Toward reliable biomarker signatures in the age of liquid biopsies - how to standardize the small RNA-Seq workflow.

Small RNA-Seq has emerged as a powerful tool in transcriptomics, gene expression profiling and biomarker discovery. Sequencing cell-free nucleic acids, particularly microRNA (miRNA), from liquid biopsies additionally provides exciting possibilities for molecular diagnostics, and might help establish disease-specific biomarker signatures. The complexity of the small RNA-Seq workflow, however, bears challenges and biases that researchers need to be aware of in order to generate high-quality data. Rigorous standardization and extensive validation are required to guarantee reliability, reproducibility and comparability of research findings. Hypotheses based on flawed experimental conditions can be inconsistent and even misleading. Comparable to the well-established MIQE guidelines for qPCR experiments, this work aims at establishing guidelines for experimental design and pre-analytical sample processing, standardization of library preparation and sequencing reactions, as well as facilitating data analysis. We highlight bottlenecks in small RNA-Seq experiments, point out the importance of stringent quality control and validation, and provide a primer for differential expression analysis and biomarker discovery. Following our recommendations will encourage better sequencing practice, increase experimental transparency and lead to more reproducible small RNA-Seq results. This will ultimately enhance the validity of biomarker signatures, and allow reliable and robust clinical predictions.

Comparison of the miRNome and piRNome of bovine blood and plasma by small RNA sequencing.

OBJECTIVES: The small RNAs of bovine plasma and whole blood were analysed using next-generation sequencing to quantify, profile and compare the microRNAs (miRNA) and piRNA signatures in both bio fluids. RESULTS: Evaluating read-count data resulted in a proportion of 5.0 ± 2.9 % of miRNAs in plasma while 38.2 ± 3.4 % were identified in whole blood. Regarding piRNAs, the percentages in both matrices were nearly the same: 1.4 ± 0.8 % of piRNAs in plasma and 1.9 ± 0.8 % in whole blood. Investigation of the ten most abundant miRNAs and piRNAs in both bio fluids revealed that two miRNAs and seven piRNAs were identical. Comparing the read-count values of these matching pairs highlighted that miRNA and piRNA levels in blood exceeded the abundance of their corresponding miRNAs and piRNAs in plasma, except liver-specific miR-122 and three piRNAs. CONCLUSIONS: The data strengthened evidence that the circulating small RNA signature in plasma is not only influenced by hematocytes and certain small RNAs could originate from other sources than cellular blood components.

Transcriptional biomarkers--high throughput screening, quantitative verification, and bioinformatical validation methods.

Molecular biomarkers found their way into many research fields, especially in molecular medicine, medical diagnostics, disease prognosis, risk assessment but also in other areas like food safety. Different definitions for the term biomarker exist, but on the whole biomarkers are measureable biological molecules that are characteristic for a specific physiological status including drug intervention, normal or pathological processes. There are various examples for molecular biomarkers that are already successfully used in clinical diagnostics, especially as prognostic or diagnostic tool for diseases. Molecular biomarkers can be identified on different molecular levels, namely the genome, the epigenome, the transcriptome, the proteome, the metabolome and the lipidome. With special "omic" technologies, nowadays often high throughput technologies, these molecular biomarkers can be identified and quantitatively measured. This article describes the different molecular levels on which biomarker research is possible including some biomarker candidates that have already been identified. Hereby the transcriptomic approach will be described in detail including available high throughput methods, molecular levels, quantitative verification, and biostatistical requirements for transcriptional biomarker identification and validation.

RNA-sequencing as useful screening tool in the combat against the misuse of anabolic agents.

