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ALK, ROS1, and RET kinase fusions are important predictive biomarkers of tyrosine kinase inhibitors (TKIs) in non‐small‐cell lung cancer (NSCLC). Analysis of cell‐free DNA (cfDNA) provides a noninvasive method to identify gene changes in tumor cells. The present study sought to use cfRNA and cfDNA for identifying fusion genes. A reliable protocol was established to detect fusion genes using cfRNA and assessed the analytical validity and clinical usefulness in 30 samples from 20 cases of fusion‐positive NSCLC. The results of cfRNA‐based assays were compared with tissue biopsy and cfDNA‐based liquid biopsy (Guardant360 plasma next‐generation sequencing [NGS] assay). The overall sensitivity of the cfRNA‐based assay was 26.7% (8/30) and that of cfDNA‐based assay was 16.7% (3/18). When analysis was limited to the samples collected at chemo‐naïve or progressive disease status and available for both assays, the sensitivity of the cfRNA‐based assay was 77.8% (7/9) and that of cfDNA‐based assay was 33.3% (3/9). Fusion gene identification in cfRNA was correlated with treatment response. These results suggest that the proposed cfRNA assay is a useful diagnostic test for patients with insufficient tissues to facilitate effective administration of first‐line treatment and is a useful tool to monitor the progression of NSCLC for consideration of second‐line treatments., cfRNA‐ and cfDNA‐based assays are evaluated in 20 cases of fusion‐positive NSCLC. cfRNA assay was superior to cfDNA assay for the detection of gene fusions. The results of the cfRNA assay were consistent with the therapeutic effect.

Many emerging technologies are reliant on circulating cell-free DNA (cfDNA) and cell-free RNA (cfRNA) applications in the clinic. However, the impact of diurnal cycles or daily meals on circulating analytes are poorly understood and may be confounding factors when developing diagnostic platforms. To begin addressing this knowledge gap, we obtained plasma from four healthy donors serially sampled five times during 12 h in a single day. For all samples, we measured concentrations of cfDNA and cfRNA using both bulk measurements and gene-specific digital droplet PCR. We found no significant variation attributed to blood draw number for the cfDNA or cfRNA. This indicated that natural diurnal cycles and meal consumption do not appear to significantly affect abundance of total cfDNA, total cfRNA, or our two selected cfRNA transcripts. Conversely, we observed significant variation between individual donors for cfDNA and one of the cfRNA transcripts. The results of this work suggest that it will be important to consider patient-specific baselines when designing reliable circulating cfDNA or cfRNA clinical assays.

Advancing knowledge of the transcriptome has revealed that circular RNAs (circRNAs) are widely expressed and evolutionarily conserved molecules that may serve relevant biological roles. More interesting is the accumulating evidence which demonstrates the implication of circRNAs in diseases, especially cancers. This revelation has helped to form the rationale for many studies exploring their utility as clinical biomarkers. CircRNAs are highly stable due to their unique structures, exhibit some tissue specificity, and are enriched in exosomes, which facilitate their detection in a range of body fluids. These properties make circRNAs ideal candidates for biomarker development in many diseases. This review will outline the discovery, biogenesis, and proposed functions of circRNAs.

Cell-free RNA (cf-RNA) is released from cells and circulated in the body via the circulatory and endocrine systems. Subsequently, cf-RNA can be extracted from different biofluids by various extraction methods. This review aims to: (1) clarify the clinical role of cf-RNA and methods of extraction, (2) introduce the evaluation parameters and methods, (3) summarize the evaluation of several common extraction methods and propose optimizing solutions for them. Eighty articles on the application of cf-RNA in diagnosing diseases and cf-RNA extraction published in journals with good impact factor (higher than 2) were then summarized and sorted into groups based on their targeted disease: (1) Cancers and (2) Neurodegenerative diseases since the cf-RNA is being studied mainly in these two fields. The general design of these studies is comparing cf-RNA to the current means of diagnosis, which are considered as standards such as endoscopy, biopsy, CSF analysis. By quantifying cf-RNA using qRT-PCR, previous studies were able to detect several cancers and neurodegenerative diseases. The key advantage of using the cf-RNA as a biomarker is reducing the risks and invasiveness without losing sensitivity since it can be extracted in the peripheral area. Furthermore, cf-RNA expression levels vary according to disease stages, suggesting its potential as prognostic markers. The main challenge in using cf-RNA is achieving the optimal yield, purity, integrity with minimal contamination. The current methods like Trizol LS, miR-Neasy, mirVANA have yielded unsatisfying results; therefore, they require modifications to optimize their results. miRCURY kit has the highest RNA yield but has been discontinued. In conclusion, cf-RNA holds excellent promises as a promising biomarker in the future. However, due to certain challenges, the use of cf-RNA is currently limited to clinical studies due to the instability in yield and integrity, requiring supporting studies and extraction methods evaluation.

