RNA Extraction Efficiency Research Articles

Whole blood RNA extraction efficiency contributes to variability in RNA sequencing data sets.

Numerous methodologies are used for blood RNA extraction, and large quantitative differences in recovered RNA content are reported. We evaluated three archived data sets to determine how extraction methodologies might influence mRNA and lncRNA sequencing results. The total quantity of RNA recovered /ml of blood affects RNA sequencing by impacting the recovery of weakly expressed mRNA, and lncRNA transcripts. Transcript expression (TPM counts) plotted in relation to transcript size (base pairs, bp) revealed a 30% loss of short to midsized transcripts in some data sets. Quantitative recovery of RNA is of considerable importance, and it should be viewed more judiciously. Transcripts common to the three data sets were subsequently normalized and transcript mean TPM counts and TPM count coefficient of variation (CV) were plotted in relation to increasing transcript size. Regression analysis of mean TPM counts versus transcript size revealed negative slopes in two of the three data sets suggesting a reduction of TPM transcript counts with increasing transcript size. In the third data set, the regression slope line of mRNA transcript TPM counts approximates zero and TPM counts increased in proportion to transcript size over a range of 200 to 30,000 bp. Similarly, transcript TPM count CV values also were uniformly distributed over the range of transcript sizes. In the other data sets, the regression CV slopes increased in relation to transcript size. The recovery of weakly expressed and /or short to midsized mRNA and lncRNA transcripts varies with different RNA extraction methodologies thereby altering the fundamental sequencing relationship between transcript size and TPM counts. Our analysis identifies differences in RNA sequencing results that are dependent upon the quantity of total RNA recovery from whole blood. We propose that incomplete RNA extraction directly impacts the recovery of mRNA and lncRNA transcripts from human blood and speculate these differences contribute to the "batch" effects commonly identified between sequencing results from different archived data sets.

Optimized procedure for high-throughput transcriptome profiling of small extracellular vesicles isolated from low volume serum samples.

Small extracellular vesicles (EVs) contain various signaling molecules, thus playing a crucial role in cell-to-cell communication and emerging as a promising source of biomarkers. However, the lack of standardized procedures impedes their translation to clinical practice. Thus, we compared different approaches for high-throughput analysis of small EVs transcriptome. Small EVs were isolated from 150 μL of serum. Quality and quantity were assessed by dynamic light scattering, transmission electron microscopy, and Western blot. Comparison of RNA extraction efficiency was performed, and expression of selected genes was analyzed by RT-qPCR. Whole transcriptome analysis was done using microarrays. Obtained data confirmed the suitability of size exclusion chromatography for isolation of small EVs. Analyses of gene expression showed the best results in case of samples isolated by Monarch Total RNA Miniprep Kit. Totally, 7,182 transcripts were identified to be deregulated between colorectal cancer patients and healthy controls. The majority of them were non-coding RNAs with more than 70 % being lncRNAs, while protein-coding genes represented the second most common gene biotype. We have optimized the protocol for isolation of small EVs and their RNA from low volume of sera and confirmed the suitability of Clariom D Pico Assays for transcriptome profiling.

Standardized evaluation of Zika nucleic acid tests used in clinical settings and blood screening.

