10 Top Tips for Working With RNA

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


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


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 


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.


[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