Since tissue material is often lacking in metastatic prostate cancer (mPCa), there is increasing interest in using liquid biopsies for treatment decision and monitoring therapy responses. The purpose of this study was to validate the usefulness of circulating tumor cells (CTCs) and plasma-derived cell-free (cf) RNA as starting material for gene expression analysis through qPCR. CTCs were identified upon prostate-specific membrane antigen and/or cytokeratin positivity after enrichment with ScreenCell (Westford, Massachusetts, USA) filters or the microfluidic ParsortixTM (Guildford, Surrey, United Kingdom) system. Overall, 50% (28/56) of the patients had ≥5 CTCs/7.5 mL of blood. However, CTC count did not correlate with Gleason score, serum PSA, or gene expression. Notably, we observed high expression of CD45 in CTC samples after enrichment, which could be successfully eliminated through picking of single cells. Gene expression in picked CTCs was, however, rather low. In cfRNA from plasma, on the other hand, gene expression levels were higher compared to those found in CTCs. Moreover, we found that PSA was significantly increased in plasma-derived cfRNA of mPCa patients compared to healthy controls. High PSA expression was also associated with poor overall survival, indicating that using cfRNA from plasma could be used as a valuable tool for molecular expression analysis.

Cell-free gene expression systems have emerged as a promising platform for field-deployed biosensing and diagnostics. When combined with programmable toehold switch-based RNA sensors, these systems can be used to detect arbitrary RNAs and freeze-dried for room temperature transport to the point-of-need. These sensors, however, have been mainly implemented using reconstituted PURE cell-free protein expression systems that are difficult to source in the Global South due to their high commercial cost and cold-chain shipping requirements. Based on preliminary demonstrations of toehold sensors working on lysates, we describe the fast prototyping of RNA toehold switch-based sensors that can be produced locally and reduce the cost of sensors by two orders of magnitude. We demonstrate that these in-house cell lysates provide sensor performance comparable to commercial PURE cell-free systems. We further optimize these lysates with a CRISPRi strategy to enhance the stability of linear DNAs by knocking-down genes responsible for linear DNA degradation. This enables the direct use of PCR products for fast screening of new designs. As a proof-of-concept, we develop novel toehold sensors for the plant pathogen Potato Virus Y (PVY), which dramatically reduces the yield of this important staple crop. The local implementation of low-cost cell-free toehold sensors could enable biosensing capacity at the regional level and lead to more decentralized models for global surveillance of infectious disease.

Although analysis of maternal plasma cell-free content has been employed for screening of genetic abnormalities within a pregnancy, limited attention has been paid to its use for the detection of adverse pregnancy outcomes (APOs) based on placental function. Here we investigated cell-free DNA and RNA content of 102 maternal and 25 cord plasma samples. Employing a novel deconvolution methodology, we found that during the first trimester, placenta-specific DNA increased prior to the subsequent development of gestational diabetes with no change in patients with preeclampsia while decreasing with maternal obesity. Moreover, using cell-free RNA sequencing, APOs revealed 71 differentially expressed genes early in pregnancy. We noticed the upregulation of S100A8, MS4A3, and MMP8 that have been already associated with APOs but also the upregulation of BCL2L15 and the downregulation of ALPL that have never been associated with APOs. We constructed a classifier with a positive predictive ability (AUC) of 0.91 for APOs, 0.86 for preeclampsia alone and 0.64 for GDM. We conclude that placenta-specific cell-free nucleic acids during early gestation provide the possibility of predicting APOs prior to the emergence of characteristic clinical features.

