RNA Extraction Methods Research Articles

Development of a Point-of-Care Microfluidic RNA Extraction Slide for Gene Expression Diagnosis after Irradiation.

In times of war, radiological/nuclear emergency scenarios have become a reemphasized threat. However, there are challenges in transferring whole-blood samples to laboratories for specialized diagnostics using RNA. This project aims to miniaturize the process of unwieldy conventional RNA extraction with its stationed technical equipment using a microfluidic-based slide (MBS) for point-of-care diagnostics. The MBS is thought to be a preliminary step toward the development of a so-called lab-on-a-chip microfluidic device. A MBS would enable early and fast field care combined with gene expression (GE) analysis for the prediction of hematologic acute radiation syndrome (HARS) severity or identification of RNA microbes. Whole blood samples from ten healthy donors were irradiated with 0, 0.5 and 4 Gy, simulating different ARS severity degrees. RNA quality and quantity of a preliminary MBS was compared with a conventional column-based (CB) RNA extraction method. GE of four HARS severity-predicting radiation-induced genes (FDXR, DDB2, POU2AF1 and WNT3) was examined employing qRT-PCR. Compared to the CB method, twice as much total RNA from whole blood could be extracted using the MBS (6.6 ± 3.2 µg vs. 12.0 ± 5.8 µg) in half of the extraction time, and all MBS RNA extracts appeared DNA-free in contrast to the CB method (30% were contaminated with DNA). Using MBS, RNA quality [RNA integrity number equivalent (RINe)] values decreased about threefold (3.3 ± 0.8 vs. 9.0 ± 0.4), indicating severe RNA degradation, while expected high-quality RINe ≥ 8 were found using column-based method. However, normalized cycle threshold (Ct) values, as well as radiation-induced GE fold-changes appeared comparable for all genes utilizing both methods, indicating that no RNA degradation took place. In summary, the preliminary MBS showed promising features such as: 1. halving the RNA extraction time without the burden of heavy technical equipment (e.g., a centrifuge); 2. absence of DNA contamination in contrast to CB RNA extraction; 3. reduction in blood required, because of twice the biological output of RNA; and 4. equal GE performance compared to CB, thus, increasing its appeal for later semi-automatic parallel field applications.

Abstract 7248: Simultaneous isolation and parallel analysis of genomic DNA and RNA for gene therapy

Abstract Introduction Viral vectors, such as adeno-associated vector (AAV), play a crucial role in delivering transgenes for gene therapy research. The success of vector transduction is vital for efficacy, which is measured through vector copy number (VCN) and transgene expression. In preclinical studies, both VCN and transgene expression are commonly tested from the same sample to ensure accuracy. We have reported a method for simultaneous high-quality genomic DNA and RNA extraction without splitting the sample, utilizing SPRI bead-based technology that has consistent performance across diverse tissue types and brain regions. Methods AAV was administered to mouse and non-human primate (NHP) models. At the conclusion of each experiment, tissue samples from the adrenal gland, brain, gonads, heart, kidney, liver, lung, skeletal muscle, spinal cord, and spleen were harvested. All tissue samples that demonstrated around 15 mm3 were snap-frozen in liquid nitrogen before nucleic acid extraction. The extraction process was carried out manually or on an automated liquid handler. The quality and quantity of DNA and RNA were then assessed from all tested tissues before downstream assay. Results Our study found that all mouse tissue types (n=72/tissue type) and NHP (n=11/tissue type) had an average gDNA and RNA yield that exceeded the minimum yield cutoff of 25 ng/μL for DNA and 6.25 ng/μL for RNA. We also analyzed DNA from tissues treated with two different AAV serotype-treated tissues in the mouse model, which showed that transduction across all the tissues was highly efficient for both AAV serotypes. Furthermore, we observed variations in transgene expression levels (copies/1 μg RNA) among different tissue types. Similar patterns of VCN and transgene expression were also observed in NHP models. Conclusions Efficient gene therapy depends on a safe and effective viral vector. The simultaneous extraction of gDNA and RNA simplifies the process, saves time, ensures quality, and facilitates integration into existing lab automation. This leads to consistent and error-resistant performance in analyzing vector copy and transgene levels. Citation Format: Han Wei. Simultaneous isolation and parallel analysis of genomic DNA and RNA for gene therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7248.