Early detection of Zika virus (ZIKV) transmission within geographic regions informs implementation of community mitigation measures such as vector reduction strategies, travel advisories, enhanced surveillance among pregnant women, and possible implementation of blood and organ donor screening or deferral. Standardized, comparative assessments of ZIKV assay and testing lab performance are important to develop optimal approaches to ZIKV diagnostic testing and surveillance. We conducted an expanded blinded panel study to characterize and compare the analytical performance of fifteen diagnostic and blood screening ZIKV NAT assays, including detection among single- and multiplex assays detecting ZIKV, dengue virus (DENV) and chikungunya virus (CHIKV). A 300 member blinded panel was constructed, consisting of 11 serial half-log dilutions ranging from ~104 to 10-1 genome equivalents/mL in 25 replicates each of the Tahitian Asian ZIKV isolate in ZIKV-negative human serum. Additionally, clinical samples from individuals with DENV-like syndrome or suspected ZIKV infection in Brazil were evaluated. The majority of assays demonstrated good specificity. Analytical sensitivities varied 1-2 logs, with a substantially higher limit of detection (LOD) in one outlier. Similar analytical sensitivity for ZIKV RNA detection in singleplex and multiplex assays of the Grifols and ThermoFisher tests were observed. Coefficient of Assay Efficiency (CE), calculated to characterize assays' RNA extraction and amplification efficiency, ranged from 0.13 for the Certest VIASURE multiplex and 0.75 for the Grifols multiplex assays. In general, assays using transcription mediated amplification (TMA) technology had greater CE compared to assays using conventional PCR technology. Donor screening NAT assays were significantly more sensitive than diagnostic RT-qPCR assays, primarily attributable to higher sample input volumes. However, ideal assays to maximize sensitivity and throughput may not be a viable option in all contexts, with other factors such as cost, instrumentation, and regulatory approval status influencing assay availability and selection, particularly in resource constrained settings.

Surfactant Treatment for Efficient Gene Detection of Enteric Viruses and Indicators in Surface Water Concentrated by Ultrafiltration.

The hollow fiber ultrafiltration (HFUF)-based microbial concentration method is widely applied for monitoring pathogenic viruses and microbial indicators in environmental water samples. However, the HFUF-based method can co-concentrate substances that interfere with downstream molecular processes-nucleic acid extraction, reverse transcription (RT), and PCR. These inhibitory substances are assumed to be hydrophobic and, therefore, expected to be excluded by a simple surfactant treatment before the silica membrane-based RNA extraction process. In this study, the efficacy and limitations of the sodium deoxycholate (SD) treatment were assessed by quantifying a process control and indigenous viruses using 42 surface water samples concentrated with HFUF. With some exceptions, which tended to be seen in samples with high turbidity (> 4.0 NTU), virus recovery by the ultrafiltration method was sufficiently high (> 10%). RNA extraction-RT-quantitative PCR (RT-qPCR) efficiency of the process control was insufficient (10%) for 30 of the 42 HFUF concentrates without any pretreatments, but it was markedly improved for 21 of the 30 inhibitory concentrates by the SD treatment. Detection rates of indigenous viruses were also improved and no substantial loss of viral RNA was observed. The SD treatment was particularly effective in mitigating RT-qPCR inhibition, although it was not effective in improving RNA extraction efficiency. The methodology is simple and easily applied. These findings indicate that SD treatment can be a good alternative to sample dilution, which is widely applied to mitigate the effect of RT-qPCR inhibition, and can be compatible with other countermeasures.

Development of Magnetic Deep Eutectic Solvent-Based Liquid-Liquid Extraction for the Selective Extraction and Separation of RNA.

Four kinds of hydrophobic magnetic deep eutectic solvents (HMDESs) were prepared and applied to RNA extraction. Based on the HMDESs, a mechanical shaking-assisted liquid-liquid extraction (MSLLE) was developed for the extraction of RNA. Factors that influence the extraction, including the extraction time, temperature, volume of HMDES, buffer types, and pH, were evaluated. After the optimization of all conditions, the RNA extraction efficiency was 82.31 ± 0.02%. RNA can be extracted from complex samples and medicinal yeast by the method proposed in this work and can be recovered from the HMDESs after being extracted.

Synthesis and Characterization of Silica Coated Magnetite Nanoparticle Clusters for Viral RNA Extraction

Magnetite nanoparticles or iron oxide nanoparticles are the most explored magnetic nanoparticles till recent times, particularly due to their attractive properties for biomedical applications such as viral RNA extraction. The physical and chemical properties of magnetite nanoparticles and their clusters largely depend on the synthesis method and chemical structure. Co-precipitation method was used to synthesize magnetite nanoparticles at varying process parameters. The nanoparticles were coated with silicon-oxide using Stober method at different deposition durations. These particles were characterized by X-Ray Diffraction, Scanning Electron Microscopy, vibrating sample Magnetometer and PCR testing to study the phases formed, morphology, size, magnetic properties and RNA extraction efficiency. The synthesized magnetite nanoparticles were in the range of 10 to 100 nm; suitable for super-para-magnetic behavior. The maximum saturation magnetization achieved for synthesized paramagnetic beads was 56 emu and RNA extraction efficiency was more than 80% as compared to commercial beads.

