Polyethylene Glycol -Mediated Exosome Isolation: A Method for Exosomal RNA Analysis.

Background: ExoRNAs offer valuable insights into their cellular origin. ExoRNA studies were faced with challenges in obtaining sufficient amounts of high-quality RNA. Herein, we aimed to compare three traditional exosome isolation methods to introduce an appropriate strategy to extract RNA from cancer-derived exosomes for further RNA analysis. Methods: Exosomes were isolated through ultracentrifugation, precipitation kit, and size exclusion column chromatography, and then characterized by DLS and TEM, followed by extracting total RNA. The quality and quantity of the extracted RNAs were assessed by a NanoDrop and 2.5% agarose gel electrophoresis. Results: Extracted exosomes displayed a similar range of size and morphology. We found that PEG-precipitation method resulted in a higher RNA yield with a 260/280 ratio of 1.9. The obtained exoRNA appeared as a smear in the agarose gel, indicative of small exoRNAs. Conclusion: We provide researchers a suitable approach to isolate exosomes based on yield and purity of exoRNA.

Role of detergents and nuclease inhibitors in the extraction of RNA from eukaryotic cells in complex matrices.

The potential for liquid biopsy samples to be used in place of more invasive tissue biopsies has become increasingly revalent as it has been found that nucleic acids (NAs) present in the blood of cancer patients originate from tumors. Nanomagnetic extraction has proven to be a highly effective means to rapidly prepare NA from clinical samples for molecular diagnostics. In this article, the lysis reaction used to extract RNA from the human epithelial melanoma cells have been optimized using silica coated superparamagnetic nanoparticles (SPM NP). The lysis buffer (LB) is composed of several agents that denature cells, i.e., surfactant and guanidinium isothiocyanate (GITC), and agents that inhibit the degradation of circulated nucleic acids (cfNAs). The surfactant Triton X-100 has been widely used in LB but has been placed on the European Union REACH list. We have compared the qRT-PCR sensitivity resulting from LBs composed of Triton X-100 to several sustainable surfactants, i.e., Tergitol 15-S-7, Tergitol 15-S-9 and Tween-20. Surprisingly, the inclusion of these surfactants in the LB was not found to significantly improve cell lysis, and subsequently the sensitivity of qRT-PCR. The role of the sample matrix was also examined by performing extractions from solutions containing up to 30 mg mL-1 serum albumin. The qRT-PCR sensitivity was found to decrease as the concentration of this protein was increased; however, this was linked to an increased RNase activity and not the concentration of the protein itself. These results lead us to recommend a reformulation of LB for clinical samples, and to conclude that sensitive qRT-PCR RNA analysis can be performed in serum with the timely addition of an RNase inhibitor.

Live-seq enables temporal transcriptomic recording of single cells.

Single-cell transcriptomics (scRNA-seq) has greatly advanced our ability to characterize cellular heterogeneity1. However, scRNA-seq requires lysing cells, which impedes further molecular or functional analyses on the same cells. Here, we established Live-seq, a single-cell transcriptome profiling approach that preserves cell viability during RNA extraction using fluidic force microscopy2,3, thus allowing to couple a cell's ground-state transcriptome to its downstream molecular or phenotypic behaviour. To benchmark Live-seq, we used cell growth, functional responses and whole-cell transcriptome read-outs to demonstrate that Live-seq can accurately stratify diverse cell types and states without inducing major cellular perturbations. As a proof of concept, we show that Live-seq can be used to directly map a cell's trajectory by sequentially profiling the transcriptomes of individual macrophages before and after lipopolysaccharide (LPS) stimulation, and of adipose stromal cells pre- and post-differentiation. In addition, we demonstrate that Live-seq can function as a transcriptomic recorder by preregistering the transcriptomes of individual macrophages that were subsequently monitored by time-lapse imaging after LPS exposure. This enabled the unsupervised, genome-wide ranking of genes on the basis of their ability to affect macrophage LPS response heterogeneity, revealing basal Nfkbia expression level and cell cycle state as important phenotypic determinants, which we experimentally validated. Thus, Live-seq can address a broad range of biological questions by transforming scRNA-seq from an end-point to a temporal analysis approach.

Examining potential confounding factors in gene expression analysis of human saliva and identifying potential housekeeping genes.

