Isohelix launches the SaliFix™ Saliva Swab DNA Collection Kit

Isohelix launches the SaliFix™ Saliva Swab DNA Collection Kit

Increasing numbers of saliva samples are being collected remotely from study participants for use in genetic testing, so the need for safe, simple and easy to use sample collection, stabilization and transportation is growing rapidly.

Studies have shown that DNA samples from swabs can degrade rapidly without the correct stabilization. SaliFix™ Collectors contain 1ml of preservation reagent that is designed to maintains DNA integrity and yields whilst preventing bacterial growth long term, yet provides easy and easy device for Saliva DNA collection, stabilization, and transport.

SaliFix™ Collectors are specifically designed for collecting Saliva samples in various environment including at home, in clinic or remotely. The foam swabs effectively maximizes saliva collection, even from donors who may find it difficult to produce saliva.

The SaliFix™ kit also includes the SwabCatcher format for improved Sample Handling

The NEW SwabCatcher tube simplifies sample processing and cuts down on cross-contamination as the cap holds the swab tightly after screwing the cap onto the tube. This removed the tedious process choice of having to cut the swab tip and or manually using tweezers to remove the swab prior to processing. This makes the tube ideal for high-throughput processing using automated instruments, and further the tubes are barcoded for easy sample tracking.

SwabCatcher tubes are leakproof and fully transport tested to 95kPa to allow mailing and storage, and the reagents are non-hazardous.

Agarose gel analyzed by electrophoresis. Saliva DNA isolated from SaliFixTM collection devices using the Isohelix Saliva-Prep-2 kit (GSPN-50), shows high quality DNA with no degradation.

DNA collected using SaliFix™ collectors is of high yield and purity, and suitable for all downstream analyses.

Saliva Swab DNA Collection Kit

  • All-in-one saliva DNA collection, stabilization, and transport device.
  • Uses the NEW Swab-Catcher for easy handling and automated-processing.
  • Simple collection at home, in clinic. a viable alternative to blood collection.
  • Instant stabilization maximises DNA yields allowing RT shipping and storage in a non-hazardous format.
  • Optimized for extraction using Isohelix DNA extraction and other kits.

Product Information

SFX/LS/1/50   SaliFix™ saliva collection tubes prefilled with 1ml SaliFix™ buffer and individually wrapped, foam saliva swabs.

Pack of 50 devices

Isohelix launches the ‘SwabCatcher’ Swab DNA/RNA Collection Kit

NEW SwabCatcher tubes are used alongside RapiDri DNA and RNA Swab Kits designed to simplify the swabs transition through sample processing. The Tubes are also available pre-filled with BuccaLyse DNA and RNA stabilization buffer for room temperature shipping.

  • No more Swab retrieval issues
  • Automation, HT and Manual compatible
  • Barcoded for traceability
  • CE-IVD Certified
  • Fully transport tested to 95kPa

Product Information

SwabCapture             L/00/50                        50 x SwabCatcher 5ml collection tubes and caps(empty)

RapiDri                        RD-01                                200 x RapiDri swabs SwabCatcher with drying pouch

BuccaLyse                  BEK/L/05/50                50 x BuccaLyse collection tubes pre-filled with 0.5ml buffer

BuccalFix                    BFX/L/05/50                50 x BuccalFix collection tubes pre-filled with 0.5ml buffer

At Isohelix we use our extensive knowledge in product design, regulatory, safety & commercial markets to design products that make it easy for our customers to collect high quality nucleic acid samples from saliva, buccal cells, and stool samples.

10 Top Tips for Working With RNA

Obtaining high-quality RNA is the first step for sensitive downstream applications such as RT-PCR, digital PCR, and RNA-seq. However, this can be tricky, as RNA is fragile and susceptible to both physical degradation and digestion by ubiquitous ribonuclease (RNAse) enzymes.

In this blog, we give you ten top tips to ensure success when working with RNA to help you get the best results from your samples.

1. Use an area of the lab dedicated to working with RNA​

RNases are ubiquitous in the environment and maintain their activity even after autoclaving, so make sure to decontaminate your reagents and equipment before using them. Set aside a separate area for working with RNA away from other work, and use separate pipettes, tips, and other consumables. Ideally, use a UV decontamination cabinet or laminar flow hood to prevent contamination from airborne micro-organisms. Your skin secretes RNases, so wear clean gloves and a clean lab coat and face mask while handling samples.

