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We present a course-embedded undergraduate research module that involves real-time polymerase chain reaction testing for the presence of SARS-CoV-2 in environmental samples. A positive control RNA was constructed and two RNA extraction methods were compared and a range of primers were available to compare. Using a combination of published protocols, we assembled a successful project that illustrated a topical exercise similar to real-world assay development. The exercise is aimed at upper-level undergraduates and requires 3 weeks of laboratory periods. The students were able to design and test experimental protocols, while learning about RNA detection. This project could be utilized in upper-level classes including molecular biology, biochemistry, biotechnology, or for independent research projects.

Background RNA detection in plasma/stool is the gold-standard for diagnosis of hepatitis E virus (HEV) infection. The impact of viral extraction methods on HEV RNA detection is poorly investigated. Methods We determined the limit of detection of the RealStar HEV RT-PCR V2.0 Kit (altona Diagnostics, RS) utilizing 3 RNA extraction methods (COBAS® AmpliPrep Total Nucleic Acid Isolation Kit, TNAi Roche; MagNA Pure 96 DNA, Viral NA SV Kit, MgP; QIAamp Viral RNA mini Kit Qiagen; VRK) in plasma and stool. The most sensitive method was evaluated in a total of 307 longitudinal samples of patients with HEV infection (acute = 18/chronic = 36) and compared to results with the former diagnostic standard of our centre (TNAi/FastTrack Diagnostic; FTD). Results The plasma-LOD was 49, 94 and 329 IU/mL for extraction with MgP, VRK and TNAi respectively. In stool, the LOD was 21 IU/mL, 528 IU/mL and indefinable for extraction with TNAi, VRK and MgP respectively. Utilizing longitudinal patient plasma samples, MgP/RS revealed 56 HEV RNA-positive samples in 158 negative samples as determined by TNAi/FTD. In stool, from 37 HEV negative samples (TNAi/FTD), 15 were positive with TNAi/RS. At end of treatment, 8 out of 27 chronically infected patients were RNA positive with MgP/RS, while classified negative with TNAi/FTD. A relapse occurred in 3 of these patients. Conclusion Different methods for RNA extraction and quantification have a significant, compartment-specific impact on the sensitivity of HEV detection. Knowledge about the favourable combinations of extraction and quantification has important implications for diagnosis and patients receiving antiviral therapy.

The SARS-CoV-2 pandemic has created an urgent need for diagnostic tests to detect viral RNA. Commercial RNA extraction kits are often expensive, in limited supply, and do not always fully inactivate the virus. Together, this calls for the development of safer methods for SARS-CoV-2 extraction that utilize readily available reagents and equipment present in most standard laboratories. We optimized and simplified a RNA extraction method combining a high molar acidic guanidinium isothiocyanate (GITC) solution, phenol and chloroform. First, we determined the GITC/RNA dilution thresholds compatible with an efficient two-step RT-qPCR for B2M mRNA in nasopharyngeal (NP) or oropharyngeal (OP) swab samples. Second, we optimized a one-step RT-qPCR against SARS-CoV-2 using NP and OP samples. We furthermore tested a SARS-CoV-2 dilution series to determine the detection threshold. The method enables downstream detection of SARS-CoV-2 by RT-qPCR with high sensitivity (~4 viral RNA copies per RT-qPCR). The protocol is simple, safe, and expands analysis capacity as the inactivated samples can be used in RT-qPCR detection tests at laboratories not otherwise classified for viral work. The method takes about 30 min from swab to PCR-ready viral RNA and circumvents the need for commercial RNA purification kits.

We have tested 16 primer mixes for RT‐LAMP targeting SARS‐CoV‐2 RNA from the available literature in three rounds of sensitivity assays. The selected RT‐LAMP primer mix has a limit of detection of six copies of viral RNA per reaction. Whole saliva, as well as saliva collected using Salivette collection tubes interfered with the RT‐LAMP analysis, but this effect was prevented with the ribonuclease inhibitor, which was confirmed on a small set of correctly diagnosed clinical samples., Summary In the fight against the recent COVID‐19 pandemics, testing is crucial. Nasopharyngeal swabs and real‐time RT‐PCR are used for the detection of the viral RNA. The collection of saliva is non‐invasive, pain‐free and does not require trained personnel. An alternative to RT‐PCR is loop‐mediated isothermal amplification coupled with reverse transcription (RT‐LAMP) that is easy to perform, quick and does not require a thermal cycler. The aim of this study was to test whether SARS‐CoV‐2 RNA can be detected directly in saliva using RT‐LAMP. We have tested 16 primer mixes from the available literature in three rounds of sensitivity assays. The selected RT‐LAMP primer mix has a limit of detection of 6 copies of viral RNA per reaction in comparison with RT‐PCR with 1 copy per reaction. Whole saliva, as well as saliva collected using Salivette collection tubes, interfered with the RT‐LAMP analysis. Neither Chelex‐100 nor protease treatment of saliva prevented the inhibitory effect of saliva. With the addition of the ribonuclease inhibitor, the sensitivity of the RT‐LAMP assay was 12 copies per reaction of RNA in Salivette® saliva samples and 6 copies per reaction of RNA in whole saliva samples. This study shows that it is possible to combine the use of saliva and RT‐LAMP for SARS‐CoV‐2 RNA detection without RNA extraction which was confirmed on a small set of correctly diagnosed clinical samples. Further studies should prove whether this protocol is suitable for point of care testing in the clinical setting.

Background Accumulating evidence shows that microRNAs are aberrantly expressed and exert essential roles in the tumorigenesis and tumor progression of non‐small cell lung cancer (NSCLC). Methods The plasma miRNAs from five healthy donors and four NSCLC patients were profiled by miRNA microarray. The differentially expressed miRNAs from 154 primary NSCLC patients and 146 healthy donors were subjected to RNA isolation and verified by quantitative PCR (qPCR). Results The miRNA microarray analysis revealed that 40 differential miRNAs between NSCLC patients and healthy donors were selected. We found that the plasma miR‐1247‐5p, miR‐301b‐3p and miR‐105‐5p levels of patients were significantly higher than those of healthy controls. The receiver operating characteristic curve (ROC) analyses revealed higher area under the ROC curve (AUC) values and higher sensitivity/specificity of carcinoembryonic antigen (CEA) in combination with miR‐1247‐5p, miR‐301b‐3p, or miR‐105‐5p were superior to that of CEA alone. Conclusions High miR‐1247‐5p, miR‐301b‐3p and miR‐105‐5p expression have been demonstrated to accelerate tumorigenesis, and these three miRNAs in plasma act as novel biomarkers for the early diagnosis of NSCLC patients. Key points Plasma miR‐1247‐5p, miR‐301b‐3p and miR‐105‐5p act as novel biomarkers for early NSCLC and NSCLC., In summary, our results indicate that miR‐1247‐5p, miR‐301b‐3p, and miR‐105‐5p are candidate biomarkers of NSCLC, which perform even better when combined with CEA levels. These findings also prompted us to hypothesize that the early dysregulation of miR‐1247‐5p, miR‐301b‐3p, and miR‐105‐5p in the NSCLC disease process, and the requirement of only a small amount of plasma, render miR‐1247‐5p, miR‐301b‐3p, and miR‐105‐5p very suitable as early warning biomarkers for NSCLC.