The abuse of anabolic substances in animal husbandry is forbidden within the EU and well controlled by detecting substance residues in different matrices. The application of newly designed drugs or substance cocktails represents big problems. Therefore developing sensitive test methods is important. The analysis of physiological changes caused by the use of anabolic agents on the molecular level, for example, by quantifying gene expression response, is a new approach to develop such screening methods. A novel technology for holistic gene expression analysis is RNA sequencing. In this study, the potential of this high-throughput method for the identification of biomarkers was evaluated. The effect of trenbolone acetate plus estradiol on gene expression in liver from Nguni heifers was analyzed with RNA sequencing. The expression of 40 selected candidate genes was verified via RT-qPCR, whereby 20 of these genes were significantly regulated. To extract the intended information from these regulated genes, biostatistical tools for pattern recognition were applied and resulted in a clear separation of the treatment groups. Those candidate genes could be verified in boars and in calves treated with anabolic substances. These results show the potential of RNA sequencing to screen for biomarker candidates to detect the abuse of anabolics. The verification of these biomarkers in boars and calves leads to the assumption that gene expression biomarkers are independent of breed or even species and that biomarkers, identified in farm animals could also act as potential biomarker candidates to detect the abuse of anabolic substances in human sports.

Changes in the miRNA profile under the influence of anabolic steroids in bovine liver.

miRNAs are regulatory RNA molecules. The analytical interest rose over the past 10 years especially in clinical diagnostics as miRNAs show specific expression patterns in several human diseases like diabetes or cancer. Therefore, it is expected that miRNA profiles might be used as biomarkers in early diagnosis. The idea of establishing biomarkers is also present in veterinary drug analysis, e.g. in the surveillance of illegal use of anabolics. Transcriptomics is a promising approach in the detection of anabolics misuse. However, miRNA expression patterns have shown their superiority over mRNA patterns in clinical diagnostics. Thus, the influence of anabolic steroids on miRNA expression in bovine liver should be investigated and an expression pattern should be validated, which might be used as a treatment biomarker. An animal experiment was conducted with 18 heifers equally allocated to a control and a treatment group, which was implanted with TBA plus E2. Liver samples were screened for miRNA expression using PCR arrays. Expression of 11 prominent miRNAs was validated via single assay qPCR. Herein, the following expression pattern could be found with an up-regulation of miR-29c and miR-103 and a down-regulation of miR-34a, miR-181c, miR-20a and miR-15a (p<0.05 each). Using principal components analysis (PCA), the control group could clearly be distinguished from the treatment group, when integrating gene expression results from both miRNA and mRNA. So, the combination of different transcribed targets (mRNA plus miRNA) might be a promising approach to find a valid expression pattern to be used for anabolic treatment screening.

Validation of extraction methods for total RNA and miRNA from bovine blood prior to quantitative gene expression analyses.

The benefit and precision of blood diagnosis by quantitative real-time PCR (qPCR) is limited by sampling procedures and RNA extraction methods. We have compared five different RNA extraction protocols from bovine blood regarding RNA and miRNA yield, quality, and most reproducible data in the qRT-PCR with the lowest point of quantification. Convincing results in terms of highest quantity, quality, and best performance for mRNA qPCR were obtained by leukocyte extraction following blood lysis as well as extraction of PAXgene stabilized blood. The best microRNA qPCR results were obtained for samples extracted by the leukocyte extraction method.

Design and optimization of reverse-transcription quantitative PCR experiments.

BACKGROUND: Quantitative PCR (qPCR) is a valuable technique for accurately and reliably profiling and quantifying gene expression. Typically, samples obtained from the organism of study have to be processed via several preparative steps before qPCR. METHOD: We estimated the errors of sample withdrawal and extraction, reverse transcription (RT), and qPCR that are introduced into measurements of mRNA concentrations. We performed hierarchically arranged experiments with 3 animals, 3 samples, 3 RT reactions, and 3 qPCRs and quantified the expression of several genes in solid tissue, blood, cell culture, and single cells. RESULTS: A nested ANOVA design was used to model the experiments, and relative and absolute errors were calculated with this model for each processing level in the hierarchical design. We found that intersubject differences became easily confounded by sample heterogeneity for single cells and solid tissue. In cell cultures and blood, the noise from the RT and qPCR steps contributed substantially to the overall error because the sampling noise was less pronounced. CONCLUSIONS: We recommend the use of sample replicates preferentially to any other replicates when working with solid tissue, cell cultures, and single cells, and we recommend the use of RT replicates when working with blood. We show how an optimal sampling plan can be calculated for a limited budget. .