In recent years, there have been major advances in the application of non-invasive techniques to predict pregnancy-related complications, for example by measuring cell-free RNA (cfRNA) in maternal blood. In contrast to cell-free DNA (cfDNA), which is already in clinical use to diagnose fetal aneuploidy, circulating RNA levels can correspond with tissue-specific gene expression and provide a snapshot of prenatal health across gestation. Here, we review the physiologic origins of cfRNA and its novel applications and corresponding challenges to monitor fetal and maternal health and predict pregnancy-related complications.

Nucleic acids include two main classes: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) [...]

Neurological diseases are responsible for approximately 6.8 million deaths every year. They affect up to 1 billion people worldwide and cause significant disability and reduced quality of life. In most neurological disorders, the diagnosis can be challenging; it frequently requires long-term investigation. Thus, the discovery of better diagnostic methods to help in the accurate and fast diagnosis of neurological disorders is crucial. Circulating nucleic acids (CNAs) are defined as any type of DNA or RNA that is present in body biofluids. They can be found within extracellular vesicles or as cell-free DNA and RNA. Currently, CNAs are being explored as potential biomarkers for diseases because they can be obtained using non-invasive methods and may reflect unique characteristics of the biological processes involved in several diseases. CNAs can be especially useful as biomarkers for conditions that involve organs or structures that are difficult to assess, such as the central nervous system. This review presents a critical assessment of the most current literature about the use of plasma and serum CNAs as biomarkers for several aspects of neurological disorders: defining a diagnosis, establishing a prognosis, and monitoring the disease progression and response to therapy. We explored the biological origin, types, and general mechanisms involved in the generation of CNAs in physiological and pathological processes, with specific attention to neurological disorders. In addition, we present some of the future applications of CNAs as non-invasive biomarkers for these diseases.

SummaryHuman plasma contains >40,000 different coding and non-coding RNAs that are potential biomarkers for human diseases. Here, we used thermostable group II intron reverse transcriptase sequencing (TGIRT-seq) combined with peak calling to simultaneously profile all RNA biotypes in apheresis-prepared human plasma pooled from healthy individuals. Extending previous TGIRT-seq analysis, we found that human plasma contains largely fragmented mRNAs from >19,000 protein-coding genes, abundant full-length, mature tRNAs and other structured small non-coding RNAs, and less abundant tRNA fragments and mature and pre-miRNAs. Many of the mRNA fragments identified by peak calling correspond to annotated protein-binding sites and/or have stable predicted secondary structures that could afford protection from plasma nucleases. Peak calling also identified novel repeat RNAs, miRNA-sized RNAs, and putatively structured intron RNAs of potential biological, evolutionary, and biomarker significance, including a family of full-length excised introns RNAs, subsets of which correspond to mirtron pre-miRNAs or agotrons.

Urothelial carcinoma of the bladder (UCB) is a common cancer worldwide with over 430,000 new cases in 2012 and 165,000 deaths and is currently the most expensive cancer per capita to treat in the U...

The rapid and accurate diagnosis of patients with minimally invasive procedures was once only found in science fiction. However, the discovery of extracellular vesicles (EVs) and their near ubiquity in body fluids, coupled with the advent of inexpensive next generation sequencing techniques and EV purification protocols, promises to make science fiction a reality. Purifying and sequencing the RNA content of EV from routine blood draws and urine samples are likely to enable pathologists and physicians to diagnose and track the progress of diseases in many inaccessible tissues in the near future. Here we present the evolutionary background of EV, summarize the biology of EV formation and cargo selection, and discuss the current barriers to making continuous liquid biopsies through the use of EV a science reality.

Nucleic acids in circulation, called cell free DNA (cfDNA) and cell free RNA (cfRNA), have recently been analyzed as suitable diagnostic and prognostic markers in cancer. There have also been several reports about the role of this type of marker in leukemia. The relevant literature was identified by a PubMed search (2000–2017) of English-language literature using the terms “cell free DNA”, “Leukemia” and Micro-RNA. Many quantitative and qualitative cfDNA biomarkers including copy number alteration, mutation, LOH and micro-RNA deregulated expression have been investigated in different studies, indicating promising results to distinguish patients from healthy individuals. The findings of this study indicate that nucleic acids in circulation have a high diagnostic and prognostic value in leukemic patients and, thus, have the potential to be used alongside the usual methods in the management of this disease.