The rapid spread of coronavirus disease 2019 (COVID-19), a disease caused by severe acute respiratory syndrome coronavirus 2, poses a huge demand for immediate diagnosis. Real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) of nasopharyngeal (NP) and oropharyngeal (OP) swabs have been used to confirm the clinical diagnosis. To avoid the risk of viral-exposure of laboratory workers, thermal inactivation is currently recommended but has unknown effects on the accuracy of the rRT-PCR results. Thirty-six NP/OP specimens were collected from COVID-19 patients and subjected to thermal inactivation (60°C for 30 min) or the RNA extraction processes to activate the form. Here, our data showed that the concentration of extracted-RNA increases upon thermal inactivation compared to the active form (p = .028). Significantly higher levels of RNA copy number were obtained in inactivated compared to the active samples for both N and ORF1ab genes (p = .009, p = .032, respectively). Thermal inactivation elevated concentration and copy number of extracted-RNA, possibly through viral-capsid degradation and/or nucleoprotein denaturation.

Peroxidase-generated proximity labeling has been widespread used to study the subcellular proteomes and protein interaction networks in living cells, but the development for study of subcellular RNA are limited. APEX-seq has emerged as a new method to study the subcellular RNA in living cells, but the labeling of RNA still have space to improve. In this work, we describe 4-thiouridine (s4U) enhanced peroxidase-generated biotinylation of RNA with high efficiency. The incorporation of s4U could introduce additional sites for RNA labeling, the enhanced biotinylation were observed on monomer, model oligo RNA and total RNA. Through s4U metabolic approach, the in vivo RNA biotinylation efficiency by peroxidase catalysis was also dramatically increased, which will benefit the RNA isolation and the study for spatial transcriptome.

Airway epithelia express intrinsic antimicrobial and nutrient-sequestering factors, which contribute to the host defense of the respiratory tract. Hepcidin is an endogenous peptide hormone that serves as a key regulator of iron metabolism, and ferroportin and ZIP8 are iron transporters. All exhibit innate antimicrobial activity. The purpose of this pilot study is to determine if molecules involved in iron regulation are expressed within sinus epithelia and to compare levels of expression between patients with and without chronic rhinosinusitis (CRS). Sinus mucosa was obtained from patients with (n = 19) and without (n = 14) CRS. Real-time polymerase chain reaction following RNA extraction was used to quantify expression of hepcidin, ferroportin, and ZIP8 mRNA. Hepcidin, ferroportin, and ZIP8 were all detected in the sinus epithelia of patients with and without CRS. However, only ZIP8 was significantly changed in CRS, with a 2.5-fold mean increase in mRNA expression relative to controls (P = .005). These findings suggest that ZIP8 may play a role in the innate epithelial defense of the paranasal sinuses.

The urgent need to implement and rapidly expand testing for severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) infection has led to the development of multiple assays. How these tests perform relative to one another is poorly understood. We evaluated the concordance between the Roche Diagnostics cobas 6800 SARS‐CoV‐2 test and a laboratory‐developed test (LDT) real‐time reverse transcription‐polymerase chain reaction based on a modified Centers for Disease Control and Prevention protocol, for the detection of SARS‐CoV‐2 in samples submitted to the Clinical Laboratories of the Mount Sinai Health System. A total of 1006 nasopharyngeal swabs in universal transport medium from persons under investigation were tested for SARS‐CoV‐2 as part of routine clinical care using the cobas SARS‐CoV‐2 test with subsequent evaluation by the LDT. Cycle threshold values were analyzed and interpreted as either positive (“detected” or “presumptive positive”), negative (not detected), inconclusive, or invalid. Statistical analysis was performed using GraphPad Prism 8. The cobas SARS‐CoV‐2 test reported 706 positive and 300 negative results. The LDT reported 640 positive, 323 negative, 34 inconclusive, and 9 invalid results. When excluding inconclusive and invalid results, the overall percent agreement between the two platforms was 95.8%. Cohen's κ coefficient was 0.904 (95% confidence interval, 0.875‐0.933), suggesting almost perfect agreement between both platforms. An overall discordance rate of 4.2% between the two systems may reflect differences in primer sequences, assay limit of detection, or other factors, highlighting the importance of comparing the performance of different testing platforms., Highlights In this study, we compared the detection of SARS‐CoV‐2 in clinical samples from patients being evaluated for CoVID‐19 infection by two different RT‐PCR assays, the cobas® 6800 SARS‐CoV‐2 test from Roche Molecular Systems and a laboratory‐developed test (LDT) using the Centers for Disease Control and Prevention 2019‐nCoV primers and probes. Overall there was excellent agreement between the two tests methods, although our results suggest that the cobas® SARSCoV‐2 test may have a lower limit of detection than the LDT.

Summary The pandemic coronavirus SARS‐CoV‐2 in the world has caused a large infected population suffering from COVID‐19. To curb the spreading of the virus, WHO urgently demanded an extension of screening and testing; thus, a rapid and simple diagnostic method is needed. We applied a reverse transcription‐loop‐mediated isothermal amplification (RT‐LAMP) to achieve the detection of SARS‐CoV‐2 in 30 min. We designed four sets of LAMP primers (6 primers in each set), targeting the viral RNA of SARS‐CoV‐2 in the regions of orf1ab, S gene and N gene. A colorimetric change was used to report the results, which enables the outcome of viral RNA amplification to be read by the naked eye without the need of expensive or dedicated instrument. The sensitivity can be 80 copies of viral RNA per ml in a sample. We validated the RT‐LAMP method in a hospital in China, employing 16 clinic samples with 8 positives and 8 negatives. The testing results are consistent with the conventional RT‐qPCR. In addition, we also show that one‐step process without RNA extraction is feasible to achieve RNA amplification directly from a sample. This rapid, simple and sensitive RT‐LAMP method paves a way for a large screening at public domain and hospitals, particularly regional hospitals and medical centres in rural areas., A rapid method for the detection of SARS‐CoV‐2 RNA virus.

Problem Current scientific guidelines recommend collecting placental specimens within two hours of delivery for gene expression analysis. However, collecting samples in a narrow time window is a challenge in the dynamic and unpredictable clinical setting, so delays in placental specimen collection are possible. The purpose of our analysis is to investigate temporal changes in placental gene expression by longitudinally sampling placentas over a 24-hour period. Method of study Eight placentas from individuals with uncomplicated, term pregnancies delivered by scheduled cesarean section were collected and sampled following placental delivery and again 1, 2, 4, 6, and 24-hours post-delivery. At each timepoint, biopsies of chorionic villous tissue were taken from 3 cotyledons to account for intra-placental heterogeneity. The 3 biopsies from each timepoint were pooled prior to RNA extraction. Expression of 382 mRNA transcripts was quantified using the nanoString nCounter system. Fold change values were calculated for each timepoint relative to delivery and a fold-change threshold of 1.25 was used to determine a meaningful change from delivery. Results Based on a fold-change threshold of 1.25, 84.3% of transcripts were stable for at least 1 hour, 80.2% were stable for at least two hours, and 20.6% of transcripts were stable through the collection at 24 hours. Conclusions Our results suggest that for some mRNA transcripts, expression changes as time to sample collection increases. We have developed a web application to allow investigators to explore transcripts relevant to their research interests and to set appropriate thresholds to aid in determining whether placentas with delayed sample collection can be included in analyses (https://placentaexpression.foundationsofhealth.org/).

