The goal of this study was to assess the biogeographic origins of African descendant Puerto Ricans and to investigate the potential for Indigenous ancestry within this community.
2ml saliva samples were collected from self-identified African descendant Puerto Ricans residing in Puerto Rico using Isohelix Genefix™ collection kits. GeneFix kits are ideal for collecting samples from study participants in remote communities as they are non-toxic, simple to use, and contain a reagent that stabilizes DNA at room temperature for up to 60 months. After sample collection, kits can be mailed to the lab for DNA extraction.
When the samples reached the lab, DNA was extracted using the Isohelix Genefix Saliva-Prep 2 DNA kit, which uses a precipitation method to isolate high yields of pure DNA. The mitochondrial genomes of these individuals were sequenced, and Y chromosome haplogroups were genotyped for samples donated from males. Summary statistics, comparative analyses, and network analysis were used to assess diversity and variation in haplogroup distribution between the samples and comparative populations.
Results from the mitochondrial haplogroups showed that 66% had African, 5% had European, and 29% had Indigenous American matrilines. The Y chromosome analysis showed that 52% had African, 28% had Western European, 16% had Eurasian, and 4% had Indigenous American patrilines.
This study successfully used Isohelix GeneFix and Saliva-Prep kits to collect and extract DNA from saliva samples from study participants across Puerto Rico. The genomic analysis of these saliva samples have contributed to understanding how African descendants and Indigenous American admixture have shaped present-day communities.
CONTACT US to find out how we can help with your sample collection and DNA extraction needs.
Isohelix will be exhibiting at ESHG 2024 June 1st – 4th in Berlin, at Booth #652. Alongside our New Product launches for DNA Collection and Isolation, we will also have our development team available to discuss any technical matters that might help in your research or diagnostics. Please visit us anytime.
The quality of DNA samples affects the quality of data produced by genomic analyses, and as analysis techniques become more sophisticated, DNA quality becomes even more important. For example, long-read sequencing enables the detection of variants that are often difficult to resolve with short reads, but it requires high-quality, high-molecular-weight input DNA to be successful.
DNA integrity must be maintained from the moment of sample collection to when the sample is processed in the lab to produce the best results. This can be particularly challenging with large genetic studies where DNA samples are collected from donors at home. Samples may be in transit for several days and exposed to wide temperature fluctuations before they reach the lab. DNA samples are particularly vulnerable to degradation in hotter, humid conditions.
The DNA integrity of a sample can be reduced during shipping and storage via several mechanisms :
Enzymes (e.g. nucleases) and chemicals present in samples may degrade DNA
Repeated freeze-thaw cycles, particularly if samples are exposed to low temperatures during shipping, may shear long pieces of DNA into shorter fragments
Microbes present in samples may grow, adding to the proportion of microbial DNA present, and also producing chemicals and enzymes that may damage target DNA.
Microbial DNA present in samples can be a particular problem for microbiome analysis. During transport and storage, some microorganisms in the sample may continue to grow at the expense of others, meaning that the sample is no longer representative of the original community.
Traditionally, freezing was the standard method for DNA preservation, but freezing presents logistical challenges, especially in field settings or when samples need to be transported over long distances. Freezing requires a lot of energy, the cold chain must be maintained, and the weight of the ice used to keep samples frozen adds to shipping costs. If the cold chain fails during shipment or storage and the sample is subjected to freeze-thaw cycles, then this can cause DNA shearing.
Fortunately, alternative techniques are available that can maintain DNA integrity without the need for freezing. In this article, we’ll explore some of the methods of maintaining DNA integrity within saliva, swabs, and stool samples.
DNA Preservation using Desiccation
Desiccation, or drying, is a simple method for DNA preservation that works well for swab samples. Silica gel or similar desiccants are used to absorb moisture from the environment surrounding the DNA sample. By removing water from the sample, desiccation inhibits the activity of nucleases and other enzymes that could degrade DNA. Desiccation is particularly useful for preserving DNA in field settings where access to freezers may be limited.
Isohelix SGC Dri-Capsules are a fast and simple way of stabilizing buccal swab samples. A silica gel capsule is placed in the collection tube sitting on top of the swab shaft, maintaining DNA integrity for up to three years.[i]
Alternatively, the Rapi-Dri swab kit from Isohelix contains an easy to use buccal swab and a quick-drying pouch with a unique matrix that rapidly stabilizes samples by removing water. The pouch then acts as a sealed shipping unit for sample transport. Sample integrity can be maintained even if samples are subjected to particularly hot and humid environments. [ii]
Stabilizing DNA using Chemical Stabilization Reagents
Chemical stabilization reagents prevent DNA degradation within saliva, swabs, and stool samples, even at ambient temperatures. Many commercially available reagents contain guanidine thiocyanate, which denatures proteins and inactivates nucleases. Although these reagents are easy to use, guanidine thiocyanate is hazardous, and so care must be taken when handling reagents containing this chemical.