Validation of RNA Extraction Methods and Suitable Reference Genes for Gene Expression Studies in Developing Fetal Human Inner Ear Tissue.

A comprehensive gene expression investigation requires high-quality RNA extraction, in sufficient amounts for real-time quantitative polymerase chain reaction and next-generation sequencing. In this work, we compared different RNA extraction methods and evaluated different reference genes for gene expression studies in the fetal human inner ear. We compared the RNA extracted from formalin-fixed paraffin-embedded tissue with fresh tissue stored at -80 °C in RNAlater solution and validated the expression stability of 12 reference genes (from gestational week 11 to 19). The RNA from fresh tissue in RNAlater resulted in higher amounts and a better quality of RNA than that from the paraffin-embedded tissue. The reference gene evaluation exhibited four stably expressed reference genes (B2M, HPRT1, GAPDH and GUSB). The selected reference genes were then used to examine the effect on the expression outcome of target genes (OTOF and TECTA), which are known to be regulated during inner ear development. The selected reference genes displayed no differences in the expression profile of OTOF and TECTA, which was confirmed by immunostaining. The results underline the importance of the choice of the RNA extraction method and reference genes used in gene expression studies.

Comparative evaluation of assay performance for SARS-CoV-2 detection in animal oral samples, lung homogenates, and phosphate-buffered saline using the TaqPath COVID-19 Combo kit.

A One Health approach has been key to monitoring the COVID-19 pandemic, as human and veterinary medical professionals jointly met the demands for an extraordinary testing effort for SARS-CoV-2. Veterinary diagnostic laboratories continue to monitor SARS-CoV-2 infection in animals, furthering the understanding of zoonotic transmission dynamics between humans and animals. A RT-PCR assay is a primary animal screening tool established within validation and verification guidelines provided by the American Association of Veterinary Laboratory Diagnosticians (AAVLD), World Organisation for Animal Health (WOAH), and the U.S. Food and Drug Administration (FDA). However, differences in sample matrices, RNA extraction methods, instrument platforms, gene targets, and cutoff values may affect test outcomes. Therefore, targeted validation for a new sample matrix used in any PCR assay is critical. We evaluated a COVID-19 assay for the detection of SARS-CoV-2 in feline and canine lung homogenates and oral swab samples. We used the commercial Applied Biosystems MagMAX Viral/Pathogen II (MVP II) nucleic acid isolation kit and TaqPath COVID-19 Combo kit, which are validated for a variety of human samples, including nasopharyngeal and oropharyngeal swab samples. Our masked test showed a high detection rate and no false-positive or false-negative results, supporting sample extension to include feline oral swab samples. Our study is a prime example of One Health, illustrating how a COVID-19 assay designed for human testing can be adapted and used to detect SARS-CoV-2 in oral swab samples from cats and likely dogs, but not lung homogenates.

Examination and comparison of the RNA extraction methods using mouse serum.

Serum microRNAs (miRNAs) are considered useful as non-invasive biomarkers for different diseases. However, the optimal method for extracting RNAs from serum is currently unknown. In the present study, several RNA extraction kits were used to examine the optimal kit. RNAs were extracted from the serum of 8-week-old C57BL/6NJcl male mice following the protocol of each RNA extraction kit. The yield of the extracted RNA samples was calculated, and an Agilent Bioanalyzer was used to assess the electrophoretic patterns. An Agilent mouse miRNA microarray was utilized to confirm the expression patterns of the extracted RNA samples. The results revealed significant differences in RNA yields from the miRNeasy Serum/Plasma Advanced kit and mirVana™ PARIS™ RNA and Native Protein Purification Kit compared with almost all other samples. Further, two peaks were determined in the miRNeasy Serum/Plasma Advanced kit using a small RNAs kit of Agilent Bioanalyzer, including one at 20-40 nucleotides (nt) and another at ~40-100 nt, whereas the other reagents had a single peak. This revealed that the extracted RNAs may differ in composition based on the RNA extraction method. Some types of miRNAs were only detected with certain RNA extraction reagents. This suggested that different RNA extraction reagents may cause differences in the types of miRNAs detected. On the other hand, the miRNAs commonly expressed by the three RNA extraction reagents are highly correlated in expression levels.