2. Decontaminate work surfaces and equipment​

Common disinfectants such as 70% Isopropanol may be insufficient for inactivating RNases. Wipe down all work surfaces, and clean pipettes with a 10% household bleach solution (or a commercially available RNAse decontamination solution), followed by wiping down with RNAse-free/DEPC-treated H2O to inactivate potential RNases. Use filtered pipette tips for liquid handling to prevent aerosols and the cross-contamination of samples. Where possible, use sterile, disposable plasticware . If you must use non-disposable plasticware, treat it with 0.1 M NaOH/1 mM EDTA and RNase-free water. Always use molecular biology grade consumables and reagents certified as RNAse/Nuclease free, as lower quality reagents may be contaminated with RNases. ISOHELIX PRODUCTS ARE PRODUCED IN A CLEAN ENVIRONMENT AND ARE CERTIFIED RNAse-FREE

3. Use an RNA stabilization reagent​

RNA collection and analysis from saliva and buccal swab samples is especially challenging, given the high quantities of enzymes in the oral cavity. To overcome this, Isohelix has developed unique, non-toxic RNA stabilization buffers and included them in RNA collection kits, immediately preserving RNA from the moment of collection :

Using an RNA stabilization reagent preserves the integrity of the RNA in your saliva and buccal swab samples during room-temperature storage and shipping.

4. Handle samples carefully

Many RNA extraction kits include guanidium isothiocyanate (GITC) during the lysis stage to denature proteins. However, GITC is a toxic reagent that can potentially react with bleach (sodium hypochlorite) to generate toxic gases.
Xtreme-RNA is free from toxic reagents such as phenol, chloroform, β-mercaptoethanol, & guanidine salts, so it is much safer to handle in the lab.

5. Choose an RNA Extraction Kit optimized for your sample type

Choose the most appropriate kit for your samples and downstream applications. For example, the Isohelix Xtreme-RNA kit is a spin column based RNA purification kit for the swift, simple preparation of total human, viral, or microbial RNA, optimized for extraction from saliva and swab samples. Extracted samples are of high purity (expected A260/280: >1.9), so they are ideal for use in downstream applications such as rt-qPCR, RNAseq and microRNA-seq. The kit is scalable and can accommodate various sample input volumes.

6. Avoid toxic reagents

Many RNA extraction kits include guanidium isothiocyanate (GITC) during the lysis stage to denature proteins. However, GITC is a toxic reagent that can potentially react with bleach (sodium hypochlorite) to generate toxic gases.
Xtreme-RNA is free from toxic reagents such as phenol, chloroform, β-mercaptoethanol, & guanidine salts, so it is much safer to handle in the lab.

7. Be aware of potential gDNA contamination

The similarity in physical and chemical properties of DNA and RNA makes gDNA contamination of isolated RNA a common problem, and non-specific amplification due to gDNA contamination of samples can lead to overestimates of transcript levels using RT-PCR gene expression analysis. Many RT-PCR assays can be designed to be gDNA insensitive using exon-spanning primers, but this is not always possible. If required, DNase digestion can be performed following the final elution of RNA using most commercially available DNAse kits.

8. Accurately quantify your RNA

Before proceeding with downstream applications, assess the quality and quantity of extracted RNA. Use spectrophotometry (e.g., NanoDrop) for concentration determination and electrophoresis or capillary electrophoresis (e.g., Agilent Bioanalyzer) to assess RNA integrity.

9. Work swiftly

RNA in inadequately maintained oral samples can be degraded by intracellular nucleases. Extract RNA as quickly as possible after taking samples, and once begun, work as fast as you can to complete sample purification. Purified samples should always be kept chilled or on ice.

10. Always freeze extracted RNA

Even trace amounts of RNase can degrade RNA in non-frozen samples. Store extracted RNA short-term at -20°C (weeks to months), but in the longer term, store RNA at -80°C (years) as the low temperature will inhibit enzyme activity, preventing sample degradation.

More Info

To find out more about Isohelix’ High Quality Products for Collecting, Stabilizing and Extracting RNA, click here RNA Products | Isohelix

Genomic Analysis with DNA from Saliva instead of blood

Blood samples have traditionally been considered the gold standard DNA source for genomic analysis, but obtaining blood samples is a painful, invasive, and costly procedure that must be performed by qualified personnel.