Isohelix stabilization reagents do not contain hazardous reagents, so donors can use them to collect samples at home and post them to the lab. DNA in samples is immediately stabilized upon contact with the reagent. Depending on the sample type there are a range of different reagents and kit formats.
Stabilization of DNA from Buccal Cells
Swabs are a straightforward method of collecting donor DNA.
For swabs that have been used to collect buccal cells, the collection tubes in the Isohelix BuccalFix DNA Stabilisation and lysis kit contain a buffer that has been specifically formulated to allow long term storage of swab samples at room temperature, enabling the subsequent isolation of high DNA yields and purity from the stabilized swabs.
Saliva samples can also be collected using swabs. To maintain DNA integrity with saliva samples, Isohelix SaliFix™ swab collectors are recommended. The kits contain SaliFix™ SwabCatcher tubes prefilled with SaliFix™ buffer that is optimized to work with saliva swab samples
For larger volumes of saliva, e.g. 1-3ml, GeneFix DNA/RNA collector tubes are pre-filled with a stabilization reagent that can stabilize DNA at room temperature for over five years.
If, however, you are interested in microbial DNA rather than human DNA, Isohelix has developed a new chemistry designed to maximize the collection and preservation of microbial DNA from human saliva samples. The reagent is included in the GeneFix Microbiome DNA Collector Kit, and can be used to provide a snapshot of the microbiome at the point of collection, stabilizing samples for over 12 months.
Stabilization of DNA from Stool Samples
Studies investigating individual gut microbiomes require the purification of microbial DNA from faecal samples. It is important that these samples are collected and stabilised correctly to ensure that the microbiome at the point of collection is analysed, and results are not biased by sample handling after collection.
Isohelix swabs, combined with StoolFix stabilization buffer, can be effectively used to collect and preserve microbial DNA from faecal samples. Samples are collected by brushing the stool lightly with each face of the swab and are stable at room temperature for up to two months.
Isohelix offers a range of DNA and RNA stabilization solutions that maintain nucleic acid integrity at room temperature for buccal cells, saliva, and stool samples. All stabilization products are optimised for use with Isohelix DNA extraction kits.
High molecular weight (HMW) DNA is important for applications that require the use of very long DNA molecules, such as long read sequencing. Long-read sequencing enables the detection of variants that are often difficult to resolve with short reads, such as structural variants, complex rearrangements, and variants in highly repetitive regions.
In order to fully take advantage of long-read sequencing, high quality, high molecular weight (HMW) input DNA is required. Traditional DNA extraction methods often result in fragmented DNA samples. If these shorter fragments are not removed, the average length of sequencing reads, and subsequent effectiveness of long-read analyses are reduced.
To address this requirement, Isohelix has developed the Mag-Filter HMW Clean Up Kit, which employs magnetic beads alongside specialized size selection chemistry to isolate high molecular weight DNA fragments from extracted DNA samples, while removing smaller DNA fragments and other contaminants from samples.
Samples prepared using the Mag-Filter HMW Clean Up Kit are perfect for NGS and long-read sequencing applications.
HMW DNA Cleanup and Size Selection
The Mag-Filter HMW kit removes low molecular weight DNA while safeguarding high molecular weight DNA integrity. High purity HWM DNA is achieved through DNA sample cleanup and size selection.
Saliva DNA samples spiked with DNA ladder and processed using the Mag-Filter Clean-Up Kit demonstrate effective removal of small DNA fragments.
HMW DNA from saliva and buccal swab samples
The Mag-Filter HMW kit is optimized for use with extracted saliva samples but can be used on DNA from a variety of sample sources, such as buccal swabs. It can also be used to increase sample purity, allowing the use of valuable samples with initially low purity that might otherwise fail quality control checks.
GFX-01
Simple to Use HMW DNA Preparation
The kit is simple to use and can cleanup samples for library prep in less than one hour. All kit components are non-hazardous, and gel separation is not required. The kit protocol can also be easily adjusted to accommodate varying sample volumes; the protocol is fully scalable, from small manually processed sample batches up to plate-based high throughput automation.
With advanced genomic analysis applications becoming routine in many labs, the need for high purity, high quality DNA and RNA samples collected using non-invasive sampling methods, such as saliva, is becoming increasingly important.
