RNA Yield Research Articles

Urinary cell mRNA profiling of kidney allograft recipients: A systematic investigation of a filtration based protocol for the simplification of urine processing

BackgroundKidney transplantation is a life-restorative therapy, but immune rejection undermines allograft survival. Urinary cell mRNA profiles offer a noninvasive means of diagnosing kidney allograft rejection, but urine processing protocols have logistical constraints. We aimed to determine whether the centrifugation-based method for urinary cell mRNA profiling could be replaced with a simpler filtration-based method without undermining quality. MethodsWe isolated RNA from urine collected from kidney allograft recipients using the Cornell centrifugation-based protocol (CCBP) or the Zymo filter-based protocol (ZFBP) and compared RNA purity and yield using a spectrophotometer or a fluorometer and measured absolute copy number of transcripts using customized real-time quantitative PCR assays. We investigated the performance characteristics of RNA isolated using ZFBP and stored either at -80 °C or at ambient temperature for 2 to 4 days and also when shipped to our Gene Expression Monitoring (GEM) Core at ambient temperature. We examined the feasibility of initial processing of urine samples by kidney allograft recipients trained by the GEM Core staff and the diagnostic utility for acute rejection, of urine processed using the ZFBP. ResultsRNA purity (P = 0.0007, Wilcoxon matched paired signed-ranks test) and yield (P < 0.0001) were higher with ZFBP vs. CCBP, and absolute copy number of 18S rRNA was similar (P = 0.79) following normalization of RNA yield by reverse transcribing a constant amount of RNA isolated using either protocol. RNA purity, yield, and absolute copy numbers of 18S rRNA, TGF-β1 mRNA and microRNA-26a were not different (P > 0.05) in the filtrates containing RNA stored either at -80 °C or at ambient temperature for 2 to 4 days or shipped overnight at ambient temperature. RNA purity, yield, and absolute copy numbers of 18S rRNA and TGF-β1 mRNA were also not different (P > 0.05) between home processed and laboratory processed urine filtrates. Urinary cell levels of mRNA for granzyme B (P = 0.01) and perforin (P = 0.0002) in the filtrates were diagnostic of acute rejection in human kidney allografts. ConclusionsUrinary cell mRNA profiling was simplified using the ZFBP without undermining RNA quality or diagnostic utility. Home processing by the kidney allograft recipients, the stability of RNA containing filtrates at ambient temperature, and the elimination of the need for centrifuges and freezers represent some of the advantages of ZFBP over the CCBP for urinary cell mRNA profiling.

Alcoholic fixation over formalin fixation: A new, safer option for morphologic and molecular analysis of tissues.

Formalin is a widely used fixative but there is potential public health risks to exposure. Besides, alcoholic fixation is advantageous over formalin fixation because of faster fixation, optimal preservation and safer workplace environment. Following fixation by EMA and 10% neutral buffered formalin (NBF), we analyzed the tissue morphology, antigenic stability, DNA and RNA quantity with quality (OD value). The findings of EMA fixing on both the tissue morphology and molecular characterization, were satisfactory. Specially, EMA was faster in penetration of tissues than NBF, fixed ideally as early as 8 h of fixation whereas improper fixation was evident for NBF. In Hematoxylin and Eosin (H & E) staining, better cellular details with stronger affinity for staining were observed. In immunohistochemistry, better antigenic stability was reported for EMA-fixed tissues. The nucleic acid analysis revealed that total genomic DNA and RNA yield from EMA fixed tissues were significantly higher (P < 0.05) with superior quality than NBF fixed tissues. Our results suggest that EMA could be a potential alternative to NBF for fixation and preservation of tissues. These data provide new insights into an option for a safer working environment to support study and research.

