA15 – 30μg DNA100 – 1000ng100 – 1000ng
ApplicationPCR, qPCR, NGS, ArraysPCR and qPCR
Stabilization Reagent CompatibleBuccalFixDri-Capsules, RapiDriGeneFixDri-Capsules, RapiDri, BuccalFix, GeneFixBuccalFix and GeneFixDri-Capsules, RapiDri, BuccalFixGeneFixDri-Capsules, RapiDriGeneFix

If you want to find out more about how Isohelix can help with your buccal DNA and RNA collection, stabilization and isolation, then CONTACT US

Top 10 Tips for Sampling DNA and RNA using Buccal Swabs

Buccal cell sampling offers a non-invasive alternative to blood collection; it is cheap, painless, and can easily be done by the subject. However, historically the problem with buccal swabs has been low yields.

The Isohelix range of Buccal Swabs has been designed to give increased yields of high-quality buccal cell and genomic DNA and RNA. Together with the Isohelix DNA Isolation and Stabilization kits, high yields of pure, intact DNA can be collected easily and quickly. Isohelix swabs are suitable for both human and veterinary use and offer significant advantages over other swab designs in terms of cell collection efficiency. Their unique swab matrix, combined with a quick-release surface, maximises yields of nucleic acids. DNA from Isohelix buccal swabs can be used for genotyping, diagnostics, paternity and heredity analysis, forensics, and population studies.

1. Maximize yield

Isohelix swabs incorporate a unique matrix that efficiently collects buccal cell samples and features a quick-release surface. Swabs are designed with a small flat head and strong handle, allowing pressure to be applied easily to the inside of the cheek without causing discomfort.

Isohelix products have been specifically developed to integrate all steps from sample collection and stabilisation to processing and isolation. An integrated workflow improves yields, reduces sample losses, and simplifies handling procedures, resulting in maximum yields and DNA/RNA quality. Buccal swab DNA extraction yields from 1 to 10µg can be routinely obtained from adult swab samples.

Isohelix Buccal Swab
BuccaFix DNA Stabilization
RapiDri Swab Kit

2. Stabilize samples

Studies have shown that DNA samples from swabs will degrade rapidly without proper stabilisation, making them useless for downstream applications, so if DNA extraction is to be done longer than 1-2 days after taking a swab sample, then stabilisation is necessary. Depending on your requirements, swabs can be frozen at -20 degrees; alternatively, several stabilization options do not require a cold chain.

If it is important to maintain the structural integrity of your DNA, then BuccalFix DNA stabilization and lysis kits are the ideal solution.

BuccalFix stabilizing buffers completely inhibit all enzymatic and microbial activity that occurs following sampling. As a result, DNA is fully stabilized, and importantly, the structure and integrity of the DNA are fully maintained for further downstream processing applications. DNA can be stored in BuccalFix at Room Temperature for periods in excess of 2 years.

Alternatively, cost-effective 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 sample transport and storage.

Finally, Isohelix Dri-Capsules with SK-1S Swab pack Cat. No: SGC-50 are silica gel capsules that offer an easy-to-use alternative to chemical stabilization for the long-term stability of the buccal DNA on the swab head before isolation. DNA stabilized using Dri-Capsules is stable for 3 years at ambient temperature. 

3. Choose the right nucleic acid extraction kit

Isohelix Buccal Cell Isolation Kits are fully optimised for use with buccal swab samples.

Depending on how your buccal cell sample has been collected and stored and the nature of your downstream analysis, there is a range of extraction kits to choose from.

BuccalFix Plus (BFP) and Buccal-Prep Plus (BPP) use a precipitation method ideal for scaling different sample volumes and is rapid and easy to automate, giving high yields and purity ratios. BuccalFix Plus is designed for use with swabs stored in BuccalFix buffers, whereas Buccal-Prep Plus is suitable for all buccal swab samples.

Silica membrane-based spin columns (e.g., as included in the Xtreme (XME) and Xtreme-RNA (XMR) kits allow the purification of very high-purity DNA or RNA that can be used for sequencing or array-based applications. Using a silica column ensures your nucleic acid is free of traces of PCR inhibitors and maintains the integrity of your DNA or RNA.