An Optimized and Cost-Effective RNA Extraction Method for Secondary Metabolite-Enriched Tissues of Norway Spruce (Picea abies)

Since the development of next-generation sequencing techniques and with the growing interest in transcriptomic studies, there is a demand for high-throughput RNA extraction techniques. General RNA extraction protocols are unreliable when it comes to the quality and quantity of isolated RNA obtained from different tissue types of different plant species. Despite Norway spruce (Picea abies) being one of the most significant and commercially valuable tree species in European forests, only limited genetic research is available. In this study, we developed a cetyltrimethylammonium bromide (CTAB) protocol by modifying the original method. We compared this CTAB protocol with other widely used methods for extracting RNA from different tissues (needle, phloem, and root) of Norway spruce, known for its richness in polyphenols, polysaccharides, and secondary metabolites. The modified CTAB method proves to be superior to the kit-based and TRIzol-based methods for extracting RNA from the metabolite-rich tissues of Norway spruce, resulting in high RNA quality and integrity values (RIN~7–9). The modified CTAB RNA extraction method is rapid, cost-effective, and relatively simple in yielding the desired RNA quality from Norway spruce tissues. It is optimal for RNA sequencing and other downstream molecular applications.

Assessment of the efficiency of virus extraction from food matrices and the frequency of occurrence of contaminated products in the retail network

Express detection of viruses, in particular, norovirus (NoV) and hepatitis A virus (HAV), is becoming an extremely important task for food safety control. This study examines various approaches to recovery of viral particles and methods for RNA extraction from food matrices to assess mengovirus extraction efficiency. Efficiency of mengovirus extraction from raspberry was 14.26%, from oysters 7.99%, from pork liver 8.33%. Assessment of RNA extraction by various methods was carried out. The highest efficiency of mengovirus extraction from pork liver (19.37%) was observed when RNA was extracted using the eGene-up semi-automatic system. The lowest extraction efficiency (5.31%) was achieved upon manual RNA extraction. When RNA was extracted from oysters, the maximum efficiency (33.35%) was ensured by the AutoPure nucleic acid extraction station and NucliSens kit, while the minimum efficiency (9.78%) was observed when using the eGene-up system. The performed monitoring of food products showed that the highest occurrence of norovirus GII was recorded in oyster samples (9.6% of tested samples); the second place was occupied by strawberry, where occurrence of norovirus GII was 6.8%. In the raspberry samples, norovirus GII was not detected.

Cross comparison of alternative diagnostic protocols including substitution to the clinical sample, RNA extraction method and nucleic acid amplification technology for COVID-19 diagnosis.

the gold-standard diagnostic protocol (GSDP) for COVID-19 consists of a nasopharyngeal swab (NPS) sample processed through traditional RNA extraction (TRE) and amplified with retrotranscription quantitative polymerase chain reaction (RT-qPCR). Multiple alternatives were developed to decrease time/cost of GSDP, including alternative clinical samples, RNA extraction methods and nucleic acid amplification. Thus, we carried out a cross comparison of various alternatives methods against GSDP and each other. we tested alternative diagnostic methods using saliva, heat-induced RNA release (HIRR) and a colorimetric retrotranscription loop-mediated isothermal amplification (RT-LAMP) as substitutions to the GSDP. RT-LAMP using NPS processed by TRE showed high sensitivity (96%) and specificity (97%), closely matching GSDP. When saliva was processed by TRE and amplified with both RT-LAMP and RT-qPCR, RT-LAMP yielded high diagnostic parameters (88%-96% sensitivity and 95%-100% specificity) compared to RT-qPCR. Nonetheless, when saliva processed by TRE and detected by RT-LAMP was compared against the GSDP, the resulting diagnostic values for sensitivity (78%) and specificity (87%) were somewhat high but still short of those of the GSDP. Finally, saliva processed with HIRR and detected via RT-LAMP was the simplest and fastest method, but its sensitivity against GSDP was too low (56%) for any clinical application. Also, in this last method, the acidity of a large percentage of saliva samples (9%-22%) affected the pH-sensitive colorimetric indicator used in the test, requiring the exclusion of these acidic samples or an extra step for pH correction. our comparison shows that RT-LAMP technology has diagnostic performance on par with RT-qPCR; likewise, saliva offers the same diagnostic functionality as NPS when subjected to a TRE method. Nonetheless, use of direct saliva after a HIRR and detected with RT-LAMP does not produce an acceptable diagnostic performance.