Optimized protocol for the extraction of RNA and DNA from frozen whole blood sample stored in a single EDTA tube

Cryopreservation of whole blood is useful for DNA collection, and clinical and basic research. Blood samples in ethylenediaminetetraacetic acid disodium salt (EDTA) tubes stored at − 80 °C are suitable for DNA extraction, but not for high-quality RNA extraction. Herein, a new methodology for high-quality RNA extraction from human blood samples is described. Quickly thawing frozen whole blood on aluminum blocks at room temperature could minimize RNA degradation, and improve RNA yield and quality compared with thawing the samples in a 37 °C water bath. Furthermore, the use of the NucleoSpin RNA kit increased RNA yield by fivefold compared with the PAXgene Blood RNA Kit. Thawing blood samples on aluminum blocks significantly increased the DNA yield by ~ 20% compared with thawing in a 37 °C water bath or on ice. Moreover, by thawing on aluminum blocks and using the NucleoSpin RNA and QIAamp DNA Blood kits, the extraction of RNA and DNA of sufficient quality and quantity was achieved from frozen EDTA whole blood samples that were stored for up to 8.5 years. Thus, extracting RNA from frozen whole blood in EDTA tubes after long-term storage is feasible. These findings may help advance gene expression analysis, as well as biomarker research for various diseases.

Real-World Performance of a Comprehensive Genomic Profiling Test Optimized for Small Tumor Samples.

PURPOSETissue-based comprehensive genomic profiling (CGP) is increasingly used for treatment selection in patients with advanced cancer; however, tissue availability may limit widespread implementation. Here, we established real-world CGP tissue availability and assessed CGP performance on consecutively received samples.MATERIALS AND METHODSWe conducted a post hoc, nonprespecified analysis of 32,048 consecutive tumor tissue samples received for StrataNGS, a multiplex polymerase chain reaction (PCR)–based comprehensive genomic profiling (PCR-CGP) test, as part of an ongoing observational trial (NCT03061305). Sample characteristics and PCR-CGP performance were assessed across all tested samples, including exception samples not meeting minimum input quality control (QC) requirements (< 20% tumor content [TC], < 2 mm2 tumor surface area [TSA], DNA or RNA yield < 1 ng/µL, or specimen age > 5 years). Tests reporting ≥ 1 prioritized alteration or meeting TC and sequencing QC were considered successful. For prostate carcinoma and lung adenocarcinoma, tests reporting ≥ 1 actionable or informative alteration or meeting TC and sequencing QC were considered actionable.RESULTSAmong 31,165 (97.2%) samples where PCR-CGP was attempted, 10.7% had < 20% TC and 59.2% were small (< 25 mm2 tumor surface area). Of 31,101 samples evaluable for input requirements, 8,089 (26.0%) were exceptions not meeting requirements. However, 94.2% of the 31,101 tested samples were successfully reported, including 80.5% of exception samples. Positive predictive value of PCR-CGP for ERBB2 amplification in exceptions and/or sequencing QC-failure breast cancer samples was 96.7%. Importantly, 84.0% of tested prostate carcinomas and 87.9% of lung adenocarcinomas yielded results informing treatment selection.CONCLUSIONMost real-world tissue samples from patients with advanced cancer desiring CGP are limited, requiring optimized CGP approaches to produce meaningful results. An optimized PCR-CGP test, coupled with an inclusive exception testing policy, delivered reportable results for > 94% of samples, potentially expanding the proportion of CGP-testable patients and impact of biomarker-guided therapies.

Assessment and Comparison of Two Different Methods to Extract Nucleic Acids From Individual Honey Bees

AbstractThe honey bee is an excellent model system to study behavioral ecology, behavioral genetics, and sociogenomics. Nucleic acid-based analyses enable a broad scope of research in functional genomics, disease diagnostics, mutant screening, and genetic breeding. Multiple levels of analysis lead to a more comprehensive understanding of the causes of phenotypic variation by integrating genomic variation, transcriptomic profiles, and epigenomic information. One limitation, however, is the sample preparation procedures to obtain high quality DNA and RNA simultaneously, particularly from small amounts of material, such as tissues of individual bees. We demonstrate that it is feasible to perform dual extractions of DNA and RNA from a single individual bee and compare the quality and quantity of the extracted nucleic acids using two different types of methods. There was a greater total yield of DNA and RNA from ethanol-based extractions with minimal differences in overall concentration in ng/uL. We describe here the first validated method for dual extraction of DNA and RNA specifically from individual honey bees (Apis mellifera).