Isolation of RNA from whole saliva, a non-invasive and easily accessible biofluid that is an attractive alternative to blood for high-throughput biodosimetry of radiological/nuclear victims might be of clinical significance for prediction and diagnosis of disease. In a previous analysis of 12 human samples we identified two challenges to measuring gene expression from total RNA: (1) the fraction of human RNA in whole saliva was low and (2) the bacterial contamination was overwhelming. To overcome these challenges, we performed selective cDNA synthesis for human RNA species only by employing poly(A)+-tail primers followed by qRT-PCR. In the current study, this approach was independently validated on 91 samples from 61 healthy donors. Additionally, we used the ratio of human to bacterial RNA to adjust the input RNA to include equal amounts of human RNA across all samples before cDNA synthesis, which then ensured comparable analysis using the same base human input material. Furthermore, we examined relative levels of ten known housekeeping genes, and assessed inter- and intra-individual differences in 61 salivary RNA isolates, while considering effects of demographical factors (e.g. sex, age), epidemiological factors comprising social habits (e.g. alcohol, cigarette consumption), oral hygiene (e.g. flossing, mouthwash), previous radiological diagnostic procedures (e.g. number of CT-scans) and saliva collection time (circadian periodic). Total human RNA amounts appeared significantly associated with age only (P ≤ 0.02). None of the chosen housekeeping genes showed significant circadian periodicity and either did not associate or were weakly associated with the 24 confounders examined, with one exception, 60% of genes were altered by mouthwash. ATP6, ACTB and B2M represented genes with the highest mean baseline expression (Ct-values ≤ 30) and were detected in all samples. Combining these housekeeping genes for normalization purposes did not decrease inter-individual variance, but increased the robustness. In summary, our work addresses critical confounders and provides important information for the successful examination of gene expression in human whole saliva.

Single-copy Loss of Rho Guanine Nucleotide Exchange Factor 10 ( arhgef10) Causes Locomotor Abnormalities in Zebrafish Larvae.

OBJECTIVE: To determine if ARHGEF10 has a haploinsufficient effect and provide evidence to evaluate the severity, if any, during prenatal consultation. METHODS: Zebrafish was used as a model for generating mutant. The pattern of arhgef10 expression in the early stages of zebrafish development was observed using whole-mount in situ hybridization (WISH). CRISPR/Cas9 was applied to generate a zebrafish model with a single-copy or homozygous arhgef10 deletion. Activity and light/dark tests were performed in arhgef10 -/-, arhgef10 +/-, and wild-type zebrafish larvae. ARHGEF10 was knocked down using small interferon RNA (siRNA) in the SH-SY5Y cell line, and cell proliferation and apoptosis were determined using the CCK-8 assay and Annexin V/PI staining, respectively. RESULTS: WISH showed that during zebrafish embryonic development arhgef10 was expressed in the midbrain and hindbrain at 36-72 h post-fertilization (hpf) and in the hemopoietic system at 36-48 hpf. The zebrafish larvae with single-copy and homozygous arhgef10 deletions had lower exercise capacity and poorer responses to environmental changes compared to wild-type zebrafish larvae. Moreover, arhgef10 -/- zebrafish had more severe symptoms than arhgef10 +/- zebrafish. Knockdown of ARHGEF10 in human neuroblastoma cells led to decreased cell proliferation and increased cell apoptosis. CONCLUSION: Based on our findings, ARHGEF10 appeared to have a haploinsufficiency effect.

RNA isolation from micro-quantity of articular cartilage for quantitative gene expression by microarray analysis.

Isolation of quality RNA from articular cartilage has been challenging due to low cellularity and the high abundance of extracellular matrix and proteoglycan proteins. Recently developed methods for isolation of high quality RNA from cartilage are more applicable to larger cartilage specimens typically weighing at least 25 mg. While these methods generate RNA suitable for analysis, they are less successful with smaller tissue inputs. For the study of small focal defect cartilage specimens an improved RNA extraction method is needed. Here we report a protocol for direct RNA isolation from less than 3 mg of wet weight rabbit articular cartilage for quantitative microarray gene profiling. This protocol is useful for identifying differentially expressed genes in chondrocytes following focal cartilage repair and can potentially be adopted for gene expression analysis of cartilage biopsy specimens from human joints.

A method for rapid flow-cytometric isolation of endothelial nuclei and RNA from archived frozen brain tissue.