Finally, magnetic-bead-based methods such as Buccal-Mag (BMG) can be used to produce high yields and purities, are fast and reliable for medium-high throughout applications, and extraction is performed in the original sample collection tube, reducing waste and cost. Buccal-Mag kits include high binding capacity magnetic beads with a large surface area for fast attachment enabling rapid isolation of intact genomic DNA.

Buccal-Prep Plus
Xtreme DNA
BuccalMag DNA
BuccalFix RNA
Xtreme RNA

4. Isolate RNA

BuccalFix RNA stabilization and lysis kits include a non-toxic buffer that stabilizes RNA and DNA for 2 weeks at room temperature. The new Isohelix Xtreme-RNA kit is a spin-column based RNA purification kit for the rapid preparation of total human, viral, or microbial RNA from stabilized saliva and swab samples. Free from toxic reagents such as phenol, chloroform, & β-mercaptoethanol, Xtreme-RNA has been fully optimised for use with GeneFix GFX & RFX saliva collectors and swabs stored in BuccalFix stabilisation solution. Extracted samples are of high purity (expected A260/280: >1.9), ideal for use in downstream applications such as rt-qPCR and gene expression studies. The kit is also scalable and can accommodate various sample input volumes.

5. Prevent buccal swab contamination

Isohelix swabs are treated with ethylene oxide and routinely tested to ensure they are not contaminated with human DNA. To prevent sample contamination, buccal swab samples should be taken >1h after food or drink, and the sampler must take care not to touch the swab head with their fingers while removing the swab from its packaging.

The RapiDri™ pouch also protects the DNA from cross-contamination by acting as a physical transport pack to protect the DNA sample. The pouch actively repels any accidental wetting from the outside due to its hydrophobic structure, preventing any backward cross-flow contamination.

Isohelix swab collection tubes SK-2S, have a unique cap designed to detach swab heads so that they fall into the tube without further handling, avoiding potential contamination from touching the swab and making it easy to handle at the DNA isolation stage.

SK-2S Buccal Swabs

6. Avoid using hazardous reagents

Collection and stabilization kit should be free from toxic reagents, such as guanidium, for safe use in the field and for shipping and handling. IsohelixTM devices are completely non-hazardous and non-toxic and do not contain ethanol or other solvents.

7. Buccal swabs for other sample types

Isohelix DNA buccal swabs have been designed for buccal applications but have also been used extensively in many other applications, including; skin, hard surface sampling, teeth and gums, tongue, and stools. MS-Mini swabs have reduced dimensions; the width of the swab has been reduced from 8mm to 6mm and employs a slightly different shaft attachment enabling yields similar to larger swabs. These smaller swabs are particularly useful for small mammals, other animals, and invertebrates. They are easy to automate for mouse genotyping.

Isohelix MS-Mini Swabs
Isohelix Transport Packs

8. Safe transport and tracking

One of the key benefits of using buccal swabs rather than blood as a source of nucleic acids is that samples can be collected remotely and mailed for analysis. If a stabilization reagent is included in the collection tube, keeping samples cold is not required, significantly reducing costs and logistical challenges associated with storage and transport. However, collection tubes must be manufactured from robust materials that can withstand the rigours of the mailing process – regulations on leak-proof sample transport specify that collection tubes must withstand defined physical pressures of 95KPA. IsohelixTM TPN-50 transport packs are pressure tested and include secure sealing strips and absorbent material that retains samples leaking from the primary tube within further layers of packaging.

9. Use high quality kits

Kits manufactured to recognised quality standards ensure safe use and high-quality nucleic acid isolation. Isohelix has ISO 9001, and ISO 13485 accreditation, and Isohelix Buccal Swab products are CE/MD and UKCA marked to ensure your samples are collected, transported, and stored securely and can be used to generate excellent results.

10. Reduce the environmental impact

Isohelix reduce the environmental impact of their products as much as possible. Hazardous reagents are avoided, swabs are manufactured using recyclable materials, and with magnetic-bead-based methods such as Buccal-Mag (BMG) extraction is performed in the original sample collection tube, reducing waste.

Buccal Swab Collection Procedure

A video showing the best way to collect a buccal swab samples using the Isohelix buccal swabs. 

Be sure to rub the swab firmly against the inside of cheeks, lower and upper lips for 1 minute to maximize sample yield.