Background Increasing evidence suggests that dysregulated long non-coding RNAs (lncRNAs) are involved in tumorigenesis and progression. RP11-284F21.9, one of the temporally expressed S-phase lncRNAs in cancer cells, was recently identified by nascent RNA capture sequencing. Methods Cal-27, Tca8113, SCC-9, HB56, and oral squamous cell carcinoma (OSCC) tissues were used in the experiment. RNA extraction, qRT-PCR, plasmid construction, cell proliferation, EdU labeling, Transwell migration, luciferase reporter, and western blotting were used to investigate the exact role and function of RP11-284F21.9 in cancer. Results RP11-284F21.9 was upregulated in human OSCC samples and cell lines. RP11-284F21.9 depletion suppressed the proliferation, migration, and invasion of OSCC cell lines. There was interaction between RP11-284F21.9, miR-383-5p, and MAL2. Increased MAL2 and decreased miR-383-5p expression were also detected in OSCC tissues and cell lines. In addition, RP11-284F21.9 knockdown could reduce MAL2 expression, while miR-383-5p inhibitors abolished this repressive effect. RP11-284F21.9 acted as a competing endogenous RNA (ceRNA) of miR-383-5p, leading to MAL2 upregulation, and subsequently promoted OSCC progression. Conclusion RP11-284F21.9/miR-383-5p represents a novel and potential therapeutic target for the treatment of OSCC.

Background Small RNA sequencing (RNA-seq) of biofluids is challenging due to the relative scarcity of microRNAs (miRNAs), limited sample volumes, and the lack of a gold standard isolation method. Additionally, few comparisons exist for the RNA isolation and sequencing methods of biofluids. Objectives We aimed to compare the performance of six commercial RNA isolation kits and two library preparation methods for small RNA-seq using canine serum and urine. Methods Serum and urine were collected from seven dogs with protein-losing nephropathy, and the samples were pooled. Total RNA from serum (2 mL) and urine (10 mL) was isolated in triplicate using three methods each for serum (Zymo Direct-zol, mirVana PARIS, miRCURY Biofluids) and urine (Qiagen exoRNeasy, Norgen Urine Exosome, miRCURY Exosome). For each sample type, the two kits yielding the highest RNA concentration were selected, and small RNA-seq was performed using TruSeq and NEXTflex library preparations. Data were analyzed by CPSS 2.0 and DESeq2. Results For serum, Zymo Direct-zol combined with NEXTflex was the only combination that enabled successful library preparation, while for urine, Qiagen exoRNeasy combined with NEXTflex outperformed other combinations for detecting miRNAs. The total number of miRNAs detected in serum and urine was 198 and up to 115, respectively. miRNA expression in serum was distinct from urine. Furthermore, the library preparation method introduced a higher variation of urine results than the RNA isolation method. Conclusions Different isolation and library preparation methods show significant differences in miRNA results that could affect biomarker discovery. Small RNA-seq provides an unbiased, global assessment to compare these methods in canine biofluids.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in both men and women in the USA. However, the underlying molecular mechanisms that drive CRC tumorigenesis are still not clear. Several studies have reported that long noncoding RNAs (lncRNAs) have important roles in tumor development. Here, we undertook a transcriptome microarray analysis in 6 pairs of CRC tissues and their corresponding adjacent normal tissues. A total of 1705 differentially expressed lncRNAs were detected in CRC tissues at stages I/II and III/IV (fold change greater than or equal to 2 or less than or equal to 0.5). Among them, we found that the lncRNA lung cancer‐associated transcript 1 (LUCAT1) was upregulated in CRC tissues and was closely associated with poor overall survival of CRC patients, through analysis of clinical data and The Cancer Genome Atlas. Functional studies indicated that LUCAT1 promoted CRC cell proliferation, apoptosis, migration, and invasion in vitro and in vivo. Furthermore, knockdown of LUCAT1 rendered CRC cells hypersensitive to oxaliplatin treatment. Mechanistically, bioinformatic analysis indicated that low expression of LUCAT1 was associated with the p53 signaling pathway. Chromatin isolation by RNA purification followed by mass spectrometry and RNA immunoprecipitation revealed that LUCAT1 bound with UBA52, which encodes ubiquitin and 60S ribosomal protein L40 (RPL40). We found that RPL40 functions in the ribosomal protein‐MDM2‐p53 pathway to regulate p53 expression. Taken together, our findings indicate that suppression of LUCAT1 induces CRC cell cycle arrest and apoptosis by binding UBA52 and activating the RPL40‐MDM2‐p53 pathway. These results implicate LUCAT1 as a potential prognostic biomarker and therapeutic target for CRC.

We developed and modified a precise, rapid, and reproducible protocol isolating high-quality RNA from tissues of multiple varieties of cassava plants (Manihot esculenta Crantz). The resulting method is suitable for use in mini, midi, and maxi preparations and rapidly achieves high total RNA yields (170-600 μg·g-1) using low-cost chemicals and consumables and with minimal contamination from polysaccharides, polyphenols, proteins, and other secondary metabolites. In particular, A 260 : A 280 ratios were > 2.0 for RNA from various tissues, and all of the present RNA samples yielded ribosomal integrity number values of greater than six. The resulting high purity and quality of isolated RNA will facilitate downstream applications (quantitative reverse transcriptase-polymerase chain reaction or RNA sequencing) in cassava molecular breeding.

Next generation sequencing has revolutionized molecular biology and has provided a mechanism for rapid DNA and RNA sequence analysis. Yet, there are few resources to introduce next generation sequencing into the undergraduate biochemistry and molecular biology curriculum. Herein, we describe the design, execution, and assessment of a four-week laboratory for junior and senior undergraduate students that focuses on bacterial gene expression changes detected by RNA sequencing (RNA-seq). In the laboratory, students analyze a bacterial RNA-seq dataset in detail and answer questions relating to the impact of DNA methylation on bacterial gene expression. In addition, students confirm key results from the RNA-seq dataset using qRT-PCR and compare their results to similar experiments in the literature. A major strength of the laboratory is the ability of students to analyze raw RNA-seq data. In addition, another strength of the laboratory is the utilization of both dry approaches (informatics and statistics) and wet approaches (RNA isolation, cDNA synthesis, and qRT-PCR) to answer bacterial gene expression questions. Assessment of the laboratory indicates that significant learning gains were achieved with respect to next generation sequencing and RNA-seq. We expect that the laboratory will be a valuable resource as is, or via modification with other datasets and projects. © 2019 International Union of Biochemistry and Molecular Biology, 47(2): 161-167, 2019.