Efficient method for isolation of high-quality RNA from Psidium guajava L. tissues.

Acquiring high-quality RNA in sufficient amounts is crucial in plant molecular biology and genetic studies. Several methods for RNA extraction from plants are available in the literature, mainly due to the great biochemical diversity present in each species and tissue, which can complicate or prevent the extraction. Psidium guajava (Myrtaceae family) is a perennial fruit tree of medicinal and economic value; nevertheless, only a few molecular studies are available for the species. One reason is the difficulty in obtaining RNA due to the content of the samples, which are rich in polyphenols, polysaccharides, and secondary metabolites. Furthermore, there are few studies available for the isolation of RNA from guava or Psidium samples, which hampers advances in the study of the genus. Here, quality and yields of RNA isolates were compared using six extraction protocols: two protocols based on the application of cetyltrimethylammonium bromide (CTAB) lysis buffer, one protocol which uses the TRIzol reagent, one which applies guanidine thiocyanate lysis buffer followed by organic phase extraction, and two commercial kits (PureLink RNA Mini Kit and RNeasy Plant Mini Kit). The CTAB-based method provided the highest RNA yields and quality for five different tissues (flower bud, immature leaf, young leaf, mature leaf, and root), genotypes, and stress conditions. For the most efficient protocol, the average yield of RNA from guava leaves was 203.06 μg/g of tissue, and the A260/A280 and A260/A230 ratios were 2.1 and 2.2, respectively. RT-qPCR analysis demonstrated that the purity of the samples was sufficient for molecular biology experiments. CTAB-based methods for RNA isolation were found to be the most efficient, providing the highest RNA yields and quality for tissues from P. guajava. Additionally, they were compatible for downstream RNA-based applications, besides being simple and cost-effective.

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint.

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

High-salt transcription of DNA cotethered with T7 RNA polymerase to beads generates increased yields of highly pure RNA

High yields of RNA are routinely prepared following the two-step approach of high-yield in vitro transcription using T7 RNA polymerase followed by extensive purification using gel separation or chromatographic methods. We recently demonstrated that in high-yield transcription reactions, as RNA accumulates in solution, T7 RNA polymerase rebinds and extends the encoded RNA (using the RNA as a template), resulting in a product pool contaminated with longer-than-desired, (partially) double-stranded impurities. Current purification methods often fail to fully eliminate these impurities, which, if present in therapeutics, can stimulate the innate immune response with potentially fatal consequences. In this work, we introduce a novel in vitro transcription method that generates high yields of encoded RNA without double-stranded impurities, reducing the need for further purification. Transcription is carried out at high-salt conditions to eliminate RNA product rebinding, while promoter DNA and T7 RNA polymerase are cotethered in close proximity on magnetic beads to drive promoter binding and transcription initiation, resulting in an increase in overall yield and purity of only the encoded RNA. A more complete elimination of double-stranded RNA during synthesis will not only reduce overall production costs, but also should ultimately enable therapies and technologies that are currently being hampered by those impurities.

THE ASSESSMENT ON RNA QUALITY AND BANANA RIPENING GENES (MaGAPDH, MaACS1, AND MaACO1) FROM FLESH TISSUES WITH DIFFERENT PRESERVATION METHODS

The preservation of banana fruit is vital to ensure ripening quality during the transportation process. The quality of banana can be defined using the gene expression marker, which needs high-quality RNA. However, when access to the laboratory is difficult, during long-distance transportation, lack of liquid nitrogen or refrigerator, we need an alternative for preservation methods, so the sample and the extracted RNA are still in excellent conditions. Aim of our research is to find the best preservation methods and test the genes expression marker of Cavendish banana flesh tissues without using the liquid nitrogen. In this study, we performed preservation methods for RNA extraction by directly storing the flesh tissues at -80°C refrigerator and combined with the use of various flesh cutting time (0, 19, and 30 minutes). We measured the total RNA yield, absorbance ratio of 260/280, and checked the expression of MaGAPDH, MaACS1, and MaACO1 genes using Reverse Transcriptase- PCR (RT-PCR). As a result, we found that the preservation methods did not affect the quality of RNA and the genes expression as well. Although the gene expression analysis need to be confirmed in future using quantitative PCR (qPCR) analysis. However, our preservation methods can be used as an alternative preservation method.

