New EXTRAClean technology enables high-purity RNA extraction

Exosomes play a key role in communication across different systems in the human body. Various cell types, including immune cells, neurons, cardiomyocytes, stem cells, and others, commonly secrete them.¹

They are present in virtually all bodily fluids ranging from plasma to serum, urine, saliva, and beyond. They can carry DNA, RNA, lipids, proteins, and metabolites according to their originating cell. ^2,3

Beyond their roles in carrying out regular physiological functions, exosomes are implicated in disease progression and the pathological development of several conditions, including heart failure, neurodegenerative diseases, liver diseases, and cancer. ^{1, 4-7}

As RNA is more indicative of disease progression and transmission, sequencing exosomal RNA delivers a deep understanding of the underlying disease.^8.9 Exosomal mRNA, miRNA, circRNA, and lncRNA, known to regulate cellular biological processes, are additionally known for their role in the progression of a range of conditions, including sarcomas, pancreatic and colon cancers, gastric tumors, prostate cancer, and lung cancer.⁹

Furthermore, exosomes contain an exceedingly small quantity of RNA, reflected in the obtained RNA yields. Sequencing such low amounts of RNA is difficult because of the limited RNA quantity and the high purity level necessary for optimal sequencing results.

In the present research, Norgen Biotek presents its EXTRAClean technology, which delivers exceedingly high-quality and pure RNA from plasma exosomes to considerably improve NGS performance.

Materials and methods

Plasma preparation and RNA isolation

Blood samples were gathered from three donors to prepare plasma in EDTA tubes (BD, Cat# 366643). Plasma was separated and processed to remove cells via centrifugation at 2500xg for 10 minutes. Cell-free plasma was moved to a fresh tube and stored at -80 °C for later use.

Intact Exosomes were purified from 0.2 mL plasma utilizing Plasma/Serum Exosome Purification Mini Kit (Norgen Biotek Corp., Cat# 57400). After extraction, exosomes underwent further processing to extract exosomal RNA utilizing Exosomal RNA Isolation Kit (Norgen Biotek Corp., Cat# 58000) and EXTRAClean Exosomal RNA Isolation Kit (Norgen Biotek Corp., Cat# 72800)

Image Credit: Norgen Biotek Corp.

Small RNA library preparation and sequencing

Image Credit: Norgen Biotek Corp.

Libraries were prepared utilizing the Small RNA Library Prep Kit for Illumina (Norgen Biotek Corp., Cat# 63600) and were quantified with the High Sensitivity DNA Assay, which ran on the Agilent 2100 Bioanalyzer System (Agilent Technologies, Cat# 5067-4626). Next, the libraries underwent further dilution to 4 nM concentration and were subsequently pooled in an equimolar ratio. Pooled libraries were then sequenced using the Illumina NextSeq 550 platform with the NextSeq 500/550 High Output Kit v2.5 (75 cycles) for a total of 51 cycles (Illumina, Cat# 20024906).

Processing and analyzing reads

Reads were verified for quality utilizing Fastqc (version 0.11.9). Qualified small RNA sequences were examined by the exceRpt Pipeline (Version 4.6.2) by aligning to the hg38 database (GRCh38.p13). Mapped read counts were normalized by quantifying reads per million (RPM) values.

Results and discussion

On average, the exosomal RNA yield gathered from 200 µL EDTA plasma utilizing the EXTRAClean version reached 11.2 ng (Avg. conc. 224.0 pg/µL). Meanwhile, the use of the control version yielded 3.9 ng (Avg. conc. 78.7 pg/ µL). The RIN values of RNA from the two versions were exceedingly low (avg. RIN; EXTRAClean – 1.9, Control – 1.0).

The RNA yield gathered utilizing the EXTRAClean kits was reduced compared to the control kits. The EXTRAClean kit has been modified to isolate superior-quality RNA with the lowest quantity of background RNA and, therefore, may represent true exosomal RNA.

Figure 1. Average read quality distribution relative to raw reads of small RNA sequencing of exosomal RNA extracted from three plasma samples using EXTRAClean Exosomal RNA Isolation Kit (Norgen Biotek Corp., Cat# 72800) and Exosomal RNA Isolation Kit (Norgen Biotek Corp., Cat# 58000). Calculations were relative to raw reads. Image Credit: Norgen Biotek Corp.