The most common method for RNA detection involves reverse transcription followed by quantitative polymerase chain reaction (RT‐qPCR) analysis. Commercial one‐step master mixes—which include both a reverse transcriptase and a thermostable polymerase and thus allow performing both the RT and qPCR steps consecutively in a sealed well—are key reagents for SARS‐CoV‐2 diagnostic testing; yet, these are typically expensive and have been affected by supply shortages in periods of high demand. As an alternative, we describe here how to express and purify Taq polymerase and M‐MLV reverse transcriptase and assemble a homemade one‐step RT‐qPCR master mix. This mix can be easily assembled from scratch in any laboratory equipped for protein purification. We also describe two simple alternative methods to prepare clinical swab samples for SARS‐CoV‐2 RNA detection by RT‐qPCR: heat‐inactivation for direct addition, and concentration of RNA by isopropanol precipitation. Finally, we describe how to perform RT‐qPCR using the homemade master mix, how to prepare in vitro−transcribed RNA standards, and how to use a fluorescence imager for endpoint detection of RT‐PCR amplification in the absence of a qPCR machine In addition to being useful for diagnostics, these versatile protocols may be adapted for nucleic acid quantification in basic research. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of a one‐step RT‐qPCR master mix using homemade enzymes Basic Protocol 2: Preparation of swab samples for direct RT‐PCR Alternate Protocol 1: Concentration of RNA from swab samples by isopropanol precipitation Basic Protocol 3: One‐step RT‐qPCR of RNA samples using a real‐time thermocycler Support Protocol: Preparation of RNA concentration standards by in vitro transcription Alternate Protocol 2: One‐step RT‐PCR using endpoint fluorescence detection

Molecular diagnosis and surveillance of pathogens such as SARS‐CoV‐2 depend on nucleic acid isolation. Pandemics at the scale of COVID‐19 can cause a global shortage of proprietary commercial reagents and BSL‐2 laboratories to safely perform testing. Therefore, alternative solutions are urgently needed to address these challenges. An open‐source method, magnetic‐nanoparticle‐aided viral RNA isolation from contagious samples (MAVRICS), built upon readily available reagents, and easily assembled in any basically equipped laboratory, is thus developed. The performance of MAVRICS is evaluated using validated pathogen detection assays and real‐world and contrived samples. Unlike conventional methods, MAVRICS works directly in samples inactivated in phenol‐chloroform (e.g., TRIzol), thus allowing infectious samples to be handled safely without biocontainment facilities. MAVRICS allows wastewater biomass immobilized on membranes to be directly inactivated and lysed in TRIzol followed by RNA extraction by magnetic nanoparticles, thereby greatly reducing biohazard risk and simplifying processing procedures. Using 39 COVID‐19 patient samples and two wastewater samples, it is shown that MAVRICS rivals commercial kits in detection of SARS‐CoV‐2, influenza viruses, and respiratory syncytial virus. Therefore, MAVRICS is safe, fast, and scalable. It is field‐deployable with minimal equipment requirements and could become an enabling technology for widespread testing and wastewater monitoring of diverse pathogens., One important bottleneck in the diagnosis and surveillance of COVID‐19 is the shortage of kits for RNA extraction. Magnetic‐nanoparticle‐aided viral RNA isolation from contagious samples (MAVRICS) is an open‐source, safe, fast, and scalable method for RNA extraction. MAVRICS rivals commercial kits but requires minimal materials, and thus could become an enabling technology for widespread community testing of diverse pathogens.

Stored biological materials should have minimal pre‐analytical variations in order to provide researchers with high‐quality samples that will give reliable and reproducible results, yet methods of storage should be easy to implement, with minimal cost and health hazard. Frozen tissue samples are a valuable biological resource. Here we compare different methods, such as liquid nitrogen (LN) or dry ice (DI), to a cheap and safe alternative using an aluminum platform (AP). Murine fresh liver and pancreas tissues were used with varying lengths of warm ischemia time. Quality assessment was based on histological evaluation, DNA and RNA extraction and quantification, and RNA degradation analysis, as well preservation of antigens for immunofluorescence, in a blinded manner. Both in superficial and deep tissue sections, based on histological assessment, AP is superior to DI, or as good as LN techniques in terms of presence of ice crystals, cutting artifacts, and overall quality/structural preservation. DNA and RNA were successfully extracted in reasonable quantities from all freezing techniques, but RNA degradation was seen for pancreas samples across all techniques. Immunofluorescence with cytokeratin8 (CK‐8), alpha smooth muscle actin (αSMA), CD3, and B220 shows equally good outcomes for AP and LN, which are better than DI. The aluminum platform is a cheap, yet reliable method to freeze samples, rapidly preserving histological, antigenic, and DNA/RNA quality. Wider testing is required across different sample types. © 2020 The Authors. Basic Protocol: Flash‐freezing fresh tissue with aluminum platform Alternate Protocol 1: Freezing fresh tissue with liquid nitrogen Alternate Protocol 2: Freezing fresh tissue with dry ice

Long read sequencing technologies now allow high-quality sequencing of RNAs (or their cDNAs) that are hundreds to thousands of nucleotides long. Long read sequences of nascent RNA provide single-nucleotide-resolution information about co-transcriptional RNA processing events-e.g., splicing, folding, and base modifications. Here, we describe how to isolate nascent RNA from mammalian cells through subcellular fractionation of chromatin-associated RNA, as well as how to deplete poly(A)+ RNA and rRNA, and, finally, how to generate a full-length cDNA library for use on long read sequencing platforms. This approach allows for an understanding of coordinated splicing status across multi-intron transcripts by revealing patterns of splicing or other RNA processing events that cannot be gained from traditional short read RNA sequencing. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Subcellular fractionation Basic Protocol 2: Nascent RNA isolation and adapter ligation Basic Protocol 3: cDNA amplicon preparation.

Analysis of RNA structuromes provides new insights into cellular processes, enabling systems biology and biotechnology researchers to calculate promoter and terminator strengths and to directly observe how differing circuit states impact host gene expression and the burdens imposed by the circuits. Such analysis, however, is crucially dependent on the availability of highly pure, intact RNA isolated from fresh or frozen cell cultures. RNA extraction from the yeast Pichia pastoris requires specific pretreatment steps to ensure the reproducibility of downstream applications, but current methods and extraction kits are generally adapted for the conventional yeast Saccharomyces cerevisiae, which has a different cell wall composition. We therefore set out to compare the efficacy of two different RNA isolation methods when applied to P. pastoris: (i) phenol/chloroform extraction and (ii) silica spin-column absorption. We compared the yield, integrity, and purity of the resulting isolated RNA from the two methods (using two different types of commercial columns for silica spin-column absorption) and further optimized them through variations in the pretreatment steps. We also assessed two different methods of cell lysis: enzyme catalytic disruption using lyticase and mechanical disruption using acid-washed glass-beads in a TissueLyser. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: RNA isolation with phenol/chloroform extraction: monophasic lysis reagent Alternate Protocol 1: RNA isolation with silica-spin column absorption: High Pure RNA Isolation Kit (Roche Life Science) Alternate Protocol 2: RNA isolation with silica-spin column absorption: RNeasy Mini Kit (Qiagen).