Metatranscriptomics of cheese microbial communities: Efficiency of RNA extraction from various cheese types and of mRNA enrichment

Microbial communities from cheeses contribute to the development of typical organoleptic properties. Metatranscriptomic analyses can be used to provide a global picture of the functioning of these communities. Our objective was to evaluate the efficiency of RNA extraction from various cheese types and to evaluate mRNA enrichment procedures for metatranscriptomic analyses. For the 32 tested cheese brands, corresponding to five cheese types, the extraction yield varied from 1 μg to 363 μg RNA per gram of cheese and, overall, the yield was lower for fresh cheeses and for the core of pressed cooked cheeses than for the other cheese types. Pressed cooked cheeses also had a lower RNA integrity than the other cheese types. For total RNA extracts from four cheeses, approximately 99% of the sequencing reads corresponded to ribosomal RNA, and mRNA enrichment by RiboPOOL and FastSelect kits decreased this percentage to a range of 75 to 97% and of 53 to 76%, respectively. Comparison of RNA libraries after mRNA enrichment with libraries of undepleted total RNA showed that the FastSelect mRNA enrichment had a lower impact on the gene expression profiles of five target cheese species than the riboPOOL kit and the oligo (dT) selection method. The procedures that we describe in the present study may be useful for metatranscriptomic analysis of various cheese types.

Development, verification, and validation of an RT-qPCR-based protocol for Yellow Fever diagnosis

IntroductionYellow fever (YF) is a public health threat with frequent outbreaks in tropical and subtropical areas, despite the existence of a safe and effective vaccine. The diagnosis of acute infection of the etiologic agent relies mainly on real-time reverse transcription-polymerase chain reaction (RT-qPCR)-based assays. ObjectivesThe aim of this study was to evaluate and compare this novel protocol for yellow fever virus (YFV) diagnosis against assays developed in-house by reference laboratories for arboviruses. MethodsWe developed a novel molecular protocol for the detection of YFV that includes an Internal Control to validate the reaction and an External Control to monitor the RNA extraction efficiency. Results and DiscussionOur assay detects one viral genome per reaction and displays no cross-reactions with dengue (1-4), Zika, or Chikungunya viruses. This novel assay yielded 95% of agreement with the reference method recommended by the Pan American Health Organization when analyzing 204 clinical samples and cultured viruses, these samples were analyzed in 3 different diagnosis centers for arboviruses in Brazil. The data suggest the use of the proposed multiplex assay protocol to do routine tests in a clinical laboratory. This product adds higher specificity and sensitivity in addition to reduced cost per test due to hands-on time and reagent spending.

Production and use of encapsidated RNA mimics as positive control reagents for SARS-CoV-2 RT-qPCR diagnostics

The current gold standard technique for SARS-CoV-2 diagnostics is hydrolysis probe-based RT-qPCR. Reliable testing requires reliable control reagents to monitor the efficiency of RNA extraction, reverse transcription and PCR amplification. Here we describe a custom RNA packaging system from the plant virus cowpea mosaic virus to produce virus-like particles that encapsidate specifically designed portions of the genome of SARS-CoV-2, the causative agent of COVID-19. These encapsidated mimics are highly stable particles which can be used either to spike patient swab samples for use as an in-tube extraction and reaction positive control in multiplex RT-qPCR, or alone as a side-by-side mock-positive control reagent. The selection of sequences in the packaged pseudogenomes ensures that these mimics are compatible with the most commonly used primer/probe combinations for SARS-CoV-2 diagnostics (including German Berlin Charité Hospital, American CDC, and Chinese CDC protocols). The plant transient expression system used to produce these encapsidated mimics is inherently low-cost, and sufficiently high-yielding that a single laboratory-scale preparation can provide enough positive control reagent for millions of tests.