Endothelial cells are important contributors to brain development, physiology, and disease. Although RNA sequencing has contributed to the understanding of brain endothelial cell diversity, bulk analysis and single-cell approaches have relied on fresh tissue digestion protocols for the isolation of single endothelial cells and flow cytometry-based sorting on surface markers or transgene expression. These approaches are limited in the analysis of the endothelium in human brain tissues, where fresh samples are difficult to obtain. Here, we developed an approach to examine endothelial RNA expression by using an endothelial-specific marker to isolate nuclei from abundant archived frozen brain tissues. We show that this approach rapidly and reliably extracts endothelial nuclei from frozen mouse brain samples, and importantly, from archived frozen human brain tissues. Furthermore, isolated RNA transcript levels are closely correlated with expression in whole cells from tissue digestion protocols and are enriched in endothelial markers and depleted of markers of other brain cell types. As high-quality RNA transcripts could be obtained from as few as 100 nuclei in archived frozen human brain tissues, we predict that this approach should be useful for both bulk analysis of endothelial RNA transcripts in human brain tissues as well as single-cell analysis of endothelial sub-populations.

Nobiletin attenuates inflammation via modulating proinflammatory and antiinflammatory cytokine expressions in an autoimmune encephalomyelitis mouse model.

The aim of this study is to investigate the potential preventive and therapeutic effects of nobiletin by evaluating the expression of cytokines associated with inflammatory reactions in an autoimmune encephalomyelitis mouse model. A total of 60 male C57BL/6 mice aged between 8 and 10 weeks were used. Mice were divided into six groups (n = 10 mice per group): control, EAE, low-prophylaxis, high-prophylaxis, low-treatment and high-treatment. Experimental autoimmune encephalomyelitis (EAE) was induced by myelin oligodendrocyte glycoprotein (MOG) and pertussis toxin. Nobiletin was administered in low (25 mg/kg) and high (50 mg/kg) doses, intraperitoneally. The prophylactic and therapeutic effects of nobiletin on brain tissue and spinal cord were evaluated by expression of interleukin-1 beta (IL-1ß), tumor necrosis factor-alpha (TNF-α), interferon gamma (IFNγ), IL-6, IL-10 and transforming growth factor-beta (TGF-ß) using immunohistochemistry and real-time polymerase chain reaction (RT-PCR). Prophylactic and therapeutic use of nobiletin inhibited EAE-induced increase of TNF-α, IL-1ß and IL-6 activities to alleviate inflammatory response in brain and spinal cord. Moreover, nobiletin supplement dramatically increased the IL-10, TGF-ß and IFNγ expressions in prophylaxis and treatment groups compared with the EAE group in the brain and spinal cord. The results obtained from this study show that prophylactic and therapeutic nobiletin modulates expressions of proinflammatory and antiinflammatory cytokines in brain and spinal cord dose-dependent manner in EAE model. These data demonstrates that nobiletin has a potential to attenuate inflammation in EAE mouse model. These experimental findings need to be supported by clinical studies.

Comparison of RNA-Based Next-Generation Sequencing Assays for the Detection of NTRK Gene Fusions.

Recently, the US Food and Drug Administration approved several targeted therapies directed against oncogenic fusions. One of the most effective such targeted therapies is Vitrakvi (larotrectinib), highly specific oral tropomyosin receptor kinase inhibitor indicated for the treatment of patients with any solid tumor harboring a fusion involving one of the neurotrophic receptor tyrosine kinase (NTRK) genes. Although several diagnostic approaches can be used to detect these NTRK fusions, RNA-based next-generation sequencing remains one of the most sensitive methods, as it can directly detect the transcribed end product of gene fusion at the mRNA level. In this study, performance characteristics of three RNA-based next-generation sequencing assays with distinct mechanisms and chemistries were investigated: anchored multiplex PCR, amplicon-based multiplex PCR, and hybrid capture-based enrichment method. Analytical sensitivity analysis shows that the amplicon-based multiplex PCR method has the lowest limit of detection. However, both hybrid-capture and anchored multiplex PCR methods can detect NTRK fusions with uncommon or novel fusion partners, which is challenging for the amplicon-based multiplex method. As for clinical sensitivity, all three methods were highly concordant in detecting NTRK fusions in patient samples. Additionally, they all presented equivalent high-level performance in specificity, suggesting that all three platforms can detect NTRK fusions in clinical samples with similar performance characteristics.

Robust Saliva-Based RNA Extraction-Free One-Step Nucleic Acid Amplification Test for Mass SARS-CoV-2 Monitoring.