Evaluation of different methods of RNA extraction from semen of cross-breed bulls (Bos Taurus)

Evaluation of different methods of RNA extraction from semen of cross-breed bulls (Bos Taurus)

Point-of-care testing for COVID-19: a simple two-step molecular diagnostic development and validation during the SARS-CoV-2 pandemic.

During the coronavirus disease 19 (COVID-19) pandemic, diagnostic testing of the general population proved challenging due to limitations of the gold-standard diagnostic procedure using reverse transcription real-time polymerase chain reaction (RT-qPCR) for large-scale testing on the centralised model, especially in low-resource areas. To address this, a point-of-care (PoC) diagnostic protocol for COVID-19 was developed, providing fast, reliable, and affordable testing, particularly for low-mid develop areas. The PoC diagnostic process combines a simple paper-based RNA extraction method housed within a 3D-printed plastic device with a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay. Nasopharyngeal/oropharyngeal swabs (NOS) and saliva samples were tested between 2020 and 2021, with the assistance of Santa Catarina's State Health Secretary, Brazil. The developed diagnostic protocol showed a limit of detection of 9,900 copies and an overall diagnostic specificity of 98% for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from 1,348 clinical analysed samples. The diagnostic sensitivity was 95% for NOS samples, 85% for early morning saliva, and 69% for indiscriminate saliva. In conclusion, the developed device successfully extracted SARS-CoV-2 viral RNA from swabs and saliva clinical samples. When combined with colorimetric RT-LAMP, it provides results within 45 min using minimal resources, thus delivering a diagnostic kit protocol that is applicable in large-scale sampling.

Optimizing total RNA extraction method for human and mice samples.

Extracting high-quality total RNA is pivotal for advanced RNA molecular studies, such as Next-generation sequencing and expression microarrays where RNA is hybridized. Despite the development of numerous extraction methods in recent decades, like the cetyl-trimethyl ammonium bromide (CTAB) and the traditional TRIzol reagent methods, their complexity and high costs often impede their application in small-scale laboratories. Therefore, a practical and economical method for RNA extraction that maintains high standards of efficiency and quality needs to be provided to optimize RNA extraction from human and mice tissues. This study proposes enhancements to the TRIzol method by incorporating guanidine isothiocyanate (GITC-T method) and sodium dodecyl sulfate (SDS-T method). We evaluated the effectiveness of these modified methods compared to the TRIzol method using a micro-volume UV-visible spectrophotometer, electrophoresis, q-PCR, RNA-Seq, and whole transcriptome sequencing. The micro-volume UV-visible spectrophotometer, electrophoresis, and RNA-Seq demonstrated that the GITC-T method yielded RNA with higher yields, integrity, and purity, while the consistency in RNA quality between the two methods was confirmed. Taking mouse cerebral cortex tissue as a sample, the yield of total RNA extracted by the GITC-T method was 1,959.06±49.68 ng/mg, while the yield of total RNA extracted by the TRIzol method was 1,673.08±86.39 ng/mg. At the same time, the OD260/280 of the total RNA samples extracted by the GITC-T method was 2.03±0.012, and the OD260/230 was 2.17±0.031, while the OD260/280 of the total RNA samples extracted by the TRIzol method was 2.013±0.041 and the OD260/230 was 2.11±0.062. Furthermore, q-PCR indicated that the GITC-T method achieved higher yields, purity, and greater transcript abundance of total RNA from the same types of animal samples than the TRIzol method. The GITC-T method not only yields higher purity and quantity of RNA but also reduces reagent consumption and overall costs, thereby presenting a more feasible option for small-scale laboratory settings.

Circulating exosomal microRNAs as biomarkers of lupus nephritis.

