RNA Extraction Kits Research Articles

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.

Full-in-House Method (FinHM) for SARS-COV-2 Automated Viral RNA Extraction, Followed by in-House ‘Primer-Probe’ Based RT-qPCR Detection; Low Cost Mass Testing

<i>Background</i>: Sever acute respiratory syndrome Coronavirus-2 (SARS-COV-2) spread prompted mass testing. The main method for testing is by any FDA approved kits for RNA extraction followed by One-Step RT-qPCR based on primer-probe assays. Yet, the high demand for these kits created a global bottleneck in the testing capacity. <i>Methods</i>: We developed a Full-In-House Method (FinHM) suitable for automated viral RNA extraction using full in-house solutions utilizing the MagMax^TM beads followed by an In-House RT-qPCR based on the CDC/WHO recommended ‘primer-probe’ assay targeting the following genes; E, RdRp2, and RdRp4. FinHM was validated by an FDA approved kit that targets S, N, and ORF1b genes made by Thermo Fisher Scientific (TF). <i>Results</i>: The sensitivity and specificity of the automated RNA extraction were evaluated on serial dilutions of in-laboratory propagated SARS-COV-2 with a successful detection down to 46 copies in both assays (P>0.05). Moreover, automated FinHM was successful in extraction of SARS-COV-2 RNA in 266 clinical samples, in which the test results replicated the FDA approved test results (>99% similarity, P>0.05). The In-House RT-qPCR assay had low limit of detection (5 RNA templates), with significant negative correlation between the Ct values and RNA titrations as shown by Pearson correlation (-0.8, -0.8 and -0.7 for E, RdRp2 and RdRp4, respectively). Finally, FinHM was also successful in extraction of SARS-COV-2-spiked plasma and patient plasma samples. <i>Conclusion</i>: We report a reliable, reproducible, specific, sensitive and low-cost platform for automated RNA extraction and detection from SARS-COV-2 and other viruses which is suitable for clinical and mass testing.

A Streamlined Approach to Rapidly Detect SARS-CoV-2 Infection Avoiding RNA Extraction: Workflow Validation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly spread worldwide from the beginning of 2020. The presence of viral RNA in samples by nucleic acid (NA) molecular analysis is the only method available to diagnose COVID-19 disease and to assess patients' viral load. Since the demand for laboratory reagents has increased, there has been a worldwide shortage of RNA extraction kits. We, therefore, developed a fast and cost-effective viral genome isolation method that, combined with quantitative RT-PCR assay, detects SARS-CoV-2 RNA in patient samples. The method relies on the addition of Proteinase K followed by a controlled heat-shock incubation and, then, E gene evaluation by RT-qPCR. It was validated for sensitivity, specificity, linearity, reproducibility, and precision. It detects as low as 10 viral copies/sample, is rapid, and has been characterized in 60 COVID-19-infected patients. Compared to automated extraction methods, our pretreatment guarantees the same positivity rate with the advantage of shortening the time of the analysis and reducing its cost. This is a rapid workflow meant to aid the healthcare system in the rapid identification of infected patients, such as during a pathogen-related outbreak. For its intrinsic characteristics, this workflow is suitable for large-scale screenings.

Stability of SARS-CoV-2 RNA in Viral Lysis Buffer Stored at Different Temperatures.

Objectives The present COVID-19 pandemic resulted in an increased need for molecular diagnostic testing. Delay in the specimen processing and suboptimal storage of suspected samples in laboratories leads to degradation of SARS-CoV-2 viral RNA. Viral lysis buffers from RNA extraction kits have the potential to stabilize RNA. Hence, this study aimed to investigate the stability of SARS-CoV-2 RNA in viral lysis buffer at different temperatures and time periods. Materials and Methods Aliquots of samples with known SARS-CoV-2 RNA were processed in viral lysis buffers simultaneously, stored separately at 2 to 8°C and 22 to 28°C for 24 hours, 48 hours and 72 hours. SARS-CoV-2 viral RNA was extracted from each aliquot and analyzed using multiplex real-time PCR. Results SARS-CoV-2 RNA in samples placed in viral lysis buffer was stable for 48 hours at both 2 to 8°C and 22 to 28°C temperatures. Slight decline in the viral RNA quantity was found on aliquots tested after 48 hours of both the temperatures. Conclusions Viral lysis buffer maintains the integrity of SARS-CoV-2 RNA for up to 48 hours even at room temperature and supports delayed diagnosis with an overwhelming sample load in testing laboratories.