From EXTRAClean kits, 15.1 million reads were produced. Of these, 0.3 million (2.24%) were reads without adapter, 3.5 million were short reads (23.64%), 0.5 million (3.62%) were reads mapped to rRNA, while 0.27 million reads (1.83%) were UniVec contaminants. Reads surpassing QC included 10.3 million reads (68.64%) and were further utilized for alignment (Figure 1).

At the same time, the control kit produced 30.6 million reads. Among these reads, 0.8 million (2.63%) did not have adapters, 2.8 million (9.19%) were short reads, 0.3 million (1.1%) were rRNA reads, and 0.1 million reads (0.43%) were UniVec contaminants. The 26.5 million (86.62%) reads that remained surpassed QC parameters (Figure 1).

Figure 2. Read length distribution obtained from sequencing of exosomal RNA extracted from three plasma samples using EXTRAClean Exosomal RNA Isolation Kit (Norgen Biotek Corp., Cat# 72800) and Exosomal RNA Isolation Kit (Norgen Biotek Corp., Cat# 58000). Calculations were relative to raw reads. Image Credit: Norgen Biotek Corp.

According to the above, the EXTRAClean kit produced a higher proportion of reads shorter than 18 nucleotides than the control kit. The EXTRAClean kits are more enriched in smaller sizes of RNA, as seen in Figure 2. The graph demonstrating the read size distribution of RNA extracted utilizing the EXTRAClean kit veers more toward the smaller sizes and is enriched close to 22 bp, attributed to miRNA.

This enrichment was additionally recorded in the miRNA analysis, as the EXTRAClean kits identified 168 miRNAs while the control kit identified 128 miRNAs. Furthermore, with Novel miRNA prediction utilizing miRDeep2 at default parameters and a cutoff score of 5, the EXTRAClean kits discovered considerably higher (p=0.0070) novel miRNA vis-a-vis the control kit (Table 1).

Table 1. Results showing comparison of Novel miRNA prediction between EXTRAClean and control kit using the miRDeep2 pipeline. Predicted miRNA with score higher than a miRDeep2 score of 5 was used. Source: Norgen Biotek Corp.

Moreover, the EXTRAClean kit could record around 7% more reads mapped to the genome compared to the control kit. This larger quantity allowed the genome-mapped reads to total 2.4 million (16.23%). Meanwhile, that of the control kit remained at 2.8 million (9.14%), demonstrating an approximately 78% rise in genome-mapped reads (Figure 3). Furthermore, the control kit (23.7 million; 77.48%) displayed a higher proportion of unmapped reads in comparison to the EXTRAClean format (7.8 million; 52.4%) (Figure 3).

Figure 3. Genome mapping relative to input reads obtained from small RNA sequencing of exosomal RNA extracted from plasma three samples using EXTRAClean Exosomal RNA Isolation Kit (Norgen Biotek Corp., Cat# 72800) and Exosomal RNA Isolation Kit (Norgen Biotek Corp., Cat# 58000). (Norgen Biotek Corp., Cat# 72800) and Exosomal RNA Isolation Kit (Norgen Biotek Corp., Cat# 58000). Image Credit: Norgen Biotek Corp.

Figure 4. Average small RNA biotype distribution relative to the input reads obtained from sequencing of exosomal RNA extracted from three plasma samples using EXTRAClean Exosomal RNA Isolation Kit (Norgen Biotek Corp., Cat# 72800) and Exosomal RNA Isolation Kit (Norgen Biotek Corp., Cat# 58000). Calculations were relative to raw reads. Image Credit: Norgen Biotek Corp.

Subsequent examination of the genome-mapped reads showed that the EXTRAClean kit produced around 12 times more miRNA reads than the control kit (EXTRAClean - 1.4 million (9.3%); Control 0.2 million (0.74%) (Figure 4). The EXTRAClean kit also produced an elevated or similar percentage of other small RNA species in comparison to the control kit (EXTRAClean vs. Control: piRNA - 0.04% vs. 0.01%; circular RNA – 0.22% vs. 0.34%).

Of interest, the EXTRAClean kits produced fewer tRNA sequences than the control kit (EXTRAClean - 0.7%; Control - 3.45%) (Figure 4).

Conclusions and summary

1. The EXTRAClean kit’s read size distribution is more enriched in miRNA species and is shifted toward smaller RNA sizes.
2. The EXTRAClean kit delivers a considerably higher proportion of reads that accord with miRNAs without compromising on other small RNA species like piRNA and circular RNA.