Fucosidosis is a rare lysosomal storage disease which has been classified into two subtypes, depending on the severity of clinical signs and symptoms. Fucosidosis patients’ skin abnormalities include angiokeratoma corporis diffusum, widespread telangiectasia, thick skin, hyperhidrosis and hypohidrosis, acrocyanosis and distal transverse nail bands. It has been described that >50% of fucosidosis patients have angiokeratoma. At molecular level, fucosidosis is caused by lysosomal alpha-L-fucosidase (FUCA1) gene mutations. Obtaining samples for functional studies has been challenging due to the inherent difficulty in finding affected individuals. The effect of FUCA1 dysfunction on gene expression is unknown. The aim of this study was to analyse, in keratinocytes, the transcriptomic effect of FUCA1 knock-down for a better understanding of skin lesions’ pathogenesis affecting fucosidosis patients. FUCA1 knock-down (siRNA) was performed in human HaCaT immortalised keratinocytes. Affymetrix arrays and qPCR were used for analysing gene expression. Bioinformatics was used for functional clustering of modified genes. In total, 387 genes showed differential expression between FUCA1 silenced and non-silenced cells (222 up-regulated and 165 down-regulated). Up-regulated genes belonged to two major groups: keratinocyte differentiation/epidermal development (n = 17) and immune response (n = 61). Several transcription factors were up-regulated in FUCA1-siRNA transfected cells. This effect might partly have been produced by abnormal transcription factor expression, that is FOXN1. We thus propose that fucosidosis-related skin lesions (eg angiokeratoma) and those of other diseases (eg psoriasis) might be caused by dysfunctions in common aetiological overlapping molecular cascades. © 2018 John Wiley and Sons A/S. Published by John Wiley and Sons Ltd

Objectives To identify differentially expressed genes between relapsed and non-relapsed clinical stage I testicular germ cell tumours (TGCTs). Materials and methods We reviewed patients with clinical stage I non-seminoma and seminoma from an institutional database (2000-2012) who were managed by active surveillance. Patients with non-relapsed non-seminoma and non-relapsed seminoma were defined as being relapse-free after 2 and 3 years of surveillance, respectively. RNA extraction and gene expression analysis was performed on archival primary tumour samples and gene-set enrichment analysis (GSEA) was conducted in order to identify differentiating biological pathways. Results A total of 57 patients (relapsed non-seminoma, n = 12; relapsed seminoma, n =15; non-relapsed non-seminoma, n = 15; non-relapsed seminoma, n = 15) were identified, with a median (range) relapse time of 5.6 (2.5-18.1) and 19.3 (4.7-65.3) months in the relapsed non-seminoma and relapsed seminoma cohorts, respectively. A total of 1 039 differentially expressed genes were identified that separated relapsed and non-relapsed groups. In patients with relapse, GSEA revealed enrichment in pathways associated with differentiation, such as skeletal development (i.e. FGFR1, BMP4, GLI2, SPARC, COL2A1), tissue (i.e. BMP4, SPARC, COL13A1) and bone remodelling (i.e. CARTPT, GLI2, MGP). A discriminative gene expression profile between relapsed and non-relapsed cases was discovered when combining non-seminoma and seminoma samples using 10- and 30-probe signatures; however, this profile was not observed in the seminoma and non-seminoma cohorts individually. Conclusion A discriminating signature for relapsed disease was identified for clinical stage I TGCT that we were not able to identify by histology alone. Further validation is required to determine if this signature provides independent prognostic information to standard pathological risk factors.

In order to study the transcriptome of individual plant cells at specific points in time, we developed protocols for fixation, embedding, and sectioning of plant tissue followed by laser capture microdissection (LCM) and processing for RNA recovery. LCM allows the isolation of individual cell types from heterogeneous tissue sections and is particularly suited to plant processing because it does not require the breakdown of cell walls. This approach allows accurate separation of a small volume of cells that can be used to study gene expression profiles in different tissues or cell layers. The technique does not require separation of cells by enzymatic digestion of any kind, does not require cell-specific reporter genes, and allows storage of fixed and embedded tissue for months before capture. The methods for fixation, embedding, sectioning, and capture of plant cells that we describe yield high-quality RNA suitable for making libraries for RNASeq. © 2018 by John Wiley & Sons, Inc.

The practice of RNA isolation in undergraduate experimental courses is rare because of the existence of robust, ubiquitous and stable ribonucleases. We reported here modifications to our original protocol for RNA isolation from plant tissues, including the recovery of nucleic acids by ethanol precipitation at 0 °C for 10 min and the assessment of RNA quality by visualizing the banding profile of the separated RNAs on a standard nondenaturing agarose gel to shorten the duration of the whole procedure and simplify the operation. As a result, the modified procedure, including RNA isolation and quality control analysis could be finished in 4 hr and divided into two sessions. Because endogenous ribonucleases released upon disruption of the organelles and vacuoles were effectively and quickly inactivated, measures were taken to protect RNA integrity throughout the whole procedure so that total RNA with high purity and integrity as well as an appropriate yield could be obtained by students. The RNA isolation protocol described here was simple, efficient, flexible, and low cost. Therefore, it is an ideal approach for undergraduates to learn about RNA techniques. The pedagogical approach of the correlation of experimental work with the rationale for the whole protocol described in this report is an effective way for undergraduates to improve their learning of the techniques of RNA isolation and analysis and the theories behind them, as well as experimental design and data analysis. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(3):253-261, 2018.

BACKGROUND Metastatic biopsies are increasingly being performed in patients with advanced prostate cancer to search for actionable targets and/or to identify emerging resistance mechanisms. Due to a predominance of bone metastases and their sclerotic nature, obtaining sufficient tissue for clinical and genomic studies is challenging. METHODS Patients with prostate cancer bone metastases were enrolled between February 2013 and March 2017 on an institutional review board-approved protocol for prospective image-guided bone biopsy. Bone biopsies and blood clots were collected fresh. Compact bone was subjected to formalin with a decalcifying agent for diagnosis; bone marrow and blood clots were frozen in optimum cutting temperature formulation for next-generation sequencing. Frozen slides were cut from optimum cutting temperature cryomolds and evaluated for tumor histology and purity. Tissue was macrodissected for DNA and RNA extraction, and whole-exome sequencing and RNA sequencing were performed. RESULTS Seventy bone biopsies from 64 patients were performed. Diagnostic material confirming prostate cancer was successful in 60 of 70 cases (85.7%). The median DNA/RNA yield was 25.5 ng/μL and 16.2 ng/μL, respectively. Whole-exome sequencing was performed successfully in 49 of 60 cases (81.7%), with additional RNA sequencing performed in 20 of 60 cases (33.3%). Recurrent alterations were as expected, including those involving the AR, PTEN, TP53, BRCA2, and SPOP genes. CONCLUSIONS This prostate cancer bone biopsy protocol ensures a valuable source for high-quality DNA and RNA for tumor sequencing and may be used to detect actionable alterations and resistance mechanisms in patients with bone metastases. Cancer 2017. © 2017 American Cancer Society.