Background Circulating tumor cells (CTC) and plasma cell-free RNA (cfRNA) can serve as biomarkers for prognosis and treatment response in lung cancer. One barrier to the selected or routine use of CTCs and plasma cfRNA in precision oncology is the limited quantity of both, and CTCs are only seen in metastatic disease. As capture of CTCs and plasma cfRNA presents an opportunity to monitor and assess malignancies without invasive procedures, we compared two methods for CTC capture and identification, and profiled mRNA from CTCs and plasma cfRNA to identify potential tumor-associated biomarkers. Methods Peripheral blood was collected from ten patients with small cell lung cancer (SCLC), ten patients with non-small cell lung cancer (NSCLC) and four healthy volunteers. Two methods were used for CTC capture: the standard epithelial cell adhesion molecule (EpCam) CellSearch kit (unicapture) and EpCAM plus HER2, EGFR and MUC-1 specific combined ferrofluid capture (quadcapture). For the quadcapture, anti-cytokeratin 7 (CK7) was additionally used to assist in CTC identification. NanoString analysis was performed on plasma cfRNA and on mRNA from combined ferrofluid isolated CTCs. Expression data was analyzed using STRING and Reactome. Results Unicapture detected CTCs in 40% of NSCLC and 60% of SCLC; whereas, quadcapture/CK7 identified CTCs in 20% of NSCLC and 80% of SCLC. Bioinformatic analysis of NanoString data identified high expression of a platelet factor 4 (PF4)-related group of transcripts. Conclusions Quadcapture ferrofluid reagent did not significantly improve CTC capture efficacy. NanoString analysis based on CTC and plasma cfRNA data highlighted an intriguing PF-4-centric network in patients with metastatic lung cancer. Electronic supplementary material The online version of this article (10.1186/s12885-019-5795-x) contains supplementary material, which is available to authorized users.

// Dan Zhou 1, 2 , Weiwei Tang 1 , Xinli Liu 1 , Han-Xiang An 1 and Yun Zhang 1, 2 1 Department of Medical Oncology, The First Affiliated Hospital of Xiamen University, Xiamen, Fujian, China 2 Xiamen Institute of Rare Earth Materials, Chinese Academy of Sciences and Key Laboratory of Design and Assembly of Functional Nanostructures, Fujian Provincial Key Laboratory of Nanomaterials, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Xiamen, Fujian, China Correspondence to: Yun Zhang, email: zhangy@fjirsm.ac.cn Han-Xiang An, email: anhanxiang@xmu.edu.cn Keywords: lung cancer, biomarker, cell-free RNA, HJURP, ADAMTS8 Received: November 30, 2016 Accepted: March 08, 2017 Published: March 30, 2017 ABSTRACT Lung cancer (LC) remains associated with significant mortality worldwide. The lack of reliable noninvasive biomarkers and targeted therapies contributes to poor survival rate. Herein, we initially took advantage of the public microarray data from Oncomine database to filter messenger RNAs (mRNAs) as potential biomarkers. Subsequently, clinical validation was applied to identify candidate noninvasive biomarkers in plasma from patients with LC. Through comprehensive analysis of transcriptional expression profiles across 12 studies, top 6 over- and underexpressed mRNAs were generated. Then, a pair of matched plasma samples from LC patient and normal control was detected by RT-PCR, and three genes with positive bands were selected for further validation. Finally, qPCR was conducted to further assess values of the three identified genes. We displayed with high confidence that two cell-free mRNAs (HJURP and ADAMTS8) were expressed at significantly levels compared to normal controls. Receiver-operating characteristic (ROC) curves on the diagnostic efficacy of plasma HJURP and ADAMTS8 mRNAs in LC diagnosis showed that the area under the ROC (AUC) was 0.6960 and 0.6877; sensitivity was 66.0% and 83.7%; specificity was 78.6% and 71.4%, respectively. Combined ROC analyses using these two biomarkers revealed an elevated AUC of 0.75. Furthermore, the higher HJURP level could be associated with early-stage LC while lower ADAMTS8 level could be correlated with non-small cell lung cancer. Collectively, circulating HJURP and ADAMTS8 mRNAs are promising noninvasive biomarkers for LC diagnosis. Our integrative strategy provides new insights into novel noninvasive biomarker identification for other types of cancer.