The EXTRAClean Exosomal RNA Isolation Kit produces high-quality RNA that improves results from small RNA sequencing and lessens the fraction of unmapped read fraction. This affects the NGS economics, rendering runs more economically viable.

References and further reading

1.  Isola, A. and Chen, S. (2016). Exosomes: The Messengers of Health and Disease. Current Neuropharmacology, 15(1), pp.157–165. https://doi.org/10.2174/1570159x14666160825160421.
2. Kalluri, R. and LeBleu, V.S. (2020). The biology, function, and Biomedical Applications of Exosomes. Science, (online) 367(6478). https://doi.org/10.1126/science.aau6977.
3. Lu, J., et al. (2023). Small RNA sequencing analysis of exosomes derived from umbilical plasma in IUGR lambs. Communications Biology, (online) 6(1). https://doi.org/10.1038/s42003-023-05276-1.
4. Halkein, J., et al. (2013). MicroRNA-146a is a therapeutic target and biomarker for peripartum cardiomyopathy. Journal of Clinical Investigation, 123(5), pp.2143–2154. https://doi.org/10.1172/jci64365.
5. Vella, L.J., et al. (2008). The role of exosomes in the processing of proteins associated with neurodegenerative diseases. European biophysics journal: EBJ, (online) 37(3), pp.323–332. https://doi.org/10.1007/s00249-007-0246-z.
6. Masyuk, A.I., Masyuk, T.V. and LaRusso, N.F. (2013). Exosomes in the pathogenesis, diagnostics and therapeutics of liver diseases. Journal of Hepatology, 59(3), pp.621–625. https://doi.org/10.1016/j.jhep.2013.03.028.
7. Hannafon, B. and Ding, W.-Q. (2013). Intercellular Communication by Exosome-Derived microRNAs in Cancer. International Journal of Molecular Sciences, 14(7), pp.14240–14269. https://doi.org/10.3390/ijms140714240.
8. Galli, M., et al. (2017). A total transcriptome profiling method for plasma-derived extracellular vesicles: applications for liquid biopsies. Scientific Reports, 7(1). https://doi.org/10.1038/s41598-017-14264-5.
9. Narang, P., Shah, M. and Beljanski, V. (2022). Exosomal RNAs in diagnosis and therapies. Non-coding RNA Research, 7(1), pp.7–15. https://doi.org/10.1016/j.ncrna.2022.01.001.

About Norgen Biotek Corp.

Norgen Biotek: Advancing science with best-in-class, scientist-backed innovations

Norgen Biotek is a fully integrated biotechnology company that focuses on providing complete workflows for molecular biology sample preparation and analysis. With a diverse portfolio of over 600 products, the company delivers high-performance, user-friendly, and cost-effective solutions.

Scientifically driven, industry trusted

Norgen kits cover a broad range of applications from collection and preservation to isolation and purification. Our expert R&D team continuously develops cutting-edge technologies that set new industry standards for RNA, DNA, protein, and exosomal isolation, ensuring superior yield, purity, and integrity from even the most challenging sample types.

Unparalleled performance

At the heart of Norgen’s success is its patented Silicon Carbide (SiC) Technology. This proprietary resin exhibits uniform binding affinity for all RNA species, regardless of molecular weight or GC content. This ensures the full diversity of small and microRNA are captured while eliminating the need for phenol extraction. This innovative technology sets Norgen kits apart from others, positioning them as leaders in RNA purification.

Comprehensive solutions for any challenge

Norgen is committed to providing high-quality kits capable of processing a wide range of sample types, from ultra-low input samples such as liquid biopsies to highly impure samples like stool or soil. Our sample collection and preservation devices for stool and saliva simplify handling by rendering samples non-infectious by preventing microbial growth and inactivating viruses, while our blood and urine preservation solutions ensure the stability of highly vulnerable cell-free nucleic acids.

To meet varying research demands, we offer multiple isolation methods including, but not limited to high-throughput and automation-ready magnetic bead-based formats. Additionally, our multiple-analyte kits enable the simultaneous purification of RNA, DNA, and proteins, maximizing data extraction from a single sample.

Norgen offers an extensive variety of TaqMan qPCR kits designed for molecular diagnostic use, including lyophilized kits for easy shipping. Library preparation kits for both DNA and RNA samples are also available to support genomic applications. Norgen recently released their EXTRAClean technology, an innovative solution that minimizes background noise while providing high-purity RNA, significantly enhancing NGS performance.

Why choose Norgen?

• Scientist-Driven Innovation – Developed by leading experts in molecular biology
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