Changes in the microRNA expression profile during blood storage.

OBJECTIVES: For several decades, autologous blood doping (ABD) in sports has been a major problem, and even today there is still no reliable method for satisfactorily detecting ABD. For this kind of doping, stored individual erythrocytes are used to increase stamina and endurance caused by a higher erythrocyte level in the athlete's body. Since there is growing evidence that these cells are enriched with microRNAs (miRNAs), this study has been carried out to discover and validate all miRNAs occurring in fresh blood as well as in stored blood. METHODS: Therefore, small RNA Next Generation Sequencing has been performed, which allows untargeted detection of all miRNAs in a blood sample. The focus of this investigation has been to find miRNA alterations in blood bags after erythrocyte processing and during storage, as compared with fresh blood directly withdrawn from subjects. Blood samples were obtained from 12 healthy, recreationally active male subjects three times before blood donation and from blood bags at several time points after blood processing. RESULTS: 189 miRNAs have been considered stable over two consecutive weeks. A further analysis revealed a complex biomarker signature of 28 miRNAs, consisting of 6 miRNAs that altered during 6 weeks of storage and 22 miRNAs that altered due to processing. CONCLUSION: These results suggest that the identified miRNA biomarker signature may be used for the detection of ABD. These 28 miRNA candidates are tested and verified currently in a follow-up study, a human transfusion clinical trial in healthy sportsmen.

The Dynamics of microRNA Transcriptome in Bovine Corpus Luteum during Its Formation, Function, and Regression.

The formation, function, and subsequent regression of the ovarian corpus luteum (CL) are dynamic processes that enable ovary cyclical activity. Studies in whole ovary tissue have found microRNAs (miRNAs) to by critical for ovary function. However, relatively little is known about the role of miRNAs in the bovine CL. Utilizing small RNA next-generation sequencing we profiled miRNA transcriptome in bovine CL during the entire physiological estrous cycle, by sampling the CL on days: d 1-2, d 3-4, and d 5-7 (early CL, eCL), d 8-12 (mid CL, mCL), d 13-16 (late CL, lCL), and d > 18 (regressed CL, rCL). We characterized patterns of miRNAs abundance and identified 42 miRNAs that were consistent significantly different expressed (DE) in the eCL relative to their expression at each of the analyzed stages (mCL, lCL, and rCL). Out of these, bta-miR-210-3p, -2898, -96, -7-5p, -183-5p, -182, and -202 showed drastic up-regulation with a fold-change of ≥2.0 and adjusted P < 0.01 in the eCL, while bta-miR-146a was downregulated at lCL and rCL vs. the eCL. Another 24, 11, and 21 miRNAs were significantly DE only between individual comparisons, eCL vs. the mCL, lCL, and rCL, respectively. Irrespective of cycle stage two miRNAs, bta-miR-21-5p and bta-miR-143 were identified as the most abundant miRNAs species and show opposing expression abundance. Whilst bta-miR-21-5p peaked in number of reads in the eCL and was significantly downregulated in the mCL and lCL, bta-miR-143 reached its peak in the rCL and is significantly downregulated in the eCL. MiRNAs with significant DE in at least one cycle stage (CL class) were further grouped into eight distinct clusters by the self-organizing tree algorithm (SOTA). Half of the clusters contain miRNAs with low-expression, whilst the other half contain miRNAs with high-expression levels during eCL. Prediction analysis for significantly DE miRNAs resulted in target genes involved with CL formation, functionalization and CL regression. This study is the most comprehensive profiling of miRNA transcriptome in bovine CL covering the entire estrous cycle and provides a compact database for further functional validation and biomarker identification relevant for CL viability and fertility.

Identification of a potential gene expression biomarker signature in bovine liver to detect the abuse of growth promoters.