For best results, combine the Isohelix Buccal Swabs with our high quality stabilization options such as BuccalFix liquid buffers and Dri-Capsules.

Buccal Collection

DNA/RNA Buccal Swabs

Full range of high-quality buccal swabs

Buccal Stabilization

Buccal DNA/RNA Stabilization Kits

Stabilization kits optimized for buccal samples

Buccal Extraction

Buccal DNA/RNA Extraction Kits

Isolation/purification kits optimized for buccal samples

If you want to find out more about how Isohelix can help with your buccal DNA and RNA collection, stabilization and isolation, then CONTACT US

Top 10 Tips for Collecting DNA and RNA from Saliva

DNA extraction from saliva has become increasingly popular in recent years. Saliva collection is safe and non-invasive, and saliva collection kits can be mailed to donors for self-collection at home.

DNA or RNA can be isolated from the donor or their oral microbiome and used for many applications, such as investigating infectious diseases, cancer, ancestry, population studies, genotyping, and gene expression studies.

Traditionally, challenges with saliva samples included microbial contamination and lower nucleic acid yields. However, there have been significant improvements in devices to collect saliva, and good yields of high quality can now be extracted[i].

Many collection, stabilization, and isolation methods are available, and variations in sample collection and handling can dramatically affect data quality. This article gives ten tips for sample collection, handling, and storage, to ensure you get the best results from your samples and avoid wasting time and money.

The saliva DNA/RNA collection and extraction workflow has the following stages:

1. Determine the nucleic acid purity required for your analyses

The first step in planning your saliva collection strategy is to determine the purity of nucleic acids you need for your analyses. If it isn’t required, there is no need to spend time and money obtaining high purity DNA or RNA.

Sensitive downstream applications such as rt-qPCR and Next Generation Sequencing (NGS) require high-purity RNA (A260/280 ratio of >1.9) and DNA (A260/280 ratio of ~1.8). However, if a less sensitive technique, such as PCR, is to be used, then a rapid sample preparation method, such as a direct-to-PCR kit, can provide a faster and more cost-effective alternative to a conventional multi-step purification method.

secondary isolation step can be used for DNA for sensitive applications if samples are contaminated due to poor sampling techniques or non-optimized DNA isolation kits.

2. Consider sample yields

As with sample purity, the required yield will depend on your downstream analyses. If you plan to store samples and revisit them for future studies, you may need higher yields of nucleic acids. 

How much DNA is in saliva?

Originally, collection devices were designed to collect 1ml or 2ml saliva, but newer devices eg GFX-03, can collect up to 3ml, if higher yields (300μg+) are required, e.g., for biobanking. Collection devices for larger volumes of saliva include higher concentrations of stabilization buffer to allow for dilution by extra saliva.

3. Make collection simple

Some donors, e.g. children or elderly patients, can find it difficult to produce saliva. Saliva production can be stimulated by gently rubbing the cheeks, or a kit including absorbent swabs to assist with collection, such as GFXA-01 can help.

Instructions for use must be clear, particularly if saliva is to be collected remotely without specialist supervision. Clear, legible markings on GeneFix™ collection tubes indicate the correct volume of saliva to be delivered, and donors need to ensure that they don’t include bubbles when measuring the liquid level in the tube.

Saliva collection process: A simple to attach funnel makes it easy to collect saliva in a tube prefilled with stabilization reagent for transport and storage of the sample.

4. Use transport packs and stabilization reagent

One of the key benefits of using saliva rather than blood as a source of nucleic acids is that samples can be collected remotely and mailed for analysis. If a stabilization reagent is included in the collection tube, keeping samples cold is not required, significantly reducing costs and logistical challenges associated with storage and transport. However, collection tubes must be manufactured from robust materials that can withstand the rigours of the mailing process – regulations on leak-proof sample transport specify that collection tubes must withstand defined physical pressures of 95KPA. 

IsohelixTM TPS-50 transport packs are pressure tested and include secure sealing strips and absorbent material that retains samples leaking from the primary tube within further layers of packaging.

5. Automate analysis for large numbers of samples

If large numbers of samples are to be analysed, automating your nucleic acid extraction workflow will significantly speed up the process and reduce hands-on time. Collection kits are available with 1D and 2D bar codes for tracking large numbers of samples.