The original single-step method is the first procedure to isolate purified total ribonucleic acid (RNA) from a variety of sources including tissues and cells from human, animal, plant, yeast, bacterial and viral origins, without the requirement of high-speed ultracentrifugation. The method is based on liquid-phase separation resulting in sequestration of pure RNA into the aqueous phase. RNA is precipitated from the aqueous phase, dissolved, reprecipitated and washed with alcohol before the final solubilisation step. The entire procedure can be completed in less than 4 h and it provides RNA that is suitable for many sensitive downstream applications such as RNase protection assays, northern blotting, sequencing studies and reverse transcription-polymerase chain reaction. This pioneering methodology has served as the impetus for the development of newer and improved RNA extraction methodology that now enable investigators to extract and purify RNA in less than 60 min. Key Concepts: Enzymes within living cells rapidly degrade RNA after a tissue sample is removed from the donor. To prevent RNA degradation, tissue samples that cannot be immediately processed must be rapidly frozen with dry ice or liquid nitrogen and stored frozen at −80 °C until the RNA can be extracted. At the time of RNA extraction, frozen cells or tissues must be immersed in the denaturing solution and rapidly homogenised before the tissue thaws in order to inactivate RNAse and avoid RNA degradation. The quality of the recovered RNA is dependent on a delicate balance of salt concentration and optimal pH. Overloading the extraction solution with too much tissue or diluting the denaturing solution beyond what is specified in the protocol will impact the quality of the resulting RNA. The extraction of RNA from samples that have a high buffering capacity, such as blood, plasma or tissue culture medium, requires greater care in order to maintain optimal salt balance and pH control. Overdrying of the RNA pellets will impede RNA solubilisation. RNA should be solubilised at a concentration that will be appropriate for meaningful spectrophotometric quantitation as well as subsequent downstream molecular biology applications. Enzymes that are involved in RNA degradation are ubiquitous and special care must be taken to avoid RNAse contamination during RNA solubilisation and storage. Keywords: ribonucleic acid; single-step method; liquid-phase separation; total RNA isolation

The combination of increased healthcare access, universal aging, and infallible therapy demands, synergistically drive the need for the development of biomaterial technologies that mitigate the challenge of biomaterial-associated infections (BAI). Staphylococcus epidermidis and Staphylococcus aureus account for the majority of BAI due to their ability to accumulate in adherent multilayered biofilm. This investigation details the development of gene expression assays to evaluate the genetic processes of attachment, accumulation, maturation, and dispersal phases of biofilms on biomaterials in vitro, while abiding by the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. The biofilm formation of S. epidermidis on polyurethane (PU) central venous catheters and S. aureus on machined titanium (Ti) was examined in terms of gene expression at early and late time points. The results provided insight into how each stage of biofilm formation is orchestrated over time on these biomaterials in vitro. Furthermore, the results suggested that mechanical RNA extraction, organic solvents, elimination of genomic DNA, and preamplification are advisable strategies to implement for biofilm gene expression analysis. It is concluded that this method can be employed for the assessment of biofilm-biomaterial interactions at the molecular level. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3400-3412, 2017.

The next-generation sequencing (NGS) based RNA sequencing (RNA-Seq) and transcriptome profiling offers an opportunity to unveil complex biological processes. Successful RNA-Seq and transcriptome profiling requires a large amount of high-quality RNA. However, NGS-quality RNA isolation is extremely difficult from recalcitrant adipose tissue (AT) with high lipid content and low cell numbers. Further, the amount and biochemical composition of AT lipid varies depending upon the animal species which can pose different degree of resistance to RNA extraction. Currently available approaches may work effectively in one species but can be almost unproductive in another species. Herein, we report a two step protocol for the extraction of NGS quality RNA from AT across a broad range of animal species.

The improvement of doping control is an ongoing race. Techniques to fight doping are usually based on the direct detection of drugs or their metabolites by analytical methods such as chromatography hyphenated to mass spectrometry after ad hoc sample preparation. Nowadays, omic methods constitute an attractive development and advances have been achieved particularly by application of molecular biology tools for detection of anabolic androgenic steroids (AAS), erythropoiesis-stimulating agent (ESA), or to control human growth hormone misuses. These interesting results across different animal species have suggested that modification of gene expression offers promising new methods of improving the window of detection of banned substances by targeting their effects on blood cell gene expression. In this context, the present study describes the possibility of using a modified version of the dedicated Human IVD (in vitro Diagnostics) PAXgene® Blood RNA Kit for horse gene expression analysis in blood collected on PAXgene® tubes applied to the horse biological passport. The commercial kit was only approved for human blood samples and has required an optimization of specific technical requirements for equine blood samples. Improvements and recommendations were achieved for sample collection, storage and RNA extraction procedure. Following these developments, RNA yield and quality were demonstrated to be suitable for downstream gene expression analysis by qPCR techniques. Copyright © 2017 John Wiley & Sons, Ltd.

Background MicroRNAs (miRNAs) can be used for early diagnosis of myocardial infarction. However, due to a lack of standardized operating procedures, their value for clinical application is low. Methods Detection of plasma miRNAs was optimized by analyzing factors influencing miRNA variance and myocardial infarction risk scores during analysis (extraction, reverse transcription, and real‐time PCR) and pre‐analysis (dietary status, anticoagulants, storage conditions, and hemolysis). Results Regarding variable factors during analysis, the centrifugal column method was superior to Trizol LS reagent when extracting miRNA from plasma. Recovery rate was highest with plasma volumes of 200 and 300 µL. During analysis, the main source of miRNA detection inaccuracy was derived from RNA extraction (mainly organic extraction), and not reverse transcription or PCR. MiRNA variance could be reduced by use of an internal reference. During analysis, 95% of risk score variation fluctuated within a range of 6.267. The variable factors pre‐analysis mainly involved dietary status, anticoagulant selection, and storage conditions. Hemolysis positively correlated with miRNA levels, but there was no significant change in risk score after internal reference calibration. Conclusion Preliminary standardization for miRNA detection provides a reference for clinical blood testing of miRNAs.

A critical bottleneck in COVID‐19 diagnosis and surveillance is the shortage of kits for RNA extraction. In article number 2000068, Mo Li and co‐workers develop a DIY, open‐source, safe, and fast method for magnetic‐nanoparticle‐aided viral RNA isolation from contagious samples (MAVRICS). MAVRICS rivals commercial kits in performance and could become an enabling technology for community testing and wastewater monitoring in the current and future pandemics.