First Report of Cucurbit Chlorotic Yellows Virus from Cucumber Plants Affected by Interveinal Yellowing Disease in Jordan

First Report of Cucurbit Chlorotic Yellows Virus from Cucumber Plants Affected by Interveinal Yellowing Disease in Jordan

Prevalence and seasonality of influenza virus among pediatric population in Nepal, 2018

Background: Influenza is one of the emerging cause of acute respiratory infection in children. Very limited information's are available on influenza morbidity and mortality in Nepal. The aim of this study was to determine the prevalence and seasonality of Influenza virus in children (below 5 years) in Nepal. Methods and materials: This was a descriptive study conducted at National Influenza Center, Nepal. A total of 424 throat swab samples were collected from children with influenza like illness from January to December 2018. Total viral RNA was extracted using mini RNA extraction kit (Qiagen, USA) and real time PCR amplification was performed according to US, CDC test protocol. Results: Out of 424 samples, 62 (14.6) samples were positive for influenza virus. Of the total, influenza type A and B were 33 (53.2%) and 29 (47%), respectively. The rate of influenza B virus infection was found higher 29 (46.8%) followed by influenza A subtype A/H3 24 (39%) and A/H1N1 pdm09 9 (14%) during the year 2018. A total of 43 (10%) samples were from SARI case, of which 12 (28%) were influenza positive. Out of 381 (90%) ILI samples, 50 (13%) samples were influenza positive. Influenza was observed year-round in Nepal, however; peak activity was found in winter season (January–March), 2018. Conclusion: Transmission of influenza virus was found year round in Nepal which was similar to subtropical countries of south-east Asia. Hence, influenza diagnostic facilities need to be expanded for timely diagnosis, pandemic preparedness and response.

Performance of concanavalin A-immobilized on polyacrylate beads for the detection of human norovirus and hepatitis A virus in fecal specimens.

Quantitative reverse transcription PCR (qRT-PCR) is a sensitive method for the detection of foodborne viruses in fecal samples. However, the performance of qRT-PCR depends on the efficiency of virus concentration methods. In this study, the effect of Concanavalin A (Con A)-immobilized on polyacrylate beads (Con A-PAB) on the qRT-PCR performance, in terms of sensitivity and specificity to detect foodborne viruses in human fecal specimens was compared with commercial viral RNA extraction kit (VRNA). The detection of foodborne viruses by qRT-PCR was validated by viral genome sequencing. Both Con A-PAB and VRNA methods were equally sensitive and specific for detecting hepatitis A virus in fecal specimens. Even though both methods showed high specificity (100% vs. 100%) for detecting human norovirus (HuNoV), Con A-PAB method exhibited higher sensitivity (100% vs. 42.9%) and accuracy (100% vs. 73.3%) compared to VRNA method. In conclusion, the application of Con A-PAB would improve the performance of qRT-PCR for the detection of HuNoV in fecal samples.

Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification.

Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor. To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples. The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44-104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%. Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.

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.

A simple RNA preparation method for SARS-CoV-2 detection by RT-qPCR

The technique RT-qPCR for viral RNA detection is the current worldwide strategy used for early detection of the novel coronavirus SARS-CoV-2. RNA extraction is a key pre-analytical step in RT-qPCR, often achieved using commercial kits. However, the magnitude of the COVID-19 pandemic is causing disruptions to the global supply chains used by many diagnostic laboratories to procure the commercial kits required for RNA extraction. Shortage in these essential reagents is even more acute in developing countries with no means to produce kits locally. We sought to find an alternative procedure to replace commercial kits using common reagents found in molecular biology laboratories. Here we report a method for RNA extraction that takes about 40 min to complete ten samples, and is not more laborious than current commercial RNA extraction kits. We demonstrate that this method can be used to process nasopharyngeal swab samples and yields RT-qPCR results comparable to those obtained with commercial kits. Most importantly, this procedure can be easily implemented in any molecular diagnostic laboratory. Frequent testing is crucial for individual patient management as well as for public health decision making in this pandemic. Implementation of this method could maintain crucial testing going despite commercial kit shortages.

Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step.

The ongoing COVID-19 pandemic has created an unprecedented need for rapid diagnostic testing. The World Health Organization (WHO) recommends a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription–quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing. The goal of this study was to determine whether SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that omits the RNA extraction step altogether. The direct RT-qPCR approach correctly identified 92% of a reference set of blinded NP samples (n = 155) demonstrated to be positive for SARS-CoV-2 RNA by traditional clinical diagnostic RT-qPCR that included an RNA extraction. Importantly, the direct method had sufficient sensitivity to reliably detect those patients with viral loads that correlate with the presence of infectious virus. Thus, this strategy has the potential to ease supply choke points to substantially expand COVID-19 testing and screening capacity and should be applicable throughout the world.

Troubleshooting of RNA isolation methods in Papanicolaou HPV cervical smears

Cervical cancer is one of the leading causes of cancer in women, worldwide. Infection with human papillomavirus (HPV) has been accepted as the primary cause for the development of invasive cervical cancer and its precursor lesions. Despite HPV infection has been proposed as an indispensable factor for cervical cancer development, only a subset of neoplastic lesions with HPV infection persist and progress to invasive cancer. This suggests us that other molecular events are also involved in cancer progression. Aim of this study was to extract mRNA from cytobrush-collected healthy and HPV infected cervical epithelial cells and investigate various RNA extraction and purification protocols for assessment of RNA yield and quality. Taking into consideration that cervical cancer screening is based on the cytology based Papanicolaou test (Pap test), main challenge is to investigate whether the samples obtained by regular Pap testing can be used for gene expression analysis. For this purpose, a total of 68 cervical specimens were previously tested for HPV infection. Following HPV testing, samples were submitted to RNA extraction and compared to the products after additional purification step involving DNase I. Products obtained after different RNA extraction and purification methods were visualized using 2% agarose gel electrophoresis. In conclusion, DNase I based RNA purification represents a necessary step for the assurance of a high-quality extracted RNA used for gene expression analysis studies. Reliance on commercial kits for RNA extraction only, without performing additional purification step can lead to errors in drawing final conclusions and/or to false negative gene expression profiling, affecting the overall diagnostic procedure. According to obtained results, the type of sampling used in this study was not suitable for the subsequent gene expression analysis.

Development of a Parallel Reaction Monitoring Mass Spectrometry Assay for the Detection of SARS-CoV-2 Spike Glycoprotein and Nucleoprotein.

There is an urgent need for robust and high-throughput methods for SARS-CoV-2 detection in suspected patient samples to facilitate disease management, surveillance, and control. Although nucleic acid detection methods such as reverse transcription polymerase chain reaction (RT-PCR) are the gold standard, during the current pandemic, the deployment of RT-PCR tests has been extremely slow, and key reagents such as PCR primers and RNA extraction kits are at critical shortages. Rapid point-of-care viral antigen detection methods have been previously employed for the diagnosis of respiratory viruses such as influenza and respiratory syncytial viruses. Therefore, the direct detection of SARS-CoV-2 viral antigens in patient samples could also be used for diagnosis of active infection, and alternative methodologies for specific and sensitive viral protein detection should be explored. Targeted mass spectrometry techniques have enabled the identification and quantitation of a defined subset of proteins/peptides at single amino acid resolution with attomole level sensitivity and high reproducibility. Herein, we report a targeted mass spectrometry assay for the detection of SARS-CoV-2 spike protein and nucleoprotein in a relevant biological matrix. Recombinant full-length spike protein and nucleoprotein were digested and proteotypic peptides were selected for parallel reaction monitoring (PRM) quantitation using a high-resolution Orbitrap instrument. A spectral library, which contained seven proteotypic peptides (four from spike protein and three from nucleoprotein) and the top three to four transitions, was generated and evaluated. From the original spectral library, we selected two best performing peptides for the final PRM assay. The assay was evaluated using mock test samples containing inactivated SARS-CoV-2 virions, added to in vitro derived mucus. The PRM assay provided a limit of detection of ∼200 attomoles and a limit of quantitation of ∼ 390 attomoles. Extrapolating from the test samples, the projected titer of virus particles necessary for the detection of SARS-CoV-2 spike and nucleoprotein detection was approximately 2 × 105 viral particles/mL, making it an attractive alternative to RT-PCR assays. Potentially, mass spectrometry-based methods for viral antigen detection may deliver higher throughput and could serve as a complementary diagnostic tool to RT-PCR. Furthermore, this assay could be used to evaluate the presence of SARS-CoV-2 in archived or recently collected biological fluids, in vitro-derived research materials, and wastewater samples.