By contrast, saliva collection is safe and non-invasive, and saliva collection kits can be mailed to donors for self-collection at home. As well as the sample source, how the sample is collected, stabilized, stored, and purified is critical to the quality and quantity of DNA that can be extracted.

Previously, challenges with DNA extracted from saliva 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 DNA can now be extracted

This article discusses the use of DNA extracted from saliva for a wide range of downstream applications

Saliva is much easier and cheaper to collect and transport than blood

Participants in large scale epidemiological studies may be reluctant to provide blood samples due to the need to travel to a health center or the painful nature of giving a sample. Recruitment and compliance rates are much higher when saliva samples are used, as saliva collection is painless and can done at home.

Traditionally blood samples were shipped and stored at -20 degrees before processing, but DNA stabilization reagents are now available for collecting, transporting, and storing whole blood. However, these reagents often contain hazardous substances such as guanidium, so care must be taken while handling them.

Saliva samples require no pre-processing and are commonly collected using non-hazardous reagents, so they can be sent via mail. Saliva collection kits can stabilize DNA at room temperature for over five years, avoiding the high cost and logistical challenges of cold chain transport.

Which cells provide the DNA extracted from saliva and blood samples?

High quality DNA extracted from peripheral blood originates from leukocytes.

Human DNA in saliva originates from epithelial cells or or leukocytes. Unlike DNA derived from blood, saliva samples can include bacterial DNA, allowing DNA extraction from the oral microbiome. If required, qPCR assays can be used to quantify either human or bacterial DNA and measure and normalize saliva DNA samples.

Isohelix estimated the relative quantities of human and microbial DNA found in samples stabilized using their GeneFix reagent, which were then stored at room temperature prior to DNA extraction [i]. Approximately 7.4% of DNA in the samples was found to be of microbial origin. In a follow up study {ii}, GeneFix saliva stability samples that had been stored for up to 48 months at room temperature were tested, and no change in the proportion of bacterial DNA in samples over the stability period was found, demonstrating the GeneFix collectors fully stabilize saliva samples, preserve DNA and prevent microbial growth.

DNA yield from saliva samples is comparable to blood for downstream applications

Figure 1 shows the DNA concentration, yield and purity from 0.5ml raw saliva isolated using the GeneFix Saliva-Prep2 DNA Kit [iii}

In a study comparing DNA extracted from saliva and blood, Looi et al [iv] (2012), the DNA yield from saliva of 7.8 µg/0.5 mL from a manual purification method was comparable to the DNA yield from blood using a salt precipitation method (7.4 µg/0.5 mL blood sample). DNA extracted from saliva and blood were both high purity (A260/280 > 1.70).

Downstream analysis using DNA from saliva

The quality and quantity of DNA required for a study depend on the downstream analysis that will be performed. Below are some recent studies demonstrating the successful use of DNA from saliva extracted using Isohelix kits.  For a more comprehensive list of publications please click here

Whole DNA on 2.2% Agarose FlashGel with 1kb markers

PCR and RT-PCR

DNA extracted from saliva is routinely used for PCR and qRT-PCR and became particularly important during the Covid 19 pandemic.

Some key examples are :

  • Potocka, Natalia, et al. “Association of ACTN3 Polymorphism with Body Somatotype and Cardiorespiratory Fitness in Young Healthy Adults.” International journal of environmental research and public health 16.9 (2019): 1489. https://doi.org/10.3390/ijerph16091489
  • Carter, Nikki, et al. “A novel automated SARS-CoV-2 saliva PCR test protects a global asymptomatic workforce.” Scientific Reports 11.1 (2021): 1-6. https://doi.org/10.1038/s41598-021-92070-w

Genotyping

The accuracy of genotyping with saliva-derived DNA has been reported as comparable to DNA derived from blood [v,vi]

There are many examples in the literature where saliva samples have been used for genotyping :

  • Campos, Adrian I., et al. “Impact of CYP2C19 metaboliser status on SSRI response: a retrospective study of 9500 participants of the Australian Genetics of Depression Study.” The Pharmacogenomics Journal 22.2 (2022): 130-135. https://doi.org/10.1038/s41397-022-00267-7
  • Potocka, Natalia, et al. “Effects of the Trp64Arg Polymorphism in the ADRB3 Gene on Body Composition, Cardiorespiratory Fitness, and Physical Activity in Healthy Adults.” Genes8 (2023): 1541. https://doi.org/10.3390/genes14081541

Next Generation Sequencing

Although the use of blood-derived DNA is the current standard for WGS, Wall et al [vii] reported no differences in sequencing quality or variant call error rate between blood and saliva samples for both whole exome sequencing (WES) and WGS.