Nucleic acid extraction from saliva can be challenging due to the presence of contaminants and inhibitors that can interfere with downstream applications. However, in recent years significant improvements have been made with devices to collect, store, and process saliva to extract DNA and RNA. Good yields of high-quality nucleic acids can now be extracted for use in sensitive downstream applications.
Variations in sample collection and handling can dramatically affect data quality. This article explains how to get the best from your saliva samples by maximizing the purity and yields of DNA and RNA with information on:
Preventing sample contamination
Saliva collection from donors
Nucleic acid stabilization
Safely shipping saliva samples
Choosing your DNA/RNA isolation kit
Isolating high molecular weight (HMW) DNA from saliva
Cleaning up your DNA after an initial extraction
Preventing sample contamination
The main sources of human DNA in saliva are epithelial cells and leukocytes. Saliva samples may also include bacterial DNA, allowing for DNA extraction and analysis of the oral microbiome. To prevent contamination from external nucleic acids and other factors that may interfere with the purity of samples, it is important to ensure that donors do not eat, drink, smoke, or chew gum for at least 30 minutes before providing a sample.
Saliva collection from donors
For collection by donors at home without medical supervision, kits such as the GeneFix™ Saliva DNA/RNA range include a simple to attach funnel that makes it easy to collect saliva in a tube prefilled with stabilization reagent for transport and storage of the sample.
GFX-03
Traditionally, collection devices were designed to collect 1ml or 2ml saliva, but newer devices such as the GFX-03, can collect up to 3ml if higher yields (300μg+) are required, e.g., for biobanking. For maximum yields, donors must ensure that they don’t include bubbles when measuring the liquid level in the tube as this can result in overfilling, or not collecting enough saliva.
Some donors, e.g., children or elderly patients, may find it difficult to produce saliva. If this is the case, saliva production can be stimulated by gently rubbing the cheeks or using a kit such as the SaliFix™ Saliva Swab DNA Collection kit which contains absorbent swabs for easy collection of smaller volumes of saliva.
Nucleic acid stabilization
One of the key benefits of using saliva as a source of nucleic acids is that samples can be collected remotely and mailed for analysis. Stabilization reagents protect sample integrity by stabilizing DNA and RNA and preventing microbial growth. GeneFix saliva collection kit tubes are prefilled with a non-hazardous guanidium-free stabilization reagent that stabilizes DNA at room temperature for 5 years and RNA for 2 months. Samples can be shipped and stored at room temperature, removing the costs and logistical challenges associated with cold chain storage and transport. For longer term storage GeneFix kits are also suitable for freezing at temperatures down to -80°C.
Safely shipping saliva samples
Shipping packaging and collection tubes must be manufactured from robust materials that can withstand the rigors of the mailing process to ensure your saliva samples arrive at the lab intact. Isohelix sample collection tubes are pressure tested to 95kPa, and Isohelix™ TPS-50 transport packs are pressure-tested and adhere to UN3373 packaging standards – including secure sealing strips and absorbent material that retains samples leaking from the primary tube within further layers of packaging, preventing sample cross-contamination.
Choosing your DNA/RNA Isolation Kit
Precipitation methods of DNA isolation such as the GeneFix™ Saliva-Prep Kit, enable the rapid and efficient extraction of high yields of high molecular weight DNA and can be easily scaled to accommodate different sample volumes. As these kits don’t require additional solvents, columns, or filtration, workflows are easy to automate, with fast handling times and reduced sample steps. The unique precipitation chemistry maximizes recovery of high molecular weight DNA, while minimizing co-precipitation of RNA and degraded low-weight DNA.
Alternatively, silica membrane-based spin columns, such as those included in the Xtreme (XME) and Xtreme-RNA (XMR) kits, allow the purification of very high-purity, high molecular weight DNA or RNA for demanding downstream applications such as next generation sequencing or microarrays.
Isolating high molecular weight (HMW) DNA from saliva
Gentle handling is key to preserving the integrity of your DNA samples and isolating HMW DNA for more demanding applications such as whole genome sequencing (WGS) or microarrays. To prevent shearing, use wide bore tips and pipette slowly, avoiding vortexing or shaking tubes.
Cleaning up your DNA after initial extraction
If, despite your best efforts, your extracted DNA is not of a sufficient concentration or purity for your downstream applications, a DNA clean up kit can be used. The Isohelix DNA clean up kit uses a simple 30 minute protocol to reclaim a “failed” sample, preserving your DNA yields and giving High Purity DNA with 260/280 ratios to 1.8.
Nanodrop Scan Before & After DCU CleanUp
Summary
By following the advice given in this article, high yields of high purity DNA can be extracted from saliva for use in a number of sensitive downstream applications.