Early diagnosis with rapid detection of the virus plays a key role in preventing the spread of infection and in treating patients effectively. In order to address the need for a straightforward detection of SARS-CoV-2 infection and assessment of viral spread, we developed rapid, sensitive, extraction-free one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) tests for detecting SARS-CoV-2 in saliva. We analyzed over 700 matched pairs of saliva and nasopharyngeal swab (NSB) specimens from asymptomatic and symptomatic individuals. Saliva, as either an oral cavity swab or passive drool, was collected in an RNA stabilization buffer. The stabilized saliva specimens were heat-treated and directly analyzed without RNA extraction. The diagnostic sensitivity of saliva-based RT-qPCR was at least 95% in individuals with subclinical infection and outperformed RT-LAMP, which had at least 70% sensitivity when compared to NSBs analyzed with a clinical RT-qPCR test. The diagnostic sensitivity for passive drool saliva was higher than that of oral cavity swab specimens (95% and 87%, respectively). A rapid, sensitive one-step extraction-free RT-qPCR test for detecting SARS-CoV-2 in passive drool saliva is operationally simple and can be easily implemented using existing testing sites, thus allowing high-throughput, rapid, and repeated testing of large populations. Furthermore, saliva testing is adequate to detect individuals in an asymptomatic screening program and can help improve voluntary screening compliance for those individuals averse to various forms of nasal collections.

Validation, Implementation, and Clinical Impact of the Oncomine Myeloid Targeted-Amplicon DNA and RNA Ion Semiconductor Sequencing Assay.

The identification of clinically significant genes recurrently mutated in myeloid malignancies necessitates expanding diagnostic testing with higher throughput, such as targeted next-generation sequencing. We present validation of the Thermo Fisher Oncomine Myeloid Next-Generation Sequencing Panel (OMP), targeting 40 genes and 29 fusion drivers recurrently mutated in myeloid malignancies. The study includes data from a sample exchange between two Canadian hospitals demonstrating high concordance for detection of DNA and RNA aberrations. Clinical validation demonstrates high accuracy, sensitivity, and specificity of the OMP, with a lower limit of detection of 5% for single-nucleotide variants and 10% for insertions/deletions. Prospective sequencing was performed for 187 samples from 168 unique patients presenting with suspected or confirmed myeloid malignancy and other hematological conditions to assess clinical impact of identifying variants. Of detected variants, 48% facilitated or clarified diagnoses, 29% affected prognoses, and 25% had the potential to influence clinical management. Of note, OMP was essential to identifying patients with premalignant clonal states likely contributing to cytopenias. We also found that the detection of even a single variant by the OMP assay, versus 0 variants, was predictive of overall survival, independent of age, sex, or diagnosis (P = 0.03). This study demonstrates that molecular profiling of myeloid malignancies with the OMP represents a promising strategy to advance molecular diagnostics.

Robotic RNA extraction for SARS-CoV-2 surveillance using saliva samples.

Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput. Using this assay, the Free Asymptomatic Saliva Testing (IGI FAST) research study on the UC Berkeley campus conducted 11,971 tests on supervised self-collected saliva samples and identified rare positive specimens containing SARS-CoV-2 RNA during a time of low infection prevalence. In an attempt to increase testing capacity, we further adapted our robotic extraction assay to process pooled saliva samples. We also benchmarked our assay against nasopharyngeal swab specimens and found saliva methods require further optimization to match this gold standard. Finally, we designed and validated a RT-qPCR test suitable for saliva self-collection. These results establish a robotic extraction-based procedure for rapid PCR-based saliva testing that is suitable for samples from both symptomatic and asymptomatic individuals.

Tumor cell invasion into Matrigel: optimized protocol for RNA extraction.

3D models are increasingly used to study mechanisms driving tumor progression and mimicking in vitro processes such as invasion and migration. However, there is a need to establish more protocols based on 3D culture systems that allow for downstream molecular biology investigations. Materials & methods: Here we present a method for optimal RNA extraction from highly aggressive primary glioma cells invading into Matrigel. The method has been established by comparing previously reported protocols, available commercial kits and optimizing specific steps for matrix dissociation, RNA separation and purification. Results and conclusion: The protocol allows RNA extraction from cells embedded into Matrigel, with optimal yield, purity and integrity suitable for subsequent sequencing analysis of both high and low molecular weight RNA.