Disruption in the delicate symphony of genes, microRNA (miRNA), or protein expression can result in the dysregulation of the immune system, leading to the devastating consequences such as lupus nephritis (LN). The capacity of exosomes to transport miRNAs between cells and modify the phenotype of recipient cells implies their involvement in persistent kidney inflammation. This study unveils identifying two previously undiscovered exosomal miRNAs in the serum of LN patients, offering potential solutions to the current challenges in LN diagnosis and management. Initially, we used a reagent-based kit to isolate serum exosomes from patients with Systemic lupus erythematosus (SLE) and used Trizol method for total RNA extraction. Subsequently, we employed small RNA sequencing to screen for differential expression profiles of exosomal small RNAs. The RT-qPCR method was used to individually validate samples in both the screening and validation cohorts, enabling the identification of candidate small RNAs; specific to LN. We assessed the diagnostic potency using receiver operating characteristic (ROC) curve, and explored the biological roles of miRNAs using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Compared to SLE patients without LN, SLE patients accompanied by LN exhibited significantly spiked levels of exosomal hsa-miR-4796-5p and hsa-miR-7974. The duo of miRNAs, hsa-miR-4796-5p and hsa-miR-7974, exhibited promising potential as biomarkers for diagnosing LN, with an AUC exceeding 0.8. Correlation analysis revealed a strong positive association between these miRNAs and proteinuria, as well as the SLE Disease Activity Index (SLEDAI) score. Moreover, the levels of two miRNAs in LN patients were significantly elevated in comparison to other autoimmune nephritis conditions, such as immunoglobulin A nephropathy (IgAN) and diabetic nephropathy (DN). Furthermore, the bioinformatics analysis indicated that this miRNAs duo can play a pivotal role in the regulation of immune processes by modulating signal pathways, such as the mTOR and PI3K-Akt signaling pathway. This study provides a new ground that serum exosomal miRNAs can effectively identify and predict LN in SLE patients.

SARS-CoV-2 Wastewater Monitoring in Thuringia, Germany: Analytical Aspects and Normalization of Results

Wastewater monitoring for SARS-CoV-2 is a valuable tool for surveillance in public health. However, reliable analytical methods and appropriate approaches for the normalization of results are important requirements for implementing state-wide monitoring programs. In times of insufficient case reporting, the evaluation of wastewater data is challenging. Between December 2021 and July 2022, we analyzed 646 samples from 23 WWTPs in Thuringia, Germany. We investigated the performance of a direct capture-based method for RNA extraction (4S-method) and evaluated four normalization methods (NH4-N, COD, Ntot, and PMMoV) in a pooled analysis using different epidemiological metrics. The performance requirements of the 4S method were well met. The method could be successfully applied to implement a state-wide wastewater monitoring program including a large number of medium and small wastewater treatment plants (<100,000 p.e) in high spatial density. Correlations between wastewater data and 7-day incidence or 7-day-hospitalization incidence were strong and independent from the normalization method. For the test positivity rate, PMMoV-normalized data showed a better correlation than data normalized with chemical markers. In times of low testing frequency and insufficient case reporting, 7-day-incidence data might become less reliable. Alternative epidemiological metrics like hospital admissions and test positivity data are increasingly important for evaluating wastewater monitoring data and normalization methods. Furthermore, future studies need to address the variance in biological replicates of wastewater.

EVALUATION OF METHODS FOR TOTAL RNA EXTRACTION FROM THE ENDOSPERM OF COCOS NUCIFERA VAR. MAKAPUNO IN VIETNAM FOR MOLECULAR ANALYSIS

Sap coconut (Cocos nucifera L. var. makapuno) in Vietnam is a mutant coconut variant; coconut water is in the state of lotus glue, and coconut rice is like cream. For high-quality transcriptome, sequencing, quality, purity and concentration of RNA are the key factors. However, coconut endosperm tissue has higher stiffness and fatness than the leaf tissue, which complicates the extraction process. Moreover, RNA is much more difficult to preserve than DNA. In this study, various RNA extraction methods were examined in Vietnamese waxy coconut endosperm tissue samples. Optimum extraction and preservation of the RNA using the simplest possible chemicals was the objective of this study. The modified CTAB method with LiCl and the TRI reagent method were tested and evaluated. The purity, concentration and quality of RNA after storage were improved. The findings indicated that the TRIsure extraction method with the addition of NaCl and β-mercaptoethanol yielded optimum RNA quality. The RNA concentration was 159 ng/µL, with a purity ratio of 1.94 ± 0.04 for A260/A280 and 1.58 ± 0.02 for A260/230. RNA samples remained stable for up to 3 weeks when stored in absolute ethanol at 8°C–10°C, which significantly reduced their degradation during transportation. This study facilitated the use of simple chemicals for high-quality RNA extraction from coconut endosperm and its preservation for applications in high throughput sequencing.

Compact Point-of-Care Device for Self-Administered HIV Viral Load Tests from Whole Blood.