INTRODUCTION: Glioblastoma (GBM) is a malignant primary brain tumour with dismal prognosis. Treatment monitoring remains a challenge in clinical routine, since brain imaging cannot reliably differentiate between true progression and treatment-associated changes. In this project, we evaluate different methods of extracellular vesicles (EV) purification, in order to specifically isolate GBM-EVs from human serum/plasma and introduce EVs, as well as cell-free RNA as possible biomarkers for treatment monitoring in GBM patients. METHODS: EVs from primary GBM cells and the Gaussia luciferase expressing Gli36-GLuc cells were isolated via size-exclusion chromatography (SEC) and ultracentrifugation. EV-surface markers were evaluated by flow cytometry. Gli36-GLuc EVs containing GLuc mRNA were spiked in healthy plasma. Thereafter, plasma EVs were isolated via ultracentrifugation, SEC and immunoprecipitation. Subsequently, RNA was isolated from vesicles and evaluated for GLuc levels via qRT-PCR. Total cell-free RNA from serum of GBM patients was tested for different mRNAs and micro-RNAs at different disease stages. RESULTS: EVs from GBM cells expressed high levels of CD29 and CD44, when compared to EVs from healthy donor plasma. Gli36-GLuc EVs spiked in healthy plasma were more effectively isolated with CD44-based immunoprecipitation than with ultracentrifugation or SEC, as shown by higher GLuc RNA levels in the corresponding vesicles. When compared to total cell-free RNA extracted from this plasma, RNA from EVs exhibited a higher GLuc yield. In cell-free RNA from GBM patients, MGMT levels alone were not capable of detecting progressive disease. CONCLUSIONS: 1. CD44 could serve as a novel, promising target for GBM-EV and be utilised for immunoprecipitation-based EV capturing. 2. Using the appropriate EV purification method possibly affects their potential as biomarkers for GBM. 3. MGMT levels alone in cell-free RNA of GBM patients did not correlate with disease status contrary to previous reports.

Cancer and fetal development share a lot. This is nothing new. The 19th century pathologist John Beard noted the similarities between a tumor and a trophoblast. Building on that, Lloyd Old, a pioneer in cancer immunology said, “If you've ever seen the trophoblast invading the uterus, it invades, spreads, creates a blood supply. It also suppresses the maternal immune system. All of those are characteristics of cancer” (1). Today, tumors are being diagnosed noninvasively through the ability to find traces of circulating malignant material in blood. The same holds true for fetal development. The ability to reliably detect fetal cell-free DNA in maternal blood is changing clinical practice. I spoke with Howard Hughes Investigator and bioengineering, physics, and applied physics professor at Stanford University, Stephen Quake, who has played a central role in these innovations. Motivated to replace invasive biopsies with blood tests, Quake developed a polymorphism-independent shotgun DNA sequencing test that could detect fetal aneuploidy from maternal plasma (2). “After 10 years, this has had an enormous impact,” he says, “three million women are tested each year, and these methods have been widely adopted in the liquid biopsy space.” Quake has his sights on another challenge around childbirth—estimating gestational age and the risk for preterm delivery. “It started with the idea of following human development, and using RNA to do that,” Quake explains. In a manuscript in which he and colleagues combined high-throughput microarrays for RNA analysis with next-generation sequencing …