1.Mucosal swabs have long been used to study various physiological processes in humans. In contrast, systematic sampling of mucosae is rare for wild animals, and except for its use as a source of DNA for population genetics, its potential as a tool to study physiologically-relevant processes of natural populations has not been explored fully. 2.We collected swabs from the rectal, genital, nasal and oral mucosae of California sea lion pups and investigated differences in RNA and protein yield, quality, and integrity between storage times and mucosal types. Downstream applications were tested on the extracted products to determine the potential value of mucosal sampling in free-ranging mammals. 3.For most samples, RNA yield was stable regardless of storage time, and RNA quality and integrity was equal for all mucosal types. Ribosomal fragments of sizes expected for mammals, yeast and bacteria were observable in genital and rectal samples, but in nasal and oral samples only bacterial ribosomal fragments were observable. Amplification of selected transcripts was successful for all samples. The protein profile was distinct between mucosae, and samples with high protein yields were useful for antibody detection. 4.Our study demonstrates that mucosal swabbing is a minimally-invasive tool that yields useful physiological data for free ranging wildlife. Minimum experience is needed to collect samples, processing is inexpensive and downstream applications for ecological studies are realistic. This article is protected by copyright. All rights reserved.

Aims Recent evidence indicates that weakly positive immunohistochemical staining of estrogen receptor (ER) is not reliably associated with a luminal subtype, with the majority re-classified as basal-like by gene expression profile. In this study we assessed the capacity of recently-identified immunohistochemical markers of basal-like subtype not dependent on ER status– positive expression of nestin or loss of inositol polyphosphate-4-phosphatase (INPP4b)– to discriminate intrinsic subtypes, focusing on clinically-problematic cases with weak ER positivity. Methods and Results Formalin-fixed paraffin embedded blocks, enriched for large proportions of ER negative and ER weakly positive breast cancers, were selected from two previous studies conducted in the period 2008-2013 and used for (1) RNA extraction for PAM50 intrinsic subtyping and (2) tissue microarray construction for immunohistochemical assessment of nestin and INPP4b. Fifty-eight cases were weakly-positive for ER (Allred 3-5), among which 28 (48%) were assigned as basal-like by PAM50 gene expression. In these 58 cases, nestin/INPP4b panel identified 23 basal-like cases with a positive predictive value of 87% (95%CI; 78%-95%) and excluded luminal subtype with a negative predictive value of 95% (95%CI; 88%-100%). Weakly positive ER patients assigned as basal-like by nestin/INPP4b definition demonstrated a median survival time of 45.8 months, significantly lower than 65 months among other ER weakly positive cases (p=0.012). Conclusions Immunohistochemical assessment of nestin and INPP4b provides an accurate, accessible and inexpensive tool to identify basal-like breast cancer subtype in the clinically problematic setting of weak ER positivity. This panel identifies poor prognosis patients who might need strong considerations for non-endocrine-based therapies. This article is protected by copyright. All rights reserved.

Our incomplete understanding of the critical changes that accompany the earliest stages of tumor initiation provides a substantial hurdle for the development of novel intervention strategies for cancer prevention. Premalignant lesions are inherently difficult to characterize given their diminutive size, creating technical obstacles for accurate genetic profiling. Here, we describe an approach combining laser-capture microdissection (LCM) with targeted RNA-sequencing to study the transcriptional state of epithelial and stromal cells during the earliest detectable stage of human colorectal neoplasia, the aberrant crypt foci (ACF). We provide a robust and reproducible workflow for RNA isolation, library preparation, and expression profiling of laser-captured cells from frozen OCT-embedded tissue specimens. It is anticipated that the methodological approach outlined in this report will provide a framework for a broad range of microgenomics analyses that can be routinely applied to many other premalignant tissues. J. Cell. Biochem. 117: 2677-2681, 2016. © 2016 Wiley Periodicals, Inc.

The spread of virus-induced infectious diseases through airborne routes of transmission is a global concern for economic and medical reasons. To study virus transmission, it is essential to have an effective aerosol collector such as the growth tube collector (GTC) system that utilizes water-based condensation for collecting virus-containing aerosols. In this work, we characterized the GTC system using bacteriophage MS2 as a surrogate for a small RNA virus. We investigated using RNA extraction and reverse transcription- polymerase chain reaction (RT-PCR) to study the total virus collection efficiency of the GTC system. Plaque assays were also used to enumerate viable viruses collected by the GTC system compared to that by a commercially available apparatus, the SKC® Biosampler. The plaque assay counts were used to enumerate viable viruses whereas RT-PCR provides a total virus count, including those viruses inactivated during collection. The effects of relative humidity (RH) and other conditions on collection efficiency were also investigated. Our results suggest that the GTC has a collection efficiency for viable viruses between 0.24 and 1.8% and a total virus collection efficiency between 18.3 and 79.0%, which is 1-2 orders of magnitude higher than that of the SKC® Biosampler. Moreover, higher RH significantly increases both the viable and total collection efficiency of the GTC, while its effect on the collection efficiency of the SKC® Biosampler is not significant.

There has been a surge of interest in the molecular biology of RNA. We describe a simple, fast, and convenient protocol for isolating total RNA from rice (Oryza sativa) seedlings and young tissues/organs. RNA obtained using this method can be used for reverse transcription-polymerase chain reaction (RT-PCR), quantitative RT-PCR (qRT-PCR), northern blot analysis, RNA-seq, microarray analysis, cDNA library construction, etc. Technical parameters critical to the success of the extraction are presented. © 2016 by John Wiley & Sons, Inc.

Exosomes are recognized as promising biomarkers for early cancer diagnosis and prognosis owing to a large amount of biological information they carried. But the key is that single type of exosomal biomarker analysis is not sufficient enough for accurate cancer diagnosis and stage monitoring due to the insufficient information and high false positive signal. To address the challenge, here simultaneous in situ detection of different types of exosomal biomarkers (surface proteins: CD81, ephrin type-A receptor 2, and carbohydrate antigen 19-9; miRNAs: miR-451a, miR-21, and miR-10b) is conducted with a 3D microfluidic chip, which works in conjunction with quantum dot (QD) labeling and vesicle fusion technology. After exosomes are efficiently captured by the microfluidic chip, the quantification of multiple exosomal proteins is achieved by using three kinds of QDs with the same excitation and different emission wavelengths, and virus-mimicking fusogenic vesicles encapsulating three exquisitely engineered molecular beacons are introduced for ultrasensitive detection of multiple exosomal miRNAs without requiring RNA extraction. Through comprehensive profiling different types of exosomal biomarkers, the false positive rate is substantially avoided and the accuracy of cancer diagnosis and stage monitoring is improved to ≈100%, which are critical to cancer effective treatment and favorable prognosis.

MicroRNAs are short non-coding RNAs that regulate gene expression at the post-transcriptional level by mRNA degradation or suppression of translation. Their stability in plasma makes them attractive biomarkers. Since many plasma microRNA isolation procedures exist and the yield can be highly variable, we recently optimized the microRNA isolation and preamplification procedure using the mirVana PARIS kit (Thermo Fisher Scientific) for miRNA quantification with TaqMan Low Density Arrays in plasma samples. The method here is slightly modified from the original procedure supplied by Thermo Fisher. Based on our findings, recommendations are the following: (1) use Arabidopsis thaliana (Ath) miR-159a as spike-in control, (2) use a 100-µl elution volume during RNA isolation, and (3) add a preamplification step without dilution of the preamplification product. In this article we provide a step-by-step microRNA isolation and quantification procedure using human plasma samples for TaqMan Low Density Arrays. © 2018 by John Wiley & Sons, Inc.