The misuse of anabolic agents in animal husbandry is a ubiquitous problem. The ban of growth promoters in food producing animals in the European Union is well controlled, but there are still application regimes, such as new designed drugs or hormone cocktails, that are difficult to detect. Therefore, the idea of identifying molecular biomarkers that are based on the physiological effect of treatment has come into focus. In a previous study we identified mRNA biomarker candidates in liver samples that enable the separation of untreated animals from animals treated with a combination of androgens plus estrogens. In the present study those candidates were validated in calves treated with a combination of progesterone plus estradiol or clenbuterol, respectively. Therefore, the candidate genes were quantified in liver samples of those calves via RT-qPCR. Using dynamic principal component analysis (PCA), a signature of 11 genes could be selected. This set of genes enabled the separation of treated and control animals independent of the applied drug. Additional quantification of these genes in a set of control samples from another animal trial resulted in a PCA that also showed a separation of those samples from treated animals. This study showed that gene expression biomarkers have a high potential to enable the detection of physiological changes caused by the application of growth-promoting substances independent of the given drug, but further studies are necessary to broaden the spectrum of anabolic substance groups for which those biomarker candidates can be used.

mRNA and microRNA purity and integrity: the key to success in expression profiling.

RNA quality control is a crucial step in guaranteeing integer nondegraded RNA and receiving meaningful results in gene expression profiling experiments, using micro-array, RT-qPCR (Reverse-Transcription quantitative PCR), or Next-Generation-Sequencing by RNA-Seq or small-RNA Seq. Therefore, assessment of RNA integrity and purity is very essential prior to gene expression analysis of sample RNA to ensure the accuracy of any downstream applications. RNA samples should be nondegraded or fragmented and free of protein, genomic DNA, nucleases, and enzymatic inhibitors. Herein we describe the current state-of-the-art RNA quality assessment by combining UV/Vis spectrophotometry and microfluidic capillary electrophoresis.

Optimization of extraction of circulating RNAs from plasma--enabling small RNA sequencing.

There are several protocols and kits for the extraction of circulating RNAs from plasma with a following quantification of specific genes via RT-qPCR. Due to the marginal amount of cell-free RNA in plasma samples, the total RNA yield is insufficient to perform Next-Generation Sequencing (NGS), the state-of-the-art technology in massive parallel sequencing that enables a comprehensive characterization of the whole transcriptome. Screening the transcriptome for biomarker signatures accelerates progress in biomarker profiling for molecular diagnostics, early disease detection or food safety. Therefore, the aim was to optimize a method that enables the extraction of sufficient amounts of total RNA from bovine plasma to generate good-quality small RNA Sequencing (small RNA-Seq) data. An increased volume of plasma (9 ml) was processed using the Qiagen miRNeasy Serum/Plasma Kit in combination with the QIAvac24 Plus system, a vacuum manifold that enables handling of high volumes during RNA isolation. 35 ng of total RNA were passed on to cDNA library preparation followed by small RNA high-throughput sequencing analysis on the Illumina HiSeq2000 platform. Raw sequencing reads were processed by a data analysis pipeline using different free software solutions. Seq-data was trimmed, quality checked, gradually selected for miRNAs/piRNAs and aligned to small RNA reference annotation indexes. Mapping to human reference indexes resulted in 4.8±2.8% of mature miRNAs and 1.4±0.8% of piRNAs and of 5.0±2.9% of mature miRNAs for bos taurus.

The physiological way: monitoring RNA expression changes as new approach to combat illegal growth promoter application.

The use of growth-promoting agents in food-producing animals is forbidden in the European Union (EU). Therefore a strict control programme has been developed, detecting residues of all known growth-promoting agents using chromatographical methods in combination with mass spectrometry or immunoassays. New designed xenobiotic substances or hormone cocktails are difficult to identify with these methods and therefore the development of new sensitive test methods is important. A promising indirect approach is the detection of physiological effects of the administered growth promoters on the molecular level using 'omic' technologies. The analysis of the transcriptome on mRNA and miRNA level and thereby identifying biomarkers for the use of anabolic agents is one possible strategy for developing a new screening method. This paper describes the technologies available for gene expression profiling and summarizes the efforts made in the analysis of the transcriptome in order to identify potential gene expression biomarkers for the use of growth promoters in cattle.

The analysis of the transcriptome as a new approach for biomarker development to trace the abuse of anabolic steroid hormones.