Non-alcoholic fatty liver disease (NAFLD) is the worldwide most common cause of chronic liver pathology, which prevalence strongly correlates with the increasing incidence of diabetes, obesity and metabolic syndrome in the general population. Simple steatosis, the earliest NAFLD stage, usually remains asymptomatic, and appropriate changes in the lifestyle, as well as the diet, can reverse the affected liver into the healthy state. The potential of simple steatosis to progress into severe fibrotic stages and to facilitate carcinogenesis necessitates timely NAFLD detection and risk stratification in community-based healthcare settings. Since their initial discovery a decade ago, extracellular circulating miRNAs have been found in all human biological fluids including blood and shown to hold great promises as non-invasive biomarkers. Normally, intracellular miRNAs participate in the regulation of gene expression, but once released by dying/dead cells they remain highly stable in the extracellular environment for prolonged periods. Therefore, circulating miRNA profiles can reflect the ongoing pathogenic processes in body’s tissues and organs, and enable highly sensitive non-invasive diagnosis of multiple disorders. A non-urgent character of the NAFLD-related decision-making justifies the use of chronic liver diseases as an excellent test case for examining the practical utility of circulating miRNAs as biomarkers for longitudinal monitoring of human health. In this review, we summarize the state-of-the-art in the field of early diagnosis of NAFLD using circulating blood miRNAs, and stress the necessity of additional experimental validation of their diagnostic potential. We further emphasize on the potential diagnostics promises of other cell-free RNA species found in human biological fluids.

Tumor heterogeneity is a major challenge and the root cause of resistance to treatment. Still, the standard diagnostic approach relies on the analysis of a single tumor sample from a local or metastatic site that is obtained at a given time point. Due to intratumoral heterogeneity and selection of subpopulations in diverse lesions this will provide only a limited characterization of the makeup of the disease. On the other hand, recent developments of nucleic acid sequence analysis allows to use minimally invasive serial blood samples to assess the mutational status and altered gene expression patterns for real time monitoring in individual patients. Here, we focus on cell-free circulating tumor-specific mutant DNA and RNA (including mRNA and non-coding RNA), as well as current limitations and challenges associated with circulating nucleic acids biomarkers.

There is limited research on cell-free RNA (cf-RNA) in the urine of cancer patients. The present study was performed to detect the cf-RNA in the urine of patients with clear cell renal cell carcinoma (ccRCC). Ninety-five urine samples from ccRCC patients and 50 urine samples from control subjects were analyzed. The cf-RNA integrity index was calculated by using quantitative real-time RT-PCR assays of the small-sized fragment (106 bp) and the big-sized fragment (416 bp) in GAPDH mRNA. The initial analysis showed that cf-RNA was stable and detectable in the urine. The mean cf-RNA integrity index was significantly lower in the urine of ccRCC patients (mean: 0.07, 95%CI: 0.05-0.10) when compared with the urine from control subjects (mean: 0.25, 95%CI: 0.16-0.33) (p < 0.001). The value of the area under the receiver-operating characteristic curve by using the cf-RNA integrity index for the diagnosis of ccRCC was 0.858 with a sensitivity of 68.0% and a specificity of 92.6%. Moreover, the small-sized VEGF mRNA fragment (98 bp) was detected in 31 of 50 urine samples of patients with ccRCC and in only 2 of 50 urine samples of control subjects (p < 0.001) while the detection of the big-sized (420 bp) VEGF mRNA fragment was an infrequent event. Our findings suggest that the small-sized cf-RNA in urine was more abundant in cancer patients. The tumor-related gene VEGF mRNA fragment was detectable in the urine of cancer patients. Our finding may provide a new molecular assay for the diagnosis of renal cell carcinoma.

Liver disease is a major cause of morbidity and mortality worldwide. As in other fields of medicine, there is a stringent need for non-invasive markers to improve patient diagnostics, monitoring and prognostic ability in liver pathology. Cell-free circulating RNA molecules have been recently acknowledged as an important source of potential medical biomarkers. However, many aspects related to the biology of these molecules remain to be elucidated. In this review, we summarize current concepts related to the origin, transportation and possible functions of cell-free RNA. We outline current development of extracellular RNA-based biomarkers in the main forms of non-inherited liver disease: chronic viral hepatitis, hepatocellular carcinoma, non-alcoholic fatty liver, hepato-toxicity, and liver transplantation. Despite recent technological advances, the lack of standardization in the assessment of these markers makes their adoption into clinical practice difficult. We thus finally review the main factors influencing quantification of circulating RNA. These factors should be considered in the reporting and interpretation of current findings, as well as in the proper planning of future studies, to improve reliability and reproducibility of results.