Digital PCR can augment the interpretation of RT-qPCR Cq values for SARS-CoV-2 diagnostics.

Coronavirus disease 2019 (COVID-19) is an infectious, acute respiratory disease caused mainly by person-to-person transmission of the coronavirus SARS-CoV-2. Its emergence has caused a world-wide acute health crisis, intensified by the challenge of reliably identifying individuals likely to transmit the disease. Diagnosis is hampered by the many unknowns surrounding this disease, including those relating to infectious viral burden. This uncertainty is exacerbated by disagreement surrounding the clinical relevance of molecular testing using reverse transcription quantitative PCR (RT-qPCR) for the presence of viral RNA, most often based on the reporting of quantification cycles (Cq), which is also termed the cycle threshold (Ct) or crossing point (Cp). Despite it being common knowledge that Cqs are relative values varying according to a wide range of different parameters, there have been efforts to use them as though they were absolute units, with Cqs below an arbitrarily determined value, deemed to signify a positive result and those above, a negative one. Our results investigated the effects of a range of common variables on Cq values. These data include a detailed analysis of the effect of different carrier molecules on RNA extraction. The impact of sample matrix of buccal swabs and saliva on RNA extraction efficiency was demonstrated in RT-qPCR and the impact of potentially inhibiting compounds in urine along with bile salts were investigated in RT-digital PCR (RT-dPCR). The latter studies were performed such that the impact on the RT step could be separated from the PCR step. In this way, the RT was shown to be more susceptible to inhibitors than the PCR. Together, these studies demonstrate that the consequent variability of test results makes subjective Cq cut-off values unsuitable for the identification of infectious individuals. We also discuss the importance of using reliable control materials for accurate quantification and highlight the substantial role played by dPCR as a method for their development.

P–021 An accurate and reliable screening for SARS-CoV–2 in human sperm samples by RT-PCR: A requirement to evaluate the viral contamination risk during SARS-CoV–2 pandemic