If you are using instruments, select a tube that fits your instruments and racking systems. Collection kits designed for high throughput workflows (Isohelix GFXH range) have tubes with locking bases and caps that can be auto de-capped.

6. Prevent sample contamination by avoiding food and drink before collection

To prevent sample contamination it is good practice to ensure that donors do not eat, drink, smoke, or chew gum for at least 30 minutes before providing a sample.

Contaminating microbial DNA in human saliva can alter the detection of variants from whole genome sequencing, but this problem can be reduced by using a bacterial decoy in the alignment process[ii].

7. Preserve samples with collection kits including stabilization buffer

You may want to extract and analyse the nucleic acids from your samples immediately, or you may prefer to process partial samples and preserve the remainder for future testing. There are kits available for processing variable sample volumes, which work well for biobanking, when samples are stored for long periods.

How long can saliva DNA last?

GeneFix saliva collection kits are able to keep DNA stabilized at room temperature for 5 years and RNA stabilized for 2 months. For long term storage, GeneFix kits are also suitable for freezing at temperatures down to -80°C.

8. Avoid using hazardous reagents

Your collection and stabilization kit should be free from toxic reagents such as guanidium, for safe use in the field and for shipping and handling. GeneFixTM devices are completely non-hazardous, non-toxic and chemically unreactive, as well as including a novel funnel design which prevents sample spillages and flow back, even if the tube is knocked over.

9. Preserve microbial profiles for microbiome studies

Stabilization reagents that preserve microbial DNA at room temperature for at least a year are available (eg MFX-01). Stabilization is instant, and the buffer prevents bacterial growth, ensuring the sample accurately reflects the oral microbiome at the moment of sampling. If you are analysing microbial DNA, the devices and buffers you use must be free from any traces of contaminating bacterial DNA.

10. Make sure to use high-quality kits

Use kits manufacture to recognised quality standards for safe use and high-quality nucleic acid isolation. Isohelix has ISO 9001, and ISO 13485 accreditation, and GeneFix products are CE/IVD and UKCA marked to ensure your samples are collected, transported, and stored securely and can be used to generate excellent results.


 [i] Hughes, S.R., Chapleau, R.R. Comparing DNA quantity and quality using saliva collection following food and beverage consumption. BMC Res Notes 12, 165 (2019). https://doi.org/10.1186/s13104-019-4211-6
[ii] Samson, C.A., Whitford, W., Snell, R.G. et al. Contaminating DNA in human saliva alters the detection of variants from whole genome sequencing. Sci Rep 10, 19255 (2020). https://doi.org/10.1038/s41598-020-76022-4

Saliva Collection

DNA/RNA Saliva Collection Kits

Full range of high-quality collection and stabilzation devices for saliva samples

Saliva Extraction

DNA/RNA Saliva Extraction Kits

Wide range of isolation/purification kits optimized for saliva samples

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Fast & Simple Post-PCR Purification

The new DCU PCR Clean-up protocol allows for fast, efficient recovery of low-quality DNA samples and purification of post-PCR samples ready for use in sequencing and other applications using an all-in-one method, without the need for columns, gel extractions or additional solvents.

The Isohelix DCU DNA CleanUp Kit is a valuable, supplementary purification step that takes less than 30 minutes to fully reclaim low purity samples without sacrificing yield, preventing costly retesting and wasting valuable laboratory time.

To highlight the effectiveness of the Isohelix DCU DNA CleanUp Kit for recovery of low-purity samples, five poor-quality DNA samples were purified using the DCU Clean-up protocol and compared before and after processing.

Results show a significant increase in sample purity after using the DCU DNA CleanUp Kit which in downstream processes would have been the difference between a failed and a successful sample.

Unlike most other purification kits, the DCU’s unique and non-hazardous purification chemistry doesn’t require additional solvents such as ethanol or isopropanol, making it safer and more cost effective.

Not only does the DCU not require single use plastic spin columns, but it also uses completely recyclable materials, meaning a much lower impact on the environment.

The DCU kit is also optimized to work with unsatisfactory buccal and saliva DNA samples, but is also compatible with DNA from other sample sources.