The abuse of anabolic steroid hormones in human sports and animal husbandry is an ubiquitous problem and therefore a tight control program in both areas is very important. Within these control programs, hormone residues are detected by immunoassays or chromatographical methods in combination with mass spectrometry. With these methods, all known substances can be detected; yet new xenobiotic growth promoters and new ways of application are difficult to detect. Therefore it is important to develop new sensitive screening methods to enable an efficient control for misused anabolic substances. The detection of their physiological action is a promising approach. Anabolic steroid hormones directly influence the expression of specific genes and thus the analysis of the transcriptome of different target tissues and matrices is of great interest. This review describes our recent efforts made concerning the analysis of gene expression changes in different tissues, different species and under different anabolic treatments.

Effect of trenbolone acetate plus estradiol on transcriptional regulation of metabolism pathways in bovine liver.

BACKGROUND: The use of anabolic steroids is forbidden for food producing animals in the EU. Owing to the advantages of anabolics for production profitability, illegal application is appealing. Anabolics are known to influence gene expression of several tissues. We focused on the liver because of its important role in nutrient and hormone metabolism. The aim of the present study was to find differentially regulated metabolic pathways, which might be used as treatment biomarkers. MATERIAL AND METHODS: A total of 18 Nguni heifers were allocated equally to a control group and a treatment group and were implanted with Revalor H. Expression of 34 target genes was measured using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). RESULTS: Upregulation of androgen receptor and insulin-like growth factor 1 (IGF-1) and downregulation of IGF-2, insulin-like growth factor binding protein 2, steroid hormone binding globulin, insulin receptor α, insulin receptor ß, tyrosine aminotransferase, 17ß-hydroxy steroid dehydrogenase 2,3-hydroxy-methylglutaryl-coenzym-A-synthase, cathepsin B, hepatocyte growth factor, steroidogenic acute regulatory protein, apolipoprotein 2 and tumor necrosis factor α was demonstrated. CONCLUSION: Several biochemical pathways showed different regulations on mRNA level under the influence of trenbolone acetate plus estradiol. The inhibition of nutrient metabolism and protein breakdown seems to support growth processes. IGF-1 plays an important role in growth and development and thus the upregulation of IGF-1 could be responsible for the stimulation of growth in treated animals. The upregulation of IGF-1 could also be revealed as a possible risk factor for the generation of artherosclerotic plaques, which are known as long-term side effects following the use of anabolic steroids. Principal components analysis of RT-qPCR results showed that both groups arrange together and can be clearly separated. Therefore, these might be used as possible biomarkers in bovine liver.

Monitoring gene expression in muscle tissue of macaca fascicularis under the influence of testosterone and SARM.

The focus of this study was to evaluate data on the gene expression profiles induced by testosterone and a selective androgen receptor modulator (SARM, TAP Pharmaceutical Products Inc., Lake Forest, IL, USA) in androgen sensitive muscle tissue to obtain a better understanding on the molecular mechanisms of action and to identify biomarkers for SARM function in primate organs. A total of 24 male cyomolgus monkeys were divided into four groups: testosterone group, SARM1 group, SARM10 group, and control group, each consisting of six animals. The testosterone group was treated i.m. with 3.0 mg/kg Testostoviron®-depot-250 (Schering, Berlin, Germany) every 2 weeks, the SARM1 and SARM10 groups with 1 mg/kg or 10 mg/kg SARM LGD2941 daily, and the control group was not treated. Muscle biopsies from musculus quadriceps and musculus triceps were collected at three time points: baseline time point before SARM application (control), on day 16, and on day 90 of treatment. A total of 30 candidate genes were selected according to their functionality by screening the actual literature and were composed to the following functional groups: cell cycle, endocrine factors, energy metabolism, muscle fiber proteins, muscle specific transcription factors, protein metabolism, and satellite cell biology. Biomarkers were identified as genes regulated from baseline in any of the three treatment groups at day 16 or day 90 using analysis of variance with baseline defined as the contrast group. Out of 23 tested candidate genes, 3 were significantly regulated in m. quadriceps after 90 days treatment; in m. triceps no significant differences were identified. Cathepsin L, calpain 3, and insulin like growth factor binding protein 3 could be identified as first biomarkers, and first physiological differences between control and treatment samples were determined. Both testosterone and SARM LGD2941 appear to have similar effects after 90 days treatment, and thus a longer-term therapy with these substances can be recommended.