Study questionHow to ensure a reliable and accurate detection of SARS-CoV–2 in seminal plasma and spermatozoa fractions of human sperm samples?Summary answerThis RT-PCR assay showed high sensibility, repeatability and reproducibility for SARS-CoV–2 detection in seminal plasma and spermatozoa fractions, with a detection limit of 17 genomes/reaction.What is known alreadySARS-CoV–2 pandemic brings numerous concerns, such as the safety of gametes for patients undergoing assisted reproductive technologies, fertility preservation or sperm donation. Transient viremia and expression of SARS-CoV–2 receptors in testis and accessory glands bring the question of the presence of the virus in sperm samples. Moreover, the contamination during sperm collection may be possible. The few available studies about this issue mostly showed the absence of SARS-CoV–2 detection in semen of COVID–19 patients, except one reported study. All these studies performed SARS-CoV–2 detection with RT-PCR assays approved for naso-pharyngeal swabs, without a process specifically validated for semen fractions.Study design, size, durationMethod validation was conducted between July 2020 and January 2021. SARS-CoV–2 direct detection was performed according to the French Society of Microbiology guidelines (SFM). Repeatability (n = 6), reproducibility (n = 3), limit of quantification (n = 2) and of detection (n = 6) were evaluated in seminal plasma (SP) and spermatozoa samples isolated after density gradient centrifugation and cryopreserved. In addition, variability of the whole analytical method efficiency was evaluated in samples of men with normal (n = 6) or altered sperm parameters (n = 6).Participants/materials, setting, methodsSamples were surplus semen obtained from men undergoing routine semen analysis after granting informed consent. Assays were performed on SP and frozen spermatozoa fractions. After automated RNA extraction (MGISP–960, MGI-Tech®), real-time RT-PCR was performed using the one-step multiplex TaqPath COVID–19 kit (ThermoFisher®) targeting three viral regions (ORF1, nucleocapsid-N and spike-S proteins). An exogenous internal control was added before RNA extraction. Positive samples and dilution ranges were prepared with a standard (SARS-CoV–2 inactivated virus, QnosticTM Randox®).Main results and the role of chanceRT-PCR assay applied for human sperm samples has been previously validated and is routinely used for SARS-CoV–2 detection in naso-pharyngeal swabs. We evaluated the efficiency of RNA extraction and RT-PCR for SARS-CoV–2 detection in semen fractions. The qualitative and quantitative performance of the whole analytical method was validated with an accuracy profile for SP and spermatozoa fractions. Overall, for repeatability, the standard deviation (SD) of the cycle threshold (Ct) was lower than 0.40 for the strong positive sample and 0.50 for the low positive one. An exception was observed for the S target of the low positive SP samples (SD = 3) which was consistent with S being the less sensitive target of the assay. For reproducibility, SD of the Ct was lower than 0.30 for the strong positive sample and 0.80 for the low positive, except for the S target of the low positive (SD = 1.5). The linearity range was determined for N target, the most sensitive target of the RT-PCR assay. It layed between 5200 and 52 SARS-CoV–2 genomes/reaction. The limit of detection of the RT-PCR assay was 17 viral genomes/reaction. Equal efficiency of the assay was observed for SP and spermatozoa independently of semen parameters (normal and altered sperm parameters). Limitations, reasons for caution: Our detection method was validated for the whole process: RNA extraction (reagents and system), RT-PCR (reagents and thermocycler QuantStudio 5TM) and for both SP and frozen spermatozoa fractions. Variability might be observed with a different extraction system or a different type of biological sample.Wider implications of the findings: This validated RT-PCR assay enables accurate and reliable screening of SARS-CoV–2 in SP and spermatozoa fractions, mandatory to investigate the presence of the virus in semen samples of patients undergoing assisted reproductive techniques, fertility preservation or sperm donation, and to ensure viral safety in the cryobanking process during covid–19 pandemic.Trial registration numberEudraCT 2020-A01409–30

Development of a quantitative real time RT-PCR assay for sensitive and rapid detection of emerging Atypical Porcine Pestivirus associated with congenital tremor in pigs

Atypical Porcine Pestivirus (APPV) is reported as the etiologic agent for type AII congenital tremors in newborn piglets. Initial PCR-based diagnostic tests to detect APPV were designed based on the limited sequence information and are not capable of detecting the majority of APPV strains. A sensitive and reliable PCR-based diagnostic test is critical for accurate detection of APPV. In this study, a quantitative reverse transcription PCR (RT-qPCR) assay was developed for reliable detection of all currently known APPV strains. The assay design also included swine 18S rRNA gene as an internal control to monitor RNA extraction efficiency. Two APPV gene fragments, one each from NS5b and NS3, were cloned and used to determine the dynamic range of detection, linearity and analytical sensitivity/limit of detection (LOD). Both individual and multiplex assays (duplex and triplex) had correlation coefficients of >0.99 and PCR amplification efficiencies of >90 %. Comparison of detection limit and analytical sensitivity between individual, and multiplex assays indicated no inhibition of PCR sensitivity upon multiplexing. The detection limit for APPV target, based on analytical sensitivity, is 7.75 copies (NS5b) and 5.2 copies (NS3) per reaction. Assay specificity was verified by testing nucleic acids of other closely related pestiviruses and clinical samples that are positive for other common swine pathogens. Assay sensitivity was also assessed on synthesized gene fragments of the most divergent China strains. Testing 339 known APPV-positive and 202 negative clinical samples demonstrated a good diagnostic sensitivity and specificity. Data from six independent runs, including 5 replicates of three clinical samples with three Ct ranges, were utilized to assess inter-assay repeatability and intra-assay reproducibility. This analysis demonstrated intra-assay/inter-assay coefficients of variation of 0.71 % and 0.01 %, respectively, with a PCR efficiency of 92.71 % for the triplex assay. Testing of 1785 clinical samples revealed ∼19 % prevalence of APPV in the US swine herds and oral fluids demonstrates to be a reliable specimen for viral detection. This multiplex RT-qPCR assay offers a rapid and reliable detection of APPV in swine herds and serves as useful tool in APPV surveillance and epidemiological investigations.

Molecular diagnosis of SARS-CoV-2 in seminal fluid

PurposeDue to relevant repercussions on reproductive medicine, we aimed to evaluate feasibility of RT-PCR as a detection method of SARS-CoV-2 RNA in seminal fluid.MethodsA qualitative determination of the RT-PCR assays in semen was performed through different approaches: (1) efficiency of RNA extraction from sperm and seminal plasma was determined using PRM1 and PRM2 mRNA and a heterologous system as control; (2) samples obtained by diluting viral preparation from a SARS-CoV-2 panel (virus cultured in Vero E6 cell lines) were tested; (3) viral presence in different fractions of seminal fluid (whole sample, seminal plasma and post-centrifugation pellet) was evaluated. Semen samples from mild and recovered COVID-19 subjects were collected by patients referring to the Infectious Disease Department of the Policlinico Umberto I Hospital - “Sapienza” University of Rome. Control subjects were recruited at the Laboratory of Seminology-Sperm Bank “Loredana Gandini'' of the same hospital.ResultsThe control panel using viral preparations diluted in saline and seminal fluid showed the capability to detect viral RNA presence with Ct values depending on the initial viral concentration. All tested semen samples were negative for SARS-CoV-2, regardless of the nasopharyngeal swab result or seminal fluid fraction.ConclusionThese preliminary data show that RT-PCR for SARS-CoV-2 RNA testing appears to be a feasible method for the molecular diagnosis of SARS-CoV-2 in seminal fluid, supported by results of the control panel. The ability to detect SARS-CoV-2 in semen is extremely important for reproductive medicine, especially in assisted reproductive technology and sperm cryopreservation.

An optimized and robust PEG precipitation method for detection of SARS-CoV-2 in wastewater

Wastewater-based epidemiology is currently being utilized to monitor the dissemination of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), on a population scale. The detection of SARS-CoV-2 in wastewater is highly influenced by methodologies used for its isolation, concentration and RNA extraction. Although various viral concentration methods are currently employed, including polyethylene glycol (PEG) precipitation, adsorption-extraction, ultracentrifugation and ultrafiltration, to our knowledge, none of these methods have been standardized for use with a variety of wastewater matrices and/or different kits for RNA extraction and quantification. To address this, wastewater with different physical characteristics was seeded with gamma-irradiated SARS-CoV-2 and used to test the efficiency of PEG precipitation and adsorption-extraction to concentrate the virus from three physiochemically different wastewater samples, sourced from three distinct wastewater plants. Efficiency of viral concentration and RNA extraction was assessed by reverse-transcriptase polymerase chain reaction and the recovery yields calculated. As co-purification of inhibitors can be problematic for subsequent detection, two commonly used commercial master mixes were assessed for their sensitivity and efficiency to detect two SARS-CoV-2 target nucleocapsid (N) gene sequences. Recovery rates varied greatly between wastewater matrices and concentration methods, with the highest and most reproducible recovery rates (46.6–56.7%) observed when SARS-CoV-2 was precipitated with PEG and detected by the Luna® Universal master mix. The adsorption-extraction method was less effective (0–21.7%). This study demonstrates that PEG precipitation is the more robust method, which translates well to varying wastewater matrices, producing consistent and reproducible recovery rates. Furthermore, it is compatible with different kits for RNA extraction and quantitation.

Effects of Warm Ischemia Time, Cryopreservation, and Grinding Methods on RNA Quality of Mouse Kidney Tissues.