A simple method for SARS-CoV-2 detection by rRT-PCR without the use of a commercial RNA extraction kit

The World Health Organization (WHO) has declared a pandemic caused by a new coronavirus named SARS-CoV-2. The growing demand for commercial kits used for automated extraction of SARS-CoV-2 RNA, a key step before rRT-PCR diagnosis, could cause a shortage of stocks that hinders the rapid processing of samples.Although the recommendation is to use automated methods for nucleic acid extraction, alternatives are necessary to replace commercial kits. However, these alternatives should be as reliable as automated methods.This work describes a simple method to detect SARS-CoV-2 from specimens collected in different preservation media. Samples were previously inactivated by heating and precipitating with a PEG/NaCl solution before rRT-PCR assays for Orf1ab, N and S genes. The new method was compared with an automated protocol of nucleic acid extraction. Both procedures showed similar analytical results. Consequently, this simple and inexpensive method is a suitable procedure for laboratory diagnosis of SARS-CoV-2 infection.

Stability of MERS-CoV RNA on spin columns of RNA extraction kit at room temperature

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging virus causing a highly fatal respiratory disease in humans. Confirmation of MERS-CoV infection and molecular study on the virus may require transportation of samples to specialized laboratories. While freezing at −80 °C is the gold standard method for RNA preservation, maintaining the integrity of viral RNA during transport will require additional precautions and, as a result, increase transport costs. We aimed at testing the stability of MERS-CoV RNA on spin columns of RNA extraction kit at room temperature for 16 weeks. Respiratory samples spiked with stock culture of MERS-CoV were extracted and loaded on QIAamp Viral RNA Mini Kit spin columns and preserved at room temperature. Amount of viral RNA was evaluated periodically by real-time quantitative reverse-transcription polymerase chain reaction. Minimal changes in cycle threshold values over the study period were noted, suggesting stability of viral RNA by this preservation method.

Use of a simplified sample processing step without RNA extraction for direct SARS-CoV-2 RT-PCR detection.

The severe acute respiratory syndrome coronavirus (SARS-CoV-2) pandemic has resulted in significant shortages of RT-PCR testing supplies including RNA extraction kits. The goal of our study was to determine if a simplified heat-RNA release method would provide comparable detection of SARS-CoV-2 without the need for nucleic acid extraction. RT-PCR results using the ChromaCode HDPCR™ SARS-CoV-2 were compared using the heat-RNA release method and an automated RNA extraction system (EMAG). The heat-RNA release method correctly identified 94 % (81/86 nasopharyngeal samples) that were positive for SARS-CoV-2. Five samples that were missed by heat-RNA release method had a mean Ct value: 35 using the automated extraction instrument, indicating a very low viral load. Our findings show that a simple heat-RNA release method is a reasonable alternative for the majority of COVID-19 positive patients and can help overcome the cost and availability issues of RNA extraction reagents.

SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP.

Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.

Validation of a single-step, single-tube reverse transcription loop-mediated isothermal amplification assay for rapid detection of SARS-CoV-2 RNA.