RNA Purity and Yield of Frozen Tumor Tissues from the Biorepository Unit, Chulabhorn Royal Academy

Background: Tumor specimens are valuable and beneficial for medical research. Effective storage is critical for the quality of frozen tumor specimens for successful outcomes. Long-term preservation of tissues can impact RNA more than DNA or protein. Thus, the quality of RNA needs to be maintained from the beginning of tissue collection until its utilization. Objective: The present study evaluated the purity and yield of tumor specimens stored at -80&deg;C in the Biorepository Unit from 2010 to 2019. Materials and Methods: Fresh frozen tumor tissues of colon, liver, cervix, and lung from several cases were annually qualified using a Nanodrop spectrophotometer. Results: We found that the optical density (OD) 260/280 ratio of RNA from all specimens remained more than 1.8. Preservative periods of 1 to 9 years did not affect purity of RNA from tissues. Additionally, RNA yields of tumor tissues were in the range of 0.89 to 15.10 &mu;g/mg depending on the tissue type. Conclusion: The results of the present study suggest that the frozen specimens from Biorepository Unit were properly preserved for the long-term and yielded quality RNA. Keywords: RNA purity, RNA yield, Tumor tissue, Biorepository

Application across species of a one health approach to liquid sample handling for respiratory based -omics analysis

Airway inflammation is highly prevalent in horses, with the majority of non-infectious cases being defined as equine asthma. Currently, cytological analysis of airway derived samples is the principal method of assessing lower airway inflammation. Samples can be obtained by tracheal wash (TW) or by lavage of the lower respiratory tract (bronchoalveolar lavage (BAL) fluid; BALF). Although BALF cytology carries significant diagnostic advantages over TW cytology for the diagnosis of equine asthma, sample acquisition is invasive, making it prohibitive for routine and sequential screening of airway health. However, recent technological advances in sample collection and processing have made it possible to determine whether a wider range of analyses might be applied to TW samples. Considering that TW samples are relatively simple to collect, minimally invasive and readily available in the horse, it was considered appropriate to investigate whether, equine tracheal secretions represent a rich source of cells and both transcriptomic and proteomic data. Similar approaches have already been applied to a comparable sample set in humans; namely, induced sputum. Sputum represents a readily available source of airway biofluids enriched in proteins, changes in the expression of which may reveal novel mechanisms in the pathogenesis of respiratory diseases, such as asthma and chronic obstructive pulmonary disease. The aim of this study was to establish a robust protocol to isolate macrophages, protein and RNA for molecular characterization of TW samples and demonstrate the applicability of sample handling to rodent and human pediatric bronchoalveolar lavage fluid isolates. TW samples provided a good quality and yield of both RNA and protein for downstream transcriptomic/proteomic analyses. The sample handling methodologies were successfully applicable to BALF for rodent and human research. TW samples represent a rich source of airway cells, and molecular analysis to facilitate and study airway inflammation, based on both transcriptomic and proteomic analysis. This study provides a necessary methodological platform for future transcriptomic and/or proteomic studies on equine lower respiratory tract secretions and BALF samples from humans and mice.

Detection of SARs-CoV-2 in wastewater using the existing environmental surveillance network: A potential supplementary system for monitoring COVID-19 transmission.