Background: High-quality RNA extraction from tissue samples is of key importance for scientific research and translational medicine. Tissue collection and preparation may affect RNA quality. In this study, we investigated effects of warm ischemia time, cryopreservation, and grinding methods on RNA quality. Methods: Total RNA was extracted from mouse kidney tissues with warm ischemia times of 0, 30, 60, 90, and 120 minutes. Half of the tissues were used to extract RNA immediately, while the others were cryopreserved in the vapor phase of liquid nitrogen for 6 months before RNA extraction. A mortar, homogenizer, and tissue lyser were used to grind tissues. RNA was extracted by TRIzol, and RNA integrity was assessed by the RNA integrity number (RIN) value. Results: For fresh tissues and frozen tissues with warm ischemia time within 60 minutes, RIN values were above 7.0 and remained above 6.0 with warm ischemia time within 120 minutes. For the same warm ischemia time, RIN values of frozen tissues were slightly lower than those of fresh tissues. No significant RIN value alterations were observed among grinding methods, but for RNA extraction efficiency, a mortar was much less efficient than the homogenizer or tissue lyser. For frozen tissues, RNA tended to degrade within 8 minutes at room temperature. Conclusions: Mouse kidney tissues with a warm ischemia time within 120 minutes are suitable for general RNA-related research. For tissues with a warm ischemia time within 60 minutes, cryopreservation may not affect RNA quality. The duration of frozen tissues held at room temperature before grinding affects the integrity of RNA, while grinding methods do not affect RNA integrity.

A comparison of the efficiency of RNA extraction from extracellular vesicles using the Qiagen RNeasy MinElute versus Enzymax LLC RNA Tini Spin columns and qPCR of miRNA.

One consequence of the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is an interruption to the supply of laboratory consumables, particularly those used for RNA extraction. This category includes column-based RNA extraction kits designed to retain short RNA species (defined as having fewer than 200 nucleotides), from small sample volumes, e.g. exosomes or extracellular vesicles (EVs). Qiagen manufactures several kits for the extraction and enrichment of short RNA species, such as microRNA (miRNA), which contain silica-membrane columns called “RNeasy MinElute Spin Columns.” These kits, which also contain buffers and collection tubes, range in price from USD380 to greater than USD1000 and have been subject to fulfillment delays. Scientists seeking to reduce single-use plastics and costs may wish to order the columns separately; however, Qiagen does not sell the RNeasy MinElute Spin Columns (in reasonable quantities) as an individual item. Thus, we sought an alternative product and found RNA Tini Spin columns from Enzymax LLC. We conducted a systematic comparison of the efficiency of RNA extraction for miRNA quantitative real-time PCR (qPCR) using the Qiagen RNeasy MinElute Spin Columns and Enzymax LLC RNA Tini Spin columns and the Qiagen total RNA extraction protocol that enriches for short RNA species. We compared the efficiency of extraction of five spike-in control miRNAs, six sample signal miRNAs, and nine low- to medium-abundance miRNAs by qPCR. The RNA was extracted from EV preparations purified from human plasma using CD81 immunoprecipitation. We report no statistically significant differences in extraction efficiencies between the two columns for any of the miRNAs examined. Therefore, we conclude that the Enzymax RNA Tini Spin columns are adequate substitutes for the Qiagen RNeasy MinElute Spin Columns for short RNA species enrichment and downstream qPCR from EVs in the miRNAs we examined.

Profiling microRNAs in uncomplicated pregnancies: Serum vs. plasma.

Blood-derived microRNAs (miRNAs/miRs) are ideal clinical biomarkers, as they can be relatively non-invasively extracted and are stable across a range of storage conditions. However, the concentration and profile of miRNAs differ between specific patient groups and starting media, which must be a key consideration before embarking upon uses for clinical applications. The optimum blood-derived starting media for biomarker discovery involving pregnant women with an uncomplicated pregnancy has not been determined. Paired serum and plasma samples were collected from 10 pregnant women with uncomplicated low-risk pregnancies at three time points: i) During the second trimester of pregnancy; ii) during the third trimester; and iii) 6 weeks post-partum. Sample miRNA content was assessed using an Agilent Bioanalyzer Small RNA chip and reverse transcription-quantitative (RT-q)PCR using four constitutively expressed miRNAs: hsa-miR-222-3p, hsa-miR-23a, hsa-miR-30e-5p and hsa-miR-451a. Quality control spike-ins measured RNA extraction (UniSp2) and cDNA extraction (cel-miR-39-3p) efficiency. MiRNA concentration and percentage were significantly higher in the serum vs. plasma samples based on data obtained from the Bioanalyzer; however, RT-qPCR failed to replicate these differences in the majority of comparisons using the ΔCq values of the four constitutively expressed miRNAs. Using the standard deviations of the ΔCq values, the consistency of serum and plasma in terms of miRNA expression levels were equivalent. Thus, clinicians and researchers should take into consideration that different miRNA quantification methods can yield contrasting results with regards to the starting media utilized. Based on the equivalent performance of serum and plasma assessed using RT-qPCR, which is less likely to be influenced by the coagulation process or degraded long RNAs, both starting media assessed in the present study are equally suitable for ongoing biomarker discovery studies involving healthy pregnant women at any gestational time point or immediately postpartum.