Human immunodeficiency virus (HIV) is a significant problem to consider as it can lead to acquired immune deficiency syndrome (AIDS). Fortunately, AIDS is manageable through antiretroviral therapy (ART). However, frequent viral load monitoring is needed to monitor the effectiveness of the therapy. The current reverse transcription-polymerase chain reaction (RT-PCR) viral load monitoring is highly effective, but is challenged by being resource-intensive and inaccessible, and its turnaround time does not meet demand. An unmet need exists for an affordable, rapid, and user-friendly point-of-care device that could revolutionize and ensure therapeutic effectiveness, particularly in resource-limited settings. In this work, we explored a point-of-care HIV viral load device to address this need. This device can perform streamlined plasma separation, viral RNA extraction, and real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) semiquantitative testing in an ultracompact device. We developed an absorption-based membrane plasma separation method suitable for finger-prick blood samples, achieving an efficiency of 80%. We also designed a syringe-based RNA extraction method for on-site plasma processing with a viral recovery efficiency of 86%. We created a portable device with a smartphone interface for real-time semiquantitative RT-LAMP, which is useful for monitoring viral load. The device uses lyophilized reagents, processed with our lyophilization method, which remain stable for 16 weeks. The device can accurately categorize viral load into low, medium, and high categories with 95% accuracy. We believe this point-of-care HIV self-test device, offering convenience and long-term storage, could aid patients in home-based ART treatment monitoring.

A Comprehensive Comparison of Rapid RNA Extraction Methods for Detection of SARS-CoV-2 as the Infectious Agent of the Upper Respiratory Tract using Direct RT-LAMP Assay.

The current COVID-19 pandemic has highlighted the need for faster and more cost-effective diagnostic methods. The RNA extraction step in current diagnostic methods, such as real-time qPCR, increases the cost and time required for testing. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is a promising technique for developing diagnostic tests with desired sensitivity and specificity without the need for RNA extraction. An RT-LAMP assay was developed to detect SARS-CoV-2 with a sensitivity of 0.5 copies of positive control plasmid per microliter in 40 min. Several rapid RNA extraction protocols were evaluated using different reagents, including bovine serum albumin, Triton X-100, Tween 20, proteinase K, guanidine hydrochloride, guanidinium isothiocyanate (GITC), and thermal treatment. Finally, the sensitivity and specificity of the developed direct RT-LAMP were determined using 150 upper respiratory tract samples. Method 10 was selected as the most efficient protocol for the RNA extraction step. The sensitivity and specificity of the developed direct RT-LAMP assay with clinical samples were estimated at 98.4% and 88.8%, respectively. These results suggest that the combination of GITC and Triton X-100 detergent is a highly efficient method for RNA extraction and direct RT-LAMP detection of SARS-CoV-2 in clinical samples, providing a valuable tool for the rapid and cost-effective diagnosis of COVID-19.

RNA-seq RNAaccess identified as the preferred method for gene expression analysis of low quality FFPE samples.

Clinical tumor tissues that are preserved as formalin-fixed paraffin-embedded (FFPE) samples result in extensive cross-linking, fragmentation, and chemical modification of RNA, posing significant challenges for RNA-seq-based gene expression profiling. This study sought to define an optimal RNA-seq protocol for FFPE samples. We employed a common RNA extraction method and then compared RNA-seq library preparation protocols including RNAaccess, RiboZero and PolyA in terms of sequencing quality and concordance of gene expression using FFPE and case-matched fresh-frozen (FF) triple-negative breast cancer (TNBC) tissues. We found that RNAaccess, a method based on exome capture, produced the most concordant results. Applying RNAaccess to FFPE gastric cancer tissues, we established a minimum RNA DV200 requirement of 10% and a RNA input amount of 10ng that generated highly reproducible gene expression data. Lastly, we demonstrated that RNAaccess and NanoString platforms produced highly concordant expression profiles from FFPE samples for shared genes; however, RNA-seq may be preferred for clinical biomarker discovery work because of the broader coverage of the transcriptome. Taken together, these results support the selection of RNA-seq RNAaccess method for gene expression profiling of FFPE samples. The minimum requirements for RNA quality and input established here may allow for inclusion of clinical FFPE samples of sub-optimal quality in gene expression analyses and ultimately increasing the statistical power of such analyses.

Comparing RNA extraction methods to face the variations in RNA quality using two human biological matrices.