The use of omic technologies for biomarker development to trace functions of anabolic agents.

The combat against misuse of growth promoting agents is a major topic in agricultural meat production and human sports. In routine screening, hormone residues of all known growth promoting agents are detected by immuno assays or chromatographical methods in combination with mass spectrometry. To overcome the detection by these routine screening methods new xenobiotic growth promoters and new ways of application were developed, e.g. the combination of different agents in hormone cocktails are employed. To enable an efficient tracing of misused anabolic substances it is necessary to develop new screening technologies for a broad range of illegal drugs including newly designed xenobiotic anabolic agents. The use of omic technologies like, transcriptomics, proteomics or metabolomics is a promising approach to discover the misuse of anabolic hormones by indirectly detecting their physiological action. With the help of biostatistical tools it is possible to extract the quested information from the data sets retrieved from the omic technologies. This review describes the potential of these omic technologies for the development of such new screening methods and presents recent literature in this field.

Identification of potential gene expression biomarkers for the surveillance of anabolic agents in bovine blood cells.

In the EU, the use of anabolic steroids in food producing animals has been forbidden since 1988. The routine methods used in practice are based on the detection of hormonal residues. To overcome these routine methods, growth-promoting agents are sometimes administered at concentrations below the detection limit and new anabolic substances are designed. Therefore, new monitoring systems are needed to overcome the misuse of anabolic agents in meat production. In this study, a new monitoring system was applied: the quantification of mRNA gene expression changes by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR). Blood was selected as ideal tissue for biomarker screening. From the literature, it is known that steroid hormones affect mRNA gene expression of the different blood cells, which can easily be taken from the living animal. In an animal trial, 18 Nguni heifers were separated to two groups of nine animals. One group served as untreated control and the other group was treated with a combination of trenbolone acetate plus estradiol for 39 days in order to allow the detection of the effect on mRNA expression in blood at three time points. Candidate genes used for developing a biomarker pattern were chosen by screening the actual literature for anabolic effects on blood cells. It could be demonstrated that the combination of trenbolone acetate plus estradiol significantly influences mRNA expression of the steroid receptors (ER-alpha and GR-alpha), the apoptosis regulator Fas, the proinflammatory interleukins IL-1alpha, IL-1beta and IL-6 and of MHCII, CK, MTPN, RBM5 and Actin-beta. Advanced statistical analysis by Principal Components Analysis (PCA) indicated that these genes represent potential biomarkers for this hormone combination in whole blood.

Influence of testosterone and a novel SARM on gene expression in whole blood of Macaca fascicularis.

Anabolic hormones, including testosterone, have been suggested as a therapy for aging-related conditions, such as osteoporosis and sarcopenia. These therapies are sometimes associated with severe androgenic side effects. A promising alternative to testosterone replacement therapy are selective androgen receptor modulators (SARMs). SARMs have the potential to mimic the desirable central and peripheral androgenic anabolic effects of testosterone without having its side effects. In this study we evaluated the effects of LGD2941, in comparison to testosterone, on mRNA expression of selected target genes in whole blood in an non-human model. The regulated genes can act as potential blood biomarker candidates in future studies with AR ligands. Cynomolgus monkeys (Macaca fascicularis) were treated either with testosterone or LGD2941 for 90 days in order to compare their effects on mRNA expression in blood. Blood samples were taken before SARM application, on day 16 and on day 90 of treatment. Gene expression of 37 candidate genes was measured using quantitative real-time RT-PCR (qRT-PCR) technology. Our study shows that both testosterone and LGD2941 influence mRNA expression of 6 selected genes out of 37 in whole blood. The apoptosis regulators CD30L, Fas, TNFR1 and TNFR2 and the interleukins IL-12B and IL-15 showