MAVRICS: A Robust and Safe Magnetic Nanoparticle based RNA Extraction Method Compatible with Phenol-chloroform Inactivated Infectious Samples v2

Diagnosis and surveillance of emerging pathogens such as SARS-CoV-2 depend on nucleic acid isolation from clinical and environmental samples. Under normal circumstances, samples would be processed using commercial proprietary reagents in Biosafety 2 (BSL-2) or higher facilities. A pandemic at the scale of COVID-19 has caused a global shortage of proprietary reagents and BSL-2 laboratories to safely perform testing. Therefore, alternative solutions are urgently needed to address these challenges. We developed an open-source method called Magneticnanoparticle-Aided Viral RNA Isolation of Contagious Samples (MAVRICS) that is built upon reagents that are either readily available or can be synthesized in any molecular biology laboratory with basic equipment. Unlike conventional methods, MAVRICS works directly in samples inactivated in acid guanidinium thiocyanate-phenol-chloroform (e.g., TRIzol), thus allowing infectious samples to be handled safely without biocontainment facilities.

An Optimised TRIzol-based Protocol for the Improvement of RNA Extraction Yield of Tomato Stem

One of the most common methods for purifying RNA is using TRIzol reagent because of its simplicity and economic feasibility. However, the drawback of this method is frequently the low quality of extracted RNA due to contaminants from the residue of phenol and guanidinium thiocyanate from the reagents. This study aimed to evaluate the improvement in the quality and concentration of RNA after the optimisation treatment. One-month-old tomato (Solanum lycopersicum) stem was used in this research. TRIzol or acid guanidinium thiocyanate-phenol-chloroform-based method was given optimisation treatments of the initial sample amount, twice chloroform extraction, overnight precipitation at low temperature, and three times final washing with ethanol. The results showed no significant improvement (p > 0.05) in the purity ratio A260/A280. At the same time, there was a significant improvement (p < 0.05) in RNA yield and purity ratio A260/A230. The quality of RNA was verified using agarose-formaldehyde electrophoresis gel. Eight of nine samples (89%) from the optimised group had better RNA integrity characterised by sharp bands for 28S and 18S rRNA. Furthermore, a representative sample from the optimised group was successfully synthesised into complementary DNA by reverse transcriptase-polymerase chain reaction (RT-PCR) with primers of the ubiquitin (UBI3) gene. To sum up, optimised TRIzol-based protocol provides meaningful insight to produce RNA with better quality and suitability for downstream applications.

Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays

Background Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings. Methods We evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C. Results SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on VeroE6 cells was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions. Conclusion eNAT and similar guanidinium thiocyanate-based media may be of value for transport, stabilization, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.

High Efficiency RNA Extraction From Sperm Cells Using Guanidinium Thiocyanate Supplemented With Tris(2-Carboxyethyl)Phosphine

The extraction of high-quality ribonucleic acid (RNA) from tissues and cells is a key step in many biological assays. Guanidinium thiocyanate-phenol-chloroform (AGPC) is a widely used and efficient method to obtain pure RNA from most tissues and cells. However, it is not efficient with some cells like sperm cells because they are resistant to chaotropic lysis solutions containing guanidinium thiocyanate such as Buffer RLT+ and Trizol. Here, we show that disulfide bonds are responsible for the chemical resistance of sperm cells to RNA extraction reagents. We show that while β-mercaptoethanol (βME) can increase sperm lysis in Buffer RLT+, it has no effect in Trizol and leaves sperm cells intact. We measured the reduction of disulfide bonds in 2,2′-dithiodipyridine (DTDP) and observed that βME has a pH-dependent activity in chaotropic solutions, suggesting that pH is a limiting factor. We identified tris(2-carboxyethyl)phosphine (TCEP) as an efficient lysis enhancer of AGPC solutions that can retain reducing activity even at acidic pH. Trizol supplemented with TCEP allows the complete and rapid lysis of sperm cells, increasing RNA yield by 100-fold and resulting in RNA with optimal quality for reverse transcription and polymerase chain reaction. Our findings highlight the importance of efficient cell lysis and extraction of various macromolecules for bulk and single-cell assays, and can be applied to other lysis-resistant cells and vesicles, thereby optimizing the amount of required starting material and animals.

Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays

BackgroundUpper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings.MethodsWe evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream RT-PCR testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C.ResultsSARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on vero-E6 cell lines was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions.ConclusioneNAT and similar guanidinium thiocyanate-based media may be of value for transport, preservation, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.

Design of surface termination for high-performance perovskite solar cells

Guanidinium thiocyanate was selected to modify the surface terminations of methylamine lead iodide (MAPbI3) perovskite films and a 21.26% PCE was acquired for a solar cell based on the MAPbI3 system, and the voltage deficit is reduced to 0.426 V.

Feasibility of phosphoproteomics on leftover samples after RNA extraction with guanidinium thiocyanate

In daily practice, different types of biomolecules are usually extracted for large-scale ‘omics’ analysis with tailored protocols. However, when sample material is limited, an all-in-one strategy is preferable. While lysis of cells and tissues with urea is the accepted standard for phosphoproteomic applications, DNA, RNA and proteins can be simultaneously extracted from small samples using acid guanidinium thiocyanate-phenol-chloroform (AGPC). Use of AGPC for mass spectrometry (MS)-based phosphoproteomics has been reported, but not benchmarked. Here we compared urea-with AGPC-based protein extraction, profiling phosphorylations in the DNA damage response pathway after ionizing irradiation of U2OS cells as proof of principle. On average we identified circa 9000 phosphosites per sample with both extraction methods. Moreover, we observed high similarity of phosphosite characteristics (e.g. 94% shared class 1 identifications) and deduced kinase activities (e.g. ATM, ATR, CHEK1/2, PRKDC). AGPC-based sample extraction can thus replace standard cell lysates for phosphoproteomic workflows and may thus be an attractive way to obtain input material for multiple omics workflows, yielding several data types from a single sample.