Membrane Particles Derived From Adipose Tissue Mesenchymal Stromal Cells Improve Endothelial Cell Barrier Integrity.

Proinflammatory stimuli lead to endothelial injury, which results in pathologies such as cardiovascular diseases, autoimmune diseases, and contributes to alloimmune responses after organ transplantation. Both mesenchymal stromal cells (MSC) and the extracellular vesicles (EV) released by them are widely studied as regenerative therapy for the endothelium. However, for therapeutic application, the manipulation of living MSC and large-scale production of EV are major challenges. Membrane particles (MP) generated from MSC may be an alternative to the use of whole MSC or EV. MP are nanovesicles artificially generated from the membranes of MSC and possess some of the therapeutic properties of MSC. In the present study we investigated whether MP conserve the beneficial MSC effects on endothelial cell repair processes under inflammatory conditions. MP were generated by hypotonic shock and extrusion of MSC membranes. The average size of MP was 120 nm, and they showed a spherical shape. The effects of two ratios of MP (50,000; 100,000 MP per target cell) on human umbilical vein endothelial cells (HUVEC) were tested in a model of inflammation induced by TNFα. Confocal microscopy and flow cytometry showed that within 24 hours >90% of HUVEC had taken up MP. Moreover, MP ended up in the lysosomes of the HUVEC. In a co-culture system of monocytes and TNFα activated HUVEC, MP did not affect monocyte adherence to HUVEC, but reduced the transmigration of monocytes across the endothelial layer from 138 ± 61 monocytes per microscopic field in TNFα activated HUVEC to 61 ± 45 monocytes. TNFα stimulation induced a 2-fold increase in the permeability of the HUVEC monolayer measured by the translocation of FITC-dextran to the lower compartment of a transwell system. At a dose of 1:100,000 MP significantly decreased endothelial permeability (1.5-fold) respect to TNFα Stimulated HUVEC. Finally, MP enhanced the angiogenic potential of HUVEC in an in vitro Matrigel assay by stimulating the formation of angiogenic structures, such as percentage of covered area, total tube length, total branching points, total loops. In conclusion, MP show regenerative effects on endothelial cells, opening a new avenue for treatment of vascular diseases where inflammatory processes damage the endothelium.

Overcoming the Challenges of High Quality RNA Extraction from Core Needle Biopsy.

The use of gene expression profiling (GEP) in cancer management is rising, as GEP can be used for disease classification and diagnosis, tailoring treatment to underlying genetic determinants of pharmacological response, monitoring of therapy response, and prognosis. However, the reliability of GEP heavily depends on the input of RNA in sufficient quantity and quality. This highlights the need for standard procedures to ensure best practices for RNA extraction from often small tumor biopsies with variable tissue handling. We optimized an RNA extraction protocol from fresh-frozen (FF) core needle biopsies (CNB) from breast cancer patients and from formalin-fixed paraffin-embedded (FFPE) tissue when FF CNB did not yield sufficient RNA. Methods to avoid ribonucleases andto homogenize or to deparaffinize tissues and the impact of tissue composition on RNA extraction were studied. Additionally, RNA's compatibility with the nanoString nCounter® technology was studied. This technology platform enables GEP using small RNA fragments. After optimization of the protocol, RNA of high quality and sufficient quantity was obtained from FF CNB in 92% of samples. For the remaining 8% of cases, FFPE material prepared by the pathology department was used for RNA extraction. Both resulting RNA end products are compatible with the nanoString nCounter® technology.

Gene expression profiling of brain endothelial cells after experimental subarachnoid haemorrhage.

Subarachnoid haemorrhage (SAH) is a type of hemorrhagic stroke that is associated with high morbidity and mortality. New effective treatments are needed to improve outcomes. The pathophysiology of SAH is complex and includes early brain injury and delayed cerebral ischemia, both of which are characterized by blood-brain barrier (BBB) impairment. We isolated brain endothelial cells (BECs) from mice subjected to SAH by injection of blood into the prechiasmatic cistern. We used gene expression profiling to identify 707 unique genes (2.8% of transcripts, 403 upregulated, 304 downregulated, 24,865 interrogated probe sets) that were significantly differentially expressed in mouse BECs after SAH. The pathway involving prostaglandin synthesis and regulation was significantly upregulated after SAH, including increased expression of the Ptgs2 gene and its corresponding COX-2 protein. Celecoxib, a selective COX-2 inhibitor, limited upregulation of Ptgs2 in BECs. In this study, we have defined the gene expression profiling of BECs after experimental SAH and provide further insight into BBB pathophysiology, which may be relevant to other neurological diseases such as traumatic brain injury, brain tumours, ischaemic stroke, multiple sclerosis, and neurodegenerative disorders.