The isolation of high‐quality RNA from the intervertebral disc and especially from the nucleus pulposus is challenging due to the low cellularity and high proteoglycan content of this tissue. In this study, we report a simple modification of the standard guanidinium thiocyanate‐phenol‐chloroform extraction method, which involves enzymatic predigestion of the tissue prior to standard RNA isolation. Yield, purity and integrity of RNA isolated from bovine nucleus pulposus, inner annulus fibrosus and outer annulus fibrosus were compared among complete matrix digestion, predigestion and pulverization, pulverization alone, and pulverization followed by on‐column purification. With predigestion, the average yield of RNA obtained from bovine nucleus pulposus was 8.82 ± 2.05 ng/mg of wet tissue with 260/280 and 260/230 optical density ratios of 1.91 ± 0.15 and 1.84 ± 0.30, respectively. RIN analysis indicated that RNA quality was best preserved with the predigestion method (RNA integrity number > 7), and the extracted RNA was suitable for real‐time polymerase chain reaction. This method is of importance for gene expression studies on intervertebral disc development, degeneration and repair, and we anticipate that it may be further applied to other tissues rich in proteoglycans.

Cell type-specific transcription is a key determinant of cell fate and function. An ongoing challenge in biology is to develop robust and stringent biochemical methods to explore gene expression with cell type specificity. This challenge has become even greater as researchers attempt to apply high-throughput RNA analysis methods under in vivo conditions. TU-tagging and EC-tagging are in vivo biosynthetic RNA tagging techniques that allow spatial and temporal specificity in RNA purification. Spatial specificity is achieved through targeted expression of pyrimidine salvage enzymes (uracil phosphoribosyltransferase and cytosine deaminase) and temporal specificity is achieved by controlling exposure to bioorthogonal substrates of these enzymes (4-thiouracil and 5-ethynylcytosine). Tagged RNAs can be purified from total RNA extracted from an animal or tissue and used in transcriptome profiling analyses. In addition to identifying cell type-specific mRNA profiles, these techniques are applicable to noncoding RNAs and can be used to measure RNA transcription and decay. Potential applications of TU-tagging and EC-tagging also include fluorescent RNA imaging and selective definition of RNA-protein interactions. TU-tagging and EC-tagging hold great promise for supporting research at the intersection of RNA biology and developmental biology. This article is categorized under: Technologies > Analysis of the Transcriptome.

Bladder cancer (BC) is a heterogeneous disease characterized by a high recurrence rate that necessitates continuous cystoscopic surveillance. MicroRNAs (miRNAs) are detectable in tissues and biofluids such as plasma/serum and urine. They represent promising biomarkers with potential not only for detecting BC but also informing on prognosis and monitoring treatment response. In this review, the many aspects of the application of next-generation sequencing (NGS) to evaluate miRNA expression in BC is discussed, including technical issues as well as a comparison with results obtained by qRT-PCR. The available studies investigating miRNA profiling in BC by NGS are described, with particular attention to the potential applicability on biofluids. Altered miRNA levels have been observed in BC tissues by NGS, but these results so far only partially overlapped among studies and with previous data obtained by qRT-PCR. The discrepancies can be ascribed to the small groups of BC patients sequenced. The few available studies on biofluids are mainly focused on implementing RNA isolation and sequencing workflow. Using NGS to analyze miRNAs in biofluids can potentially provide results comparable to tissues with no invasive procedures for the patients. In particular, the analyses performed on exosomes/microvesicles appear to be more informative. Thanks to the improvement of both wet-lab procedures and pipelines/tools for data analyses, NGS studies on biofluids will be performed on a larger scale. MiRNAs detected in urine and serum/plasma will demonstrate their potentiality to describe the variegated scenario of BC and to become relevant clinical markers.

Ingredients and complexity of food matrices are inhibitory factors for gene expression studies of bacterial pathogens directly from food matrix. The aim of this study was to develop a simple and fast method for RNA extraction from ground beef matrix as well as stx genes expression of Escherichia coli O157:H7 by relative quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). Inoculated ground beef samples were kept 7 days in refrigerated storage (4 ± 1C) and bacterial RNA was extracted according to the described method at 0, 2, 5 and 7 days after storage. Results indicate that refrigerated storage temperature decreased gene expression of stx1A (−3.35 relative changes on day 7) and stx2A (−2.9 relative changes on day 7) in ground beef matrix. In the current study, a method requiring minimal equipment and limited interference for RNA extraction from bacteria associated with a meat matrix was evaluated (developed, invested, etc.). Results of the study suggest that the quantitative real-time RT-PCR method could be used as a reliable and sensitive method for gene expression studies. Practical Applications Polymerase chain reaction (PCR), known as a sensitive and specific method, was extensively used for the detection of genes involved in stress and virulence of bacteria. In this way, real-time PCR shows a good sensitivity and diversity for detection and gene expression studies of pathogens. This technique frequently has been used for the study of bacterial behavior in broth or food-like broth models. Complexity of food matrix and presence of proteins, and also other PCR-inhibiting factors, restricted gene expression studies in food matrix. There has been great interest in the survival and pathogenicity of Escherichia coli O157:H7 because it is a great concern of ground meat industry. Shiga toxins (Stx1 and Stx2) as manifest characteristics of E. coli O157:H7 are known as significant virulence factors in the pathogenicity of bacterium so its behavior and gene expression profile in meat matrix and during cold chain were evaluated.

Flow cytometry (FCM) has become a well-established method for analysis of both intracellular and cell-surface proteins, while quantitative RT-PCR (RT-qPCR) is used to determine gene expression with high sensitivity and specificity. Combining these two methods would be of great value. The effects of intracellular staining on RNA integrity and RT-qPCR sensitivity and quality have not, however, been fully examined. We, therefore, intended to assess these effects further. Cells from the human lung cancer cell line A549 were fixed, permeabilized and sorted by FCM. Sorted cells were analyzed using RT-qPCR. RNA integrity was determined by RNA quality indicator analysis. A549 cells were then mixed with cells of the mouse cardiomyocyte cell line HL-1. A549 cells were identified by the cell surface marker ABCG2, while HL-1 cells were identified by intracellular cTnT. Cells were sorted and analyzed by RT-qPCR. Finally, cell cultures from human atrial biopsies were used to evaluate the effects of fixation and permeabilization on RT-qPCR analysis of nonimmortalized cells stored prior to analysis by FCM. A large amount of RNA could be extracted even when cells had been fixed and permeabilized. Permeabilization resulted in increased RNA degradation and a moderate decrease in RT-qPCR sensitivity. Gene expression levels were also affected to a moderate extent. Sorted populations from the mixed A549 and HL-1 cell samples showed gene expression patterns that corresponded to FCM data. When samples were stored before FCM sorting, the RT-qPCR analysis could still be performed with high sensitivity and quality. In summary, our results show that intracellular FCM may be performed with only minor impairment of the RT-qPCR sensitivity and quality when analyzing sorted cells; however, these effects should be considered when comparing RT-qPCR data of not fixed samples with those of fixed and permeabilized samples.