Introduction The SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has necessitated the exploration of alternative diagnostic options to alleviate supply chain issues.Aim To establish and validate a reverse transcription loop-mediated isothermal amplification (RT- LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs.Methodology We used a commercial RT-LAMP mastermix from OptiGene in combination with a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A single-tube, single-step fluorescence assay was implemented whereby 1 µl of universal transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing the requirement for RNA purification. Amplification and detection could be conducted in any thermocycler capable of holding 65 °C for 30 min and measure fluorescence in the FAM channel at 1 min intervals.Results Assay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed assay sensitivity and specificity of 87 and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical samples of 14 min (sd±7 min). Using dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 Tissue Culture Infectious Dose 50 per ml (TCID50 ml−1), with satisfactory assay sensitivity and specificity. A comparison of 20 clinical specimens between four laboratories showed excellent interlaboratory concordance; performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay.Conclusion With a simplified workflow, The N1 gene Single Tube Optigene LAMP assay (N1-STOP-LAMP) is a powerful, scalable option for specific and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium against COVID-19.

Microbial Diversity and Physicochemical Characteristics of the Maotai-Flavored Liquor Fermentation Process.

The grains fermented in Maotai-flavored liquor can be classified into two groups: high-temperature fermented grains generated by stacking and grains fermented in pits. The Maotai-flavor making process enriches a special microbiota from the fermented grains, which makes related studies more difficult. The use of modern molecular techniques to detect and analyze diversity and changes in total bacterial (16S rRNA sequencing) and fungal (internal transcribed spacer (ITS) sequencing) counts have helped to overcome the shortcomings of traditional technological research. The total RNA extracted by Fe₃O₄-SiO₂ nanoparticles was retrieved into DNA by MagBeads Total RNA Extraction Kit. However, discrepancies exist in the microbial community structures at different fermentation periods. Dominant bacteria include Escherichia-Shigella, Lactobacillus, Clostridium, and Streptococcus species and dominant fungi include Alternaria, Ciliophora, Pyrenochaetopsis, Cyphellophora, Aspergillus, Issatchenkia, Pichia, Candida, and Zygosaccharomyces species. Analyses of the enzymatic activity of samples at different fermentation stages have revealed that some existing bacilli influence amylase activity. Here, to investigate flavor changes during each fermentation round, the liquor quality at all rounds was comprehensively assessed based on the corresponding physicochemical properties, which were summarized by sensory evaluation. These results suggested that the liquor quality in the third, fourth, and fifth fermentation rounds was superior.

Optimization of microRNA Acquirement from Seminal Plasma and Identification of Diminished Seminal microRNA-34b as Indicator of Low Semen Concentration.

About 10–15% of couples who want to conceive suffer from subfertility, while in 30% of these cases, a male factor plays a role. Levels of particular microRNAs in seminal plasma, including those involved in spermatogenesis, may serve as an indicative parameter for subfertility. We first optimized a protocol for acquiring microRNAs from seminal plasma. Next, using a test-validation strategy in a male cohort, we aimed to identify microRNAs of which the levels are related to semen motility and concentration. By qPCR, 742 microRNAs were profiled in three normozoospermic samples, three seminal samples with a low semen motility (asthenozoospermia), and three with a low semen concentration (oligozoospermia). MicroRNAs showing significant differences between groups were further validated in a second cohort consisting of 40 samples with normozoospermia (control group), 47 samples with asthenozoospermia, and 19 samples with oligozoospermia (of which 74% also low motility). Highest microRNA yields were obtained with the Biofluids RNA extraction kit, with inclusion of MS2 RNA carrier and proteinase K treatment to the protocol, and when 50 µL of seminal plasma was used as input. Exosome isolation prior to RNA extraction did not lead to enhanced yields. In the test cohort, 236 microRNAs could be detected, of which 54 microRNAs showed a difference between groups. Five microRNAs were analyzed in the validation cohort. MiR-34b-5p levels in the control group were significantly higher compared to the asthenozoospermia group (p < 0.05) and compared to the oligozoospermia group (p < 0.001). We optimized microRNA acquirement from seminal plasma and identified microRNA levels in relation to semen concentration and motility. As recent human and mouse studies show that the miR-34 family is a marker of low semen concentration and is crucial in spermatogenesis, seminal plasma miR-34b-5p may represent a suitable candidate to study further as a marker of male subfertility.