Sticky problems: extraction of nucleic acids from molluscs.

Traditional molecular methods and omics-techniques across molluscan taxonomy increasingly inform biology of Mollusca. Recovery of DNA and RNA for such studies is challenged by common biological properties of the highly diverse molluscs. Molluscan biomineralization, adhesive structures and mucus involve polyphenolic proteins and mucopolysaccharides that hinder DNA extraction or copurify to inhibit enzyme-catalysed molecular procedures. DNA extraction methods that employ the detergent hexadecyltrimethylammoniumbromide (CTAB) to remove these contaminants importantly facilitate molecular-level study of molluscs. Molluscan pigments may stain DNA samples and interfere with spectrophotometry, necessitating gel electrophoresis or fluorometry for accurate quantification. RNA can reliably be extracted but the 'hidden break' in 28S rRNA of molluscs (like most protostomes) causes 18S and 28S rRNA fragments to co-migrate electrophoretically. This challenges the standard quality control based on the ratio of 18S and 28S rRNA, developed for deuterostome animals. High-AT content in molluscan rRNA prevents the effective purification of polyadenylated mRNA. Awareness of these matters aids the continuous expansion of molecular malacology, enabling work also with museum specimens and next-generation sequencing, with the latter imposing unprecedented demands on DNA quality. Alternative methods to extract nucleic acids from molluscs are available from literature and, importantly, from communications with others who study the molecular biology of molluscs. This article is part of the Theo Murphy meeting issue 'Molluscan genomics: broad insights and future directions for a neglected phylum'.

Two novel qualitative transcriptional signatures robustly applicable to non-research-oriented colorectal cancer samples with low-quality RNA.

Currently, due to the low quality of RNA caused by degradation or low abundance, the accuracy of gene expression measurements by transcriptome sequencing (RNA-seq) is very challenging for non-research-oriented clinical samples, majority of which are preserved in hospitals or tissue banks worldwide with complete pathological information and follow-up data. Molecular signatures consisting of several genes are rarely applied to such samples. To utilize these resources effectively, 45 stage II non-research-oriented samples which were formalin-fixed paraffin-embedded (FFPE) colorectal carcinoma samples (CRC) using RNA-seq have been analysed. Our results showed that although gene expression measurements were significantly affected, most cancer features, based on the relative expression orderings (REOs) of gene pairs, were well preserved. We then developed two REO-based signatures, which consisted of 136 gene pairs for early diagnosis of CRC, and 4500 gene pairs for predicting post-surgery relapse risk of stage II and III CRC. The performance of our signatures, which included hundreds or thousands of gene pairs, was more robust for non-research-oriented clinical samples, compared to that of two published concise REO-based signatures. In conclusion, REO-based signatures with relatively more gene pairs could be robustly applied to non-research-oriented CRC samples.

Purification of Total RNA from Stomach Tissue Biopsies.

In order to further our understanding of the physiological consequences of Helicobacter pylori infection , analysis of clinical tissue specimens is required. To this end, RNA is frequently isolated from stomach biopsies of H. pylori-infected patients and compared to samples from uninfected controls to monitor gene expression using molecular methods such as reverse-transcription real-time PCR, microarrays, and next-generation sequencing. The successful purification of sufficient quantities of high-quality RNA is essential for accurate and reproducible downstream analysis. This chapter describes the key steps for high-quality RNA purification from human tissue samples, including sample collection and storage, tissue disruption and lysis, RNA purification, and assessment of RNA yield and quality.

Transcript-Based Detection of Circulating Melanoma Cells.

Circulating tumor cells (CTCs) are cancer cells shed by the primary tumor or its metastases that circulate in the peripheral blood. CTCs are potential seeds for metastases, and their detection may allow early uncovering of metastatic dissemination and disease prognostication. To fully ascertain the biomarker potential of melanoma CTCs, sensitive and reliable methods are required. Melanoma-specific transcript analysis has been widely utilized as a standard approach for measuring the presence of CTCs. Here we describe a method for the analysis of CTCs through the detection of specific transcripts in CTC-enriched fractions.