There are several examples in the literature of DNA from saliva being used for NGS :

  • Hansen, Marcus Høy, and Charlotte Guldborg Nyvold. “Replicate whole-genome next-generation sequencing data derived from Caucasian donor saliva samples.” Data in Brief 38 (2021): 107349. https://doi.org/10.1016/j.dib.2021.107349
  • Gopinath, Divya, et al. “Salivary bacterial shifts in oral leukoplakia resemble the dysbiotic oral cancer bacteriome.” Journal of oral microbiology 13.1 (2021): 1857998. https://doi.org/10.1080/20002297.2020.1857998

Methylation-based studies

There are some challenges with using DNA extracted from saliva for methylation-based analyses, e.g., cellular heterogeneity in salivary DNA, saliva samples can include bacterial DNA, and saliva samples are fragmented making long-range PCR or long read sequencing difficult [viii].

However, there are several examples in the literature of DNA from saliva being used for methylation studies, e.g., :

  • Ruffell, Simon GD, et al. “Ceremonial Ayahuasca in Amazonian Retreats—Mental Health and Epigenetic Outcomes From a Six-Month Naturalistic Study.” Frontiers in Psychiatry 12 (2021): 898. https://doi.org/10.3389/fpsyt.2021.687615
  • Zhang, Jun, et al. “Exploring Effect of Postdischarge Developmental Support Program on Preterm Infant Neurodevelopment and BDNF Gene DNA Methylation.” Advances in Neonatal Care (2022): 10-1097i 

Summary

Saliva collection is a robust, non-invasive and low cost method of gathering samples that is particularly useful for large-scale epidemiological and other genetic studies where recruitment rates are much higher when saliva samples are used, rather than blood.
Saliva DNA can be used for a wide range of analyses, including PCR, RTPCR, Genotyping Arrays, and Next Generation Sequencing.

References

[i] “Existing Human and Bacterial DNA Content in Human Saliva Samples,”  March 2023

[ii] “Does Bacterial Growth Occur in Isohelix GeneFixTM stabilized Saliva Samples”  Tech Note January 2020

[iii] “New Saliva-Prep2 Isolates High Purity Genomic DNA using GeneFixTM Collectors”, Isoghelix Application Note GSPN: January 2019

[iv] Looi ML, Zakaria H, Osman J, Jamal R. Quantity and quality assessment of DNA extracted from saliva and blood. Clin Lab. 2012;58(3-4):307-12. PMID: 22582505

[v] Genetic epidemiology : Ng DP, Koh D, Choo S, Chia KS. Saliva as a viable alternative source of human genomic DNA in genetic epidemiology. Clin Chim Acta. 2006;367(1–2):81–5.

[vi] Gudiseva HV, Hansen M, Gutierrez L, Collins DW, He J, Verkuil LD, et al. Saliva DNA quality and genotyping efficiency in a predominantly elderly population. BMC Med. Genom. [Internet]. (2016). 2016 Apr 7

[vii] Wall JD, Tang LF, Zerbe B, Kvale MN, Kwok PY, Schaefer C, et al. Estimating genotype error rates from high-coverage next-generation sequence data. Genome Res. 2014;24(11):1734–9

[viii] Nishitani S, Parets SE, Haas BW, Smith AK. DNA methylation analysis from saliva samples for epidemiological studies. Epigenetics. 2018;13(4):352-362. doi: 10.1080/15592294.2018.1461295. Epub 2018 Aug 1. PMID: 29912612; PMCID: PMC6140812

If you want to find out more about how Isohelix can help with stabilizing and extracting your saliva samples, then CONTACT US

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)
MFX-01
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.

References

[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)

Summary

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
Dri-Capsules
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