The ongoing COVID-19 pandemic is caused by SARs-CoV-2. The virus is transmitted from person to person through droplet infections i.e. when infected person is in close contact with another person. In January 2020, first report of detection of SARS-CoV-2 in faeces, has made it clear that human wastewater might contain this virus. This may illustrate the probability of environmentally facilitated transmission, mainly the sewage, however, environmental conditions that could facilitate faecal oral transmission is not yet clear. We used existing Pakistan polio environment surveillance network to investigate presence of SARs-CoV-2 using three commercially available kits and E-Gene detection published assay for surety and confirmatory of positivity. A Two-phase separation method is used for sample clarification and concentration. An additional high-speed centrifugation (14000Xg for 30 min) step was introduced, prior RNA extraction, to increase viral RNA yield resulting a decrease in Cq value. A total of 78 wastewater samples collected from 38 districts across Pakistan, 74 wastewater samples from existing polio environment surveillance sites, 3 from drains of COVID-19 infected areas and 1 from COVID 19 quarantine center drainage, were tested for presence of SARs-CoV-2. 21 wastewater samples (27%) from 13 districts turned to be positive on RT-qPCR. SARs-COV-2 RNA positive samples from areas with COVID 19 patients and quarantine center strengthen the findings and use of wastewater surveillance in future. Furthermore, sequence data of partial ORF 1a generated from COVID 19 patient quarantine center drainage sample also reinforce our findings that SARs-CoV-2 can be detected in wastewater. This study finding indicates that SARs-CoV-2 detection through wastewater surveillance has an epidemiologic potential that can be used as supplementary system to monitor viral tracking and circulation in cities with lower COVID-19 testing capacity or heavily populated areas where door-to-door tracing may not be possible. However, attention is needed on virus concentration and detection assay to increase the sensitivity. Development of highly sensitive assay will be an indicator for virus monitoring and to provide early warning signs.

Microrna Regulation of Nodule Zone-Specific Gene Expression In Soybean

Microrna Regulation of Nodule Zone-Specific Gene Expression In Soybean

Do Tissues Fixed in a Non-crosslinking Fixative Require a Dedicated Formalin-free Processor?

We evaluate the consequences of processing alcohol-fixed tissue in a processor previously used for formalin-fixed tissue. Biospecimens fixed in PAXgene Tissue Fixative were cut into three pieces then processed in a flushed tissue processor previously used for formalin-fixed, paraffin-embedded (FFPE) blocks (neutral buffered formalin [NBF]+ve), a formalin-free system (NBF-ve), or left unprocessed. Histomorphology and immunohistochemistry were compared using hematoxylin/eosin staining and antibodies for MLH-1, Ki-67, and CK-7. Nucleic acid was extracted using the PAXgene Tissue RNA/DNA kits and an FFPE RNA extraction kit. RNA integrity was assessed using RNA integrity number (RIN), reverse transcription polymerase chain reaction (RT-PCR) (four amplicons), and quantitative RT-PCR (three genes). For DNA, multiplex PCR, quantitative PCR, DNA integrity number, and gel electrophoresis were used. Compared with NBF-ve, RNA from NBF+ve blocks had 88% lower yield and poorer purity; average RIN reduced from 5.0 to 3.8, amplicon length was 408 base pairs shorter, and Cq numbers were 1.9-2.4 higher. Using the FFPE extraction kit rescued yield and purity, but RIN further declined by 1.1 units. Differences between NBF+ve and NBF-ve in respect of DNA, histomorphology, and immunohistochemistry were either non-existent or small in magnitude. Formalin contamination of a tissue processor and its reagents therefore critically reduce RNA yield and integrity. We discuss the available options users can adopt to ameliorate this problem.

Isolation of intact extracellular vesicles from cryopreserved samples.

Extracellular vesicles (EVs) have emerged as promising candidates in biomarker discovery and diagnostics. Protected by the lipid bilayer, the molecular content of EVs in diverse biofluids are protected from RNases and proteases in the surrounding environment that may rapidly degrade targets of interests. Nonetheless, cryopreservation of EV-containing samples to -80°C may expose the lipid bilayer to physical and biological stressors which may result in cryoinjury and contribute to changes in EV yield, function, or molecular cargo. In the present work, we systematically evaluate the effect of cryopreservation at -80°C for a relatively short duration of storage (up to 12 days) on plasma- and media-derived EV particle count and/or RNA yield/quality, as compared to paired fresh controls. On average, we found that the plasma-derived EV concentration of stored samples decreased to 23% of fresh samples. Further, this significant decrease in EV particle count was matched with a corresponding significant decrease in RNA yield whereby plasma-derived stored samples contained only 47-52% of the total RNA from fresh samples, depending on the extraction method used. Similarly, media-derived EVs showed a statistically significant decrease in RNA yield whereby stored samples were 58% of the total RNA from fresh samples. In contrast, we did not obtain clear evidence of decreased RNA quality through analysis of RNA traces. These results suggest that samples stored for up to 12 days can indeed produce high-quality RNA; however, we note that when directly comparing fresh versus cryopreserved samples without cryoprotective agents there are significant losses in total RNA. Finally, we demonstrate that the addition of the commonly used cryoprotectant agent, DMSO, alongside greater control of the rate of cooling/warming, can rescue EVs from damaging ice formation and improve RNA yield.