In addition, the DCU includes a NEW method to purify samples following PCR without the need for time-consuming gel extractions; removing primers, polymerases, salts, and other impurities from amplified DNA, ready to use in sequencing and other downstream applications.”

Microbiome and it’s Role in Sake Brewing

A recent preprint study (A dark matter in sake brewing: Origin of microbes producing a Kimoto-style fermentation starter,Kohei Ito et alusing Isohelix SK-3S Buccal Swabs and Isohelix BuccalFix Stabilization Buffer has investigated how microbes present in the environment influenced and contributed to the brewing process of the Japanese alcoholic drink, sake.

Microbe Managing

Sake brewing is an inherently microbial-driven process, and requires careful management in order to encourage the proliferation of microbes that contribute to the process while minimising the influence of microbes that may cause spoilage.

On the Surface

Using Isohelix SK-3S Buccal Swabs and BuccalFix DNA Stabilization Buffer, the study collected surface samples from various locations within the brewery, such as floors, brewing materials, and brewing equipment. DNA from the samples were extracted and underwent microbiome analysis using 16S gene sequencing.

Full of Flavour

The study found that microbes present in raw materials and brewing equipment both contributed to the microbial profile of the brewing starter culture used for fermentation. Beneficial microbes from the environment, such as lactic acid bacteria, help to control the brewing process, preventing the growth of undesirable microbes while in turn contributing to the flavour profile of the final product. This study also demonstrates the suitability and efficacy of Isohelix’s swab and stabilization products for use in environmental microbiome investigations.

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Featured Products

Isohelix SK-3S Buccal Swab

SK-3S Is An Easy To Use Buccal Swab That Feature Our Unique Swab Matrix

Isohelix BuccalFix Stabilization Tubes

BuccalFix Fully Stabilizes the Structure & Integrity of DNA for Further Downstream Applications

Isohelix Files US FDA 510k Pre-Submission for GeneFix Saliva Collectors

Isohelix is pleased to announce that it has commenced engagement with the U.S. Food and Drug Administration (FDA) to progress the full regulatory approval of the GeneFix® Saliva DNA Collectors into the USA for their use in molecular and genetic diagnostics.

Featured Products

GeneFix Saliva Collectors

The GFX DNA/RNA Saliva Collectors are easy to use, high quality devices for the collection and stabilization of saliva samples.

Wine Tasting & Microbial Diversity

A study using Isohelix SK-2S Buccal Swabs has discovered that wine tasting has a washing effect on the tongue which causes lower microbiome diversity in professional wine tasters. This also affects how people taste, explaining why people can drink the same bottle of wine and perceive different flavours!

Washing Effect

Using the Isohelix SK-2S Buccal Swabs, the study collected DNA samples from the tongues of both professional wine tasters & non-professionals to compare the microbiome profiles between the two groups. They found that samples collected after wine tasting sessions had a lower microbial diversity than those taken prior to the tasting event. Therefore the study concluded that wine tasting sessions have a washing effect on the tongue, directly affecting microbiome diversity.

Sparkling Wine

As well as discovering the washing effect that wine has on the microbial diversity on the tongue, the study also discovered that this effect is increased when tasting sparkling wines! The comparison of bacterial diversity between samples collected before and after wine tasting, along with the presence of new bacterial taxa, indicated the direct effect of wine on the microbiome of frequent wine tasters, particularly in those tasting sparkling wines.

Putting Taste to the Test

In addition, results from an accompanying questionnaire found that having a long career in wine tasting effected taste perception compared to non-wine tasters, particularly in bitter or acidic food & drink. Results from these data suggest that changes in the microbial profile of the tongue have an effect on how we taste, giving some explanation as to why people can drink the same bottle of wine and perceive different flavours!

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Featured Products

Isohelix SK-2S Buccal Swab

SK-2S Is An Easy To Use Buccal Swab That Includes a Special 2ml Tube For Swab Head Storage

Isohelix Initiates the Full IVD/Medical Device Registration of its Buccal and Saliva Collectors in Switzerland

Isohelix is pleased to announce that it has engaged ‘AJW Technology’ as the Swiss Representative for the ‘GeneFiX DNA/RNA Collectors’ and ‘Isohelix DNA/RNA Buccal Swabs’ to ensure a smooth device registration and supply of these products into the Swiss market.