GeneFix offers superior performance and high yields when compared with alternative collectors :
2023 was a busy year for Isohelix™ and our customers. In this blog, we present examples of how studies reported in peer-reviewed publications used Isohelix™ products to solve their sampling challenges.
We’ve highlighted four studies where Isohelix™ technologies were used to make groundbreaking discoveries in the world of biomedical research. In each of these studies, Isohelix™ sample collection products were used to collect DNA samples which were then analyzed using highly sensitive genetic analyses such as single nucleotide polymorphism (SNP) genotyping, genome wide association studies (GWAS), and next generation sequencing (NGS) analyses.
GeneFix™ Saliva Collection Devices are used to collect samples for a large population-based study in Australia
The Hong Kong Genome Project uses GeneFix™ Saliva Collection Devices to collect saliva samples and the RapiDri Pouch kit for buccal swabs.
Isohelix™ saliva collection kits are used to collect saliva from a large cohort of children in the USA
Isohelix™ Buccal Swabs are used to swab surfaces to collect DNA from bacterial communities in subway facilities
STUDY 1 : Preliminary results from the Australian Genetics of Bipolar Disorder Study: A nation-wide cohort[i]
The Australian Genetics of Bipolar Disorder (BD) Study investigates a nation-wide cohort of adults living with bipolar disorder. The study aims to identify genetic risk factors influencing BD, medication treatment response, and adverse drug reactions (ADRs). Key goals are to characterize the patterns and costs of healthcare usage in BD and to examine the relationship between genetic risk and symptom severity. This article describes the study design and sample characteristics and summarises data collected on BD symptoms, psychiatric comorbidities, and medication use.
A significant strength of the study is its sample size; it’s the largest population-based study of adults living with BD in Australia. Study participants are based throughout Australia, with many living in rural or remote communities. Saliva samples are collected using Isohelix™GeneFix™GFX-02 2 mL Saliva Collection Devices which stabilize DNA and RNA at room temperature for up to 60 months, then mailed to QIMR Berghofer via prepaid Australia Post. Following DNA extraction, genotyping is conducted using the Illumina Global Screening Array V.2.0.
GFX-02
The study demonstrates that Australians with BD are motivated to participate in genetically focused research studies if given the opportunity and provided with simple and effective sample donation protocols. GeneFix™ Saliva Collectors are ideal for this type of project as their reagents are non-toxic, and the collectors are easy to use as they have a unique funnel design that prevents buffer spillages or flow-back.
The results highlight the high rate of comorbidities and adverse drug reactions among adults living with bipolar disorder in the general Australian population and pave the way for future genomic analyses to identify genetic variants influencing pharmacotherapy treatment response and side effects.
STUDY 2 : The Hong Kong genome project: building genome sequencing capacity and capability for advancing genomic science in Hong Kong[ii]
The Hong Kong Genome Project (HKGP) is the first large-scale genome sequencing (GS) project in the Hong Kong Special Administrative Region. The project aims to sequence 45,000-50,000 genomes in five years, providing participants with diagnoses and personalized treatments and driving the application and integration of genomic medicine into routine clinical care.
For each participant, 6 mL of blood is obtained and stored in two 3-mL EDTA-containing anticoagulation tubes. For buccal swabs, the RapiDri Pouch kit is used to collect cells from inside the cheek, and saliva samples are collected using the GeneFix™ Saliva DNA Collection and Stabilization Kit. DNA from saliva samples has been shown to be comparable to DNA from blood in many downstream applications[iii] and DNA from saliva samples collected using GeneFix™ kits is stable at room temperature for over five years, avoiding the high cost and logistical challenges of cold chain transport.
RapiDri Swab
This paper describes the establishment of a robust genome sequencing workflow for the project. The laboratory analyses approximately 350-500 samples per week. During the first 24 months of the project, 12,937 participants and their family members were recruited, and the laboratory has been adapted to facilitate the latest technologies, e.g., long read sequencing and multi-omics.
STUDY 3 : Avoidant Restrictive Food Intake Disorder Genes and Environment (ARFID-GEN): study protocol[iv]
This paper describes the Avoidant Restrictive Food Intake Disorder – Genes and Environment study (ARFID-GEN), which is designed to expand the discovery of genetic and environmental contributions to ARFID risk. The goal of the study is to deliver actionable findings that can be transformed into clinically meaningful insights. This will be achieved by advancing the understanding of the genetic architecture of ARFID relative to other eating disorders and other psychiatric, neurodevelopmental, and metabolic disorders and traits.