Reference Gene Optimization for Ganglia of the Sympathetic Nervous System in Male and Female Mice after Exposure to Cold Stress

The sympathetic branch of the autonomic nervous system (ANS) regulates the body’s response to psychogenic and systemic stress. In response to stress, endocrine pathways are activated to rebalance physiological changes and restore homeostasis. Gene expression analysis through quantitative real-time PCR (qPCR) is a key method to assess differential gene expression in tissue and cell samples, with high sensitivity and specificity. To assess differential gene expression between samples, qPCR data must be normalized to two or more reference genes to account for technical variation in nucleic acid loading between samples, ensuring data represents target gene expression due to experimental treatments (1). Reference genes must be selected and tested for each experimental paradigm, showing stability of expression between samples and treatments. Reference genes are usually constitutively expressed genes required for cellular maintenance, such as well known “housekeeping” genes or genes encoding ribosomal RNAs. Ganglia of the sympathetic nervous system (SNS) are small in size, and thus total yields of RNA for qPCR experiments are limited. Our group has established a protocol for the isolation of adrenal glands, superior cervical, celiac, and stellate ganglia using a dissecting scope and anatomical landmarks in mice. Here we present optimization data assessing five commonly used reference genes for normalization of qPCR data in three ganglia of the SNS (superior cervical, stellate, and celiac) and adrenal glands. We have evaluated their suitability as reference genes in these tissues after acclimation to cold (4°C) stress compared to the same tissues from animals housed at thermoneutrality (30°C). Through qPCR reaction optimization and subsequent calculations of geNorm stability-measure M, we present optimized primer sequences and reaction conditions, and provide recommendations for combinations of three or more reference genes to use for normalization in gene expression analysis experiments in adrenal gland, superior cervical, stellate or celiac ganglia from male and female mice exposed to thermoneutrality or cold. This research will be valuable to groups interested in SNS physiology and facilitate a streamlined approach to gene expression analysis given the low RNA availability from these minute tissue samples, saving valuable sample, costs and time in carrying out qPCR experiments. Reference: (1) Bustin et al., Clin Chem. 2009 Jan 27;55(4):611-622.

Gene expression of Candida albicans strains isolates from patients with denture stomatitis submitted to treatments with photodynamic therapy and nystatin

The study evaluated the effect of antimicrobial photodynamic therapy (aPDT) and nystatin (NYS) in the expression of genes (ACT1, ALS1, CAP1, CAT1, EFG1, HWP1, LIP3, PLB1, SAP1, and SOD1) involved in the virulence of Candida albicans strains recovered from patients with denture stomatitis (DS). These strains were isolated from the patients before (initial) and after treatment (final), and 45 days after the treatments (follow-up). For gene expression analyses, RNA was isolated from the clinical strains, followed by cDNA synthesis and qPCR using specific primers for each target gene. The samples that present integrity were pooled to increase the RNA yield. In the end, four patients treated with aPDT and five patients treated with NYS had the clinical isolates of C. albicans submitted to gene expression evaluation. The data demonstrated a statistical difference in the expression of PLB1 and ACT1 for the different therapies (aPDT versus NYS). Also, there was a statistical difference in the expression of CAT1, SOD1, and LIP3 at the time intervals assessed (initial, final, and follow-up). In contrast, no statistical difference was found in the expression of ALS1, HWP1, EFG1, CAP1, CAT1, SOD1, LIP3, and SAP1 between the therapies, while no significant difference was detected at the time intervals evaluated for ALS1, HWP1, EFG1, CAP1, and SAP1. Therefore, the topical treatments for DS with aPDT or NYS did not effect the expression of most C. albicans virulence genes evaluated.