Circulating nucleic acids are present in the blood of humans and other vertebrates. During the last 10 years researchers actively studied cell-free nucleic acids present in plasma or serum with great expectations of their use as potential biomarkers for cancer and other pathologic conditions. In the present manuscript the main findings related to the principal characteristics of circulating nucleic acids, the hypothesis on their origin and some methodological considerations on sample collection and extraction as well as on some innovative assay methods have been summarized. Recent reports on the importance of circulating nucleic acids in the intercellular exchange of genetic information between eukaryotic cells have been reviewed.

e16613Background: In prostate cancer (PC), the androgen-receptor isoform that is encoded by the splice variant 7 (AR-V7) is characterized by an absence of the ligand-binding domain that is the targ...

The use of cell-free nucleic acids in the circulation of pregnant women for noninvasive prenatal diagnosis is arguably one of the hottest current topics in prenatal medicine. Between 1997 and the present era this field has gone from basic research to clinical application for diagnosis of fetal gender and Rhesus D status. Over the next few years it is likely that noninvasive prenatal diagnosis for Down syndrome will also be possible. Here we summarize current and future clinical applications of analyzing cell-free fetal DNA and RNA in both maternal and neonatal body fluids, including maternal plasma, serum, whole blood, amniotic fluid, and neonatal saliva. We describe methods to evaluate normal and abnormal fetal and neonatal development using gene expression microarrays. We also discuss the ways in which differentially-regulated gene lists can advance knowledge of both fetal and neonatal biology, as well as suggest novel possibilities for fetal and neonatal treatment.

Background Since there is no validated assay to monitor disease in children with neuroblastoma (NB), we tested whether NB specific cell-free RNA could be detected in their plasma samples. Moreover, with the aim of reducing patients' discomfort, we compared this assay to a recently standardized procedure that uses a larger amount of whole blood. Procedures Using conditions that excluded RNA recovery from contaminating tumor cells, the total amount of cell-free RNA present in healthy children and patients with NB was quantified. Expression of tyrosine hydroxylase (TH) was assayed by quantitative RT-PCR. Results In patients with NB the amount of cell-free RNA was higher than in healthy children. However, it was less and more degraded than in healthy adults. The median amount of cell-free RNA that was reverse transcribed, measured through the use of standard curves for reference genes, was 0.03 (range 0–30) pg of input RNA, that is, always less than 1/10,000 of that reverse transcribed from total RNA extracted from whole cells. Despite the presence of disease and the positive results obtained with RNA extracted from peripheral blood cells, few cell-free RNA samples tested positive by the TH assay. Similar results were obtained also with TH primers specifically designed to amplify 50 bp RNA fragments. Conclusion These findings suggest that for monitoring disease status detection of cell-free tumor-specific RNAs in patients with NB is not a reliable alternative to whole cell RNA. Pediatr Blood Cancer 2010;54:897–903 © 2010 Wiley-Liss, Inc.

Background Cell-free RNA (cfRNA) naturally occurs in blood and has clinical significance. Accurate quantification of these extracellular RNAs in whole blood is hindered by the simultaneous unintended release of cellular RNA and degradation of cfRNA after blood draw. An appropriate blood collection device is needed to stabilize cfRNA during blood processing, transportation and storage, which will ensure cfRNA test reliability. In this study we compared a novel blood collection device against traditional K3EDTA tubes for its ability to stabilize cfRNA in blood when subjected to conditions that can occur during sample storage and shipping. Findings Shipping blood samples drawn into K3EDTA tubes showed a significant increase in mRNA copy numbers for β-actin, c-fos, and 18S rRNA in plasma. In contrast, shipping blood drawn into Cell-Free RNA BCT™s (BCTs) showed only a slight change in mRNA copy numbers for circulating β-actin, c-fos, and 18S rRNA. Moreover, blood stored in K3EDTA tubes at 6°C, 22°C and 30°C for 3 days showed a significant increase in mRNA copy numbers for c-fos and β-actin, whereas samples stored in BCTs only showed a slight increase. Conclusion Our results show that BCTs minimize increases in background RNA levels caused by temperature fluctuations or agitation that can occur during blood sample storage and shipping. This novel blood collection tube could provide a method for obtaining high quality stabilized cfRNA samples for rare RNA target detection and determining accurate cfRNA concentrations.