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).

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.

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)

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.

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.

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.

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. Isohelix^TM 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.

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

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 (A_260/280 ratio of >1.9) and DNA (A_260/280 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.

A 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. GeneFix^TM 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.

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