High-Quality Nucleic Acid Isolation from Hard-to-Lyse Bacterial Strains Using PMAP-36, a Broad-Spectrum Antimicrobial Peptide.

The efficiency of existing cell lysis methods to isolate nucleic acids from diverse bacteria varies depending on cell wall structures. This study tested a novel idea of using broad-spectrum antimicrobial peptides to improve the lytic efficiency of hard-to-lyse bacteria and characterized their differences. The lysis conditions of Staphylococcus aureus using recombinant porcine myeloid antimicrobial peptide 36 (PMAP-36), a broad-spectrum pig cathelicidin, was optimized, and RNA isolation was performed with cultured pellets of ten bacterial species using various membranolytic proteins. Additionally, three other antimicrobial peptides, protegrin-1 (PG-1), melittin, and nisin, were evaluated for their suitability as the membranolytic agents of bacteria. However, PMAP-36 use resulted in the most successful outcomes in RNA isolation from diverse bacterial species. The amount of total RNA obtained using PMAP-36 increased by ~2-fold compared to lysozyme in Salmonella typhimurium. Streptococci species were refractory to all lytic proteins tested, although the RNA yield from PMAP-36 treatment was slightly higher than that from other methods. PMAP-36 use produced high-quality RNA, and reverse transcription PCR showed the efficient amplification of the 16S rRNA gene from all tested strains. Additionally, the results of genomic DNA isolation were similar to those of RNA isolation. Thus, our findings present an additional option for high quality and unbiased nucleic acid isolation from microbiomes or challenging bacterial strains.

Active Microbiome Structure and Functional Analyses of Freshwater Benthic Biofilm Samples Influenced by RNA Extraction Methods.

Advances in high-throughput sequencing technologies have enabled extensive studies of freshwater biofilms and significant breakthroughs in biofilm meta-omics. To date, however, no standardized protocols have been developed for the effective isolation of RNA from freshwater benthic biofilms. In this study, we compared column-based kit RNA extraction with five RNAzol-based extractions, differentiated by various protocol modifications. The RNA products were then evaluated to determine their integrity, purity and yield and were subjected to meta-transcriptomic sequencing and analysis. Significant discrepancies in the relative abundance of active communities and structures of eukaryotic, bacterial, archaebacterial, and viral communities were observed as direct outcomes of the tested RNA extraction methods. The column isolation-based group was characterized by the highest relative abundance of Archaea and Eukaryota, while the organic isolation-based groups commonly had the highest relative abundances of Prokaryota (bacteria). Kit extraction methods provided the best outcomes in terms of high-quality RNA yield and integrity. However, these methods were deemed questionable for studies of active bacterial communities and may contribute a significant degree of bias to the interpretation of downstream meta-transcriptomic analyses.

A head-to-head comparison of ribodepletion and polyA selection approaches for Caenorhabditis elegans low input RNA-sequencing libraries.

A recent and powerful technique is to obtain transcriptomes from rare cell populations, such as single neurons in Caenorhabditis elegans, by enriching dissociated cells using fluorescent sorting. However, these cell samples often have low yields of RNA that present challenges in library preparation. This can lead to PCR duplicates, noisy gene expression for lowly expressed genes, and other issues that limit endpoint analysis. Furthermore, some common resources, such as sequence-specific kits for removing ribosomal RNA, are not optimized for nonmammalian samples. To advance library construction for such challenging samples, we compared two approaches for building RNAseq libraries from less than 10 nanograms of C. elegans RNA: SMARTSeq V4 (Takara), a widely used kit for selecting poly-adenylated transcripts; and SoLo Ovation (Tecan Genomics), a newly developed ribodepletion-based approach. For ribodepletion, we used a custom kit of 200 probes designed to match C. elegans rRNA gene sequences. We found that SoLo Ovation, in combination with our custom C. elegans probe set for rRNA depletion, detects an expanded set of noncoding RNAs, shows reduced noise in lowly expressed genes, and more accurately counts expression of long genes. The approach described here should be broadly useful for similar efforts to analyze transcriptomics when RNA is limiting.