3,000 children and adults with ARFID from the United States are included in the study. Efficient online phenotyping of children and adults with ARFID including environmental exposures is combined with at-home saliva sampling, and samples are genotyped to analyze single nucleotide polymorphism (SNP) – based heritability, genome wide association studies, genetic correlations (rgs), and copy number variations (CNVs).
SK-2S
Eligible children are asked to complete a questionnaire on their symptoms and provide a saliva sample using Isohelix™ saliva collection kits, which are non-toxic and easy for children to use at home as they have a unique funnel design that prevents buffer spillages or flow-back. The kits are provided with return packaging for mailing to the lab for analysis.
The aim is to identify genome-wide significant loci for ARFID, informative rgs, implicated CNVs, and environmental precipitants. Ultimately, the work will hopefully yield information on critical biological pathways that may point toward drug discovery or repurposing that could aid in reversing the tenacity and lethality of these illnesses.
STUDY 4 : Investigation of Surface Bacterial Diversities and Compositions in the Global Subway Facilities[v]
This study explored surface bacterial communities in four global subway facilities using 16S rRNA gene amplicon sequencing of DNA extracted from swabs.
Isohelix™ Buccal swabs can be used to collect surface DNA from environmental sources as well as from buccal samples. Indoor surface samples for Busan City were collected twice per site using Isohelix™ DNA/RNA buccal swabs (SK-2S, Isohelix) moistened with sterile 1 mL of phosphate-buffered saline. Samples were collected for 3 min from each site in a 100 cm2 area as recommended in a previous study[vi]. The microbiome was analysed based on the 16S rRNA gene.
SK-2S
Samples collected in Korea were analysed alongside comparable samples obtained from previous studies in Boston[vii], Moscow[viii], and Mexico City[ix].
The study showed that the surface bacterial diversities and taxonomic profiling of the studied subway station surface environments were similar. Most of the predominant phylotypes were Gram-positive microorganisms that probably originated from human and outdoor sources.
Summary
The above studies are just a few of the projects that have used Isohelix™ sample collection and stabilization products to collect samples from research study participants or the environment, for genomic analysis.
Tell us about how Isohelix™ products helped your study by emailing us at info@isohelix.com!
References
[i] Lind, Penelope A., et al. “Preliminary results from the Australian Genetics of Bipolar Disorder Study: A nation-wide cohort.” Australian & New Zealand Journal of Psychiatry 57.11 (2023): 1428-1442. https://journals.sagepub.com/doi/epub/10.1177/00048674231195571
[ii] Chu, Annie TW, et al. “The Hong Kong genome project: building genome sequencing capacity and capability for advancing genomic science in Hong Kong.” J Transl Genet Genom 2023;7:196-212 https://www.oaepublish.com/articles/jtgg.2023.22
[v] Kim J, Bae S, Park S, Shukla SK, Yoo K. Investigation of Surface Bacterial Diversities and Compositions in the Global Subway Facilities. Atmosphere. 2023; 14(1):140. https://doi.org/10.3390/atmos14010140
[vi] Jansson, L.; Akel, Y.; Eriksson, R.; Lavander, M.; Hedman, J. Impact of swab material on microbial surface sampling. J. Microbiol. Methods 2020
[vii] Hsu, T.; Joice, R.; Vallarino, J.; Abu-Ali, G.; Hartmann, E.M.; Shafquat, A.; DuLong, C.; Baranowski, C.; Gevers, D.; Green, J.L. Urban transit system microbial communities differ by surface type and interaction with humans and the environment. Msystems 2016, 1, e00018-16
[viii] Klimenko, N.S.; Tyakht, A.V.; Toshchakov, S.V.; Shevchenko, M.A.; Korzhenkov, A.A.; Afshinnekoo, E.; Mason, C.E.; Alexeev, D.G. Co-occurrence patterns of bacteria within microbiome of Moscow subway. Comput. Struct. Biotechnol. J. 2020, 18, 314–322
[ix] Hernández, A.M.; Vargas-Robles, D.; Alcaraz, L.D.; Peimbert, M. Station and train surface microbiomes of Mexico City’s metro (subway/underground). Sci. Rep.2020,10, 8798
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.
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 SwabCatcher collection tubes pre-filled with 0.5ml buffer
BuccalFix BFX/L/05/50 50 x BuccalFix SwabCatcher 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.
Isohelix will be present at the upcoming Festival of Genomics & Biodata in London, Booth #79, from 24th to the 25th of January. Join us to explore our latest products in Sample Collection, Stabilization, and Purification technologies.
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 :
GeneFix RFX– stabilizes RNA in saliva samples for up to two months at room temperature.
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
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