SARS-CoV-2: Comparative analysis of different RNA extraction methods

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of the COVID-19 pandemic. Although other diagnostic methods have been introduced, detection of viral genes on oro- and nasopharyngeal swabs by reverse-transcription real time-PCR (rRT-PCR) assays is still the gold standard. Efficient viral RNA extraction is a prerequisite for downstream performance of rRT-PCR assays. Currently, several automatic methods that include RNA extraction are available. However, due to the growing demand, a shortage in kit supplies could be experienced in several labs. For these reasons, the use of different commercial or in-house protocols for RNA extraction may increase the possibility to analyze high number of samples. Herein, we compared the efficiency of RNA extraction of three different commercial kits and an in-house extraction protocol using synthetic ssRNA standards of SARS-CoV-2 as well as in oro-nasopharyngeal swabs from six COVID-19-positive patients. It was concluded that tested commercial kits can be used with some modifications for the detection of the SARS-CoV-2 genome by rRT-PCR approaches, although with some differences in RNA yields. Conversely, EXTRAzol reagent was the less efficient due to the phase separation principle at the basis of RNA extraction. Overall, this study offers alternative suitable methods to manually extract RNA that can be taken into account for SARS-CoV-2 detection.

Dendritic Fibrous Nanosilica (DFNS) for RNA Extraction from Cells.

Efficient RNA extraction is critical for all downstream molecular applications and techniques. Despite the availability of several commercial kits, there is an enormous scope to develop novel materials that have high binding and elution capacities. Here, we show that RNA from the cells can be extracted by dendritic fibrous nanosilica (DFNS) with higher efficiency than commercially available silicas. This could be because of the unique fibrous morphology, high accessible surface area, and nanosize particles of DFNS. We studied various fundamental aspects, including the role of particle size, morphology, surface area, and charge on the silica surface in RNA extraction efficiency. Fourier transform infrared (FTIR) spectroscopy studies revealed the interaction of functional groups of RNA with the silica surface, causing selective binding. Due to the sustainable synthesis protocol of DFNS and the simplicity of various buffers and washing solutions used, this RNA extraction kit can be assembled in any lab. In addition to the fundamental aspects of DFNS-RNA interactions, this study has the potential to initiate the development of indigenous DFNS-based kits for RNA extraction.

Comparison of RNA Extraction Methods of Norovirus Evaluated by Real-Time PCR

RNA extraction is essential for reverse transcription (RT) PCR or RT real-time PCR to detect many viral pathogens in various samples. Owing to the lack of cell culture methods of norovirus, only molecular methods have been applied to the detection of norovirus. In the notification of the Ministry of Health, Labour and Welfare, a column method is exemplified for the extraction of RNA from various samples, so extraction of nucleic acid by column method is performed generally. We compared a column method and a magnetic bead method in terms of the RNA extraction efficiency from fecal, oyster and sewage. RT real-time PCR was used for the evaluation of RNA extraction efficiency. The norovirus concentration in oyster and sewage samples was significantly higher when the magnetic bead method was used (p<0.01). It was found that the magnetic bead method is superior for RNA extraction from oyster and sewage samples, and it gives higher RNA yield than the column method.