Nucleic acids, RNA among them, are widely used in biomedicine and Biotechnology. Because of their susceptibility to degradation by RNases, the handling and extraction process of RNA from cells and tissues require specialized personnel and standardized methods to guarantee high purity and integrity. Due to the diversity of techniques found in the market, a comparative study between different RNA extraction methods is useful to facilitate the best choice for the researcher or in research service platforms such as biobanks to see the traceability of the samples. In this study, we have compared seven different RNA extraction methods: manual (TRIzol™), semiautomated (QIAGEN™, Bio-Rad, Monarch®, and Canvax™), and fully automated (QIAcube™ and Maxwell®) processes, from two biological matrices: human Jurkat T cells and peripheral blood mononuclear cells (PBMC). Results showed marked differences in the RNA quality and functionality according to the method employed for RNA extraction and the matrix used. QIAcube™ and semi-automated extraction methods were perceived as the best options because of their lower variability, good functionality, and lower cost (P < 0.001). These data contribute to facilitating researchers or research service platforms (Biobanks) in decision-making practices and emphasize the relevance of the selection of the RNA extraction method in each experimental procedure or traceability study to guarantee both quality standards and its reproducibility.

Metabolically Active Microbial Communities in Oilfields: A Systematic Review and Synthesis of RNA Preservation, Extraction, and Sequencing Methods

Characterizing metabolically active microorganisms using RNA-based methods is a crucial tool for monitoring and mitigating operational issues, such as oil biodegradation and biocorrosion of pipelines in the oil and gas industry. Our review, a pioneering study, addresses the main methods used to preserve, isolate, and sequence RNA from oilfield samples and describes the most abundant metabolically active genera studied. Using the MEDLINE/PubMed, PubMed Central, Scopus, and Web of Science databases, 2.561 potentially eligible records were identified. After screening, 20 studies were included in our review, underscoring the scarcity of studies related to the subject. Data were extracted and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA). These studies evaluated different samples, including produced water (PW), injection water (IW), solid deposits (SD), oil (OIL), and oily sludge (OS) collected from oilfields located in Australia, China, India, Mexico, and the United Arab Emirates. Environmental samples accounted for 55% of the studies, while enriched cultures and microbial consortia represented 35% and 15% of studies, respectively. PW was the most frequently studied sample, comprising 72% of all samples. Filtration and centrifugation were the only processes employed to concentrate the biomass present in samples. For RNA preservation, the most used method was a solution composed of 95:5 v/v ethanol/TRIzol, while for RNA isolation, the TRIzol reagent was the most cited. The Sanger sequencing method was used in all studies evaluating functional genes (alkB, dsrA, aprA, assA, and mcrA), and the Next-Generation Sequencing (NGS) method was employed in studies for sequencing transcripts of the 16S rRNA gene and metatranscriptomes. Pseudomonas (16S rRNA = PW: 2%; IW: 8%; metatranscriptome = PW: 20%) and Acinetobacter (16S rRNA = PW: 1%; IW: 4%; metatranscriptome = PW: 17%) were the most abundant genera. This study outlined the primary methods employed in researching metabolically active microorganisms. These data provide a foundation for future research. However, it is essential to note that we cannot yet determine the most effective method. We hope that this study will inspire further research related to the standardization of RNA preservation, extraction, and sequencing methods and significantly contribute to our understanding of active microbial communities in oilfields.

Transcriptome analysis of maize explant for anther culture: comparison of three different RNA extraction methods

Breeding is one method to develop better varieties. This method can be accelerated by using haploid technology. The availability of haploid inducer lines in maize makes in-vivo techniques preferred, especially for temperate maize. Developing in-vitro through anther culture in haploid breeding is still needed since haploid inducer lines are still limited in tropical maize. Like many other crops facing recalcitrancy problems, maize also experiences the same, so cracking recalcitrant traits using transcriptomic approaches is necessary. In order to enhance the success of the sequencing, obtaining a high quantity and quality of RNA product is needed since it will affect the data. This study aimed to determine the best RNA extraction methods using three different kits of maize explant for anther culture with high purity and concentrations. RNA extraction in this study was performed using Tripure, RibospinTM, Promega, and Tripure kits. The results showed that all All the kits were able to produce high concetration of RNA, where the Ribospin (Geneall) performed the best (both for the concentration and purity). Meanwhile Tripure kit were only able to produced highest concentration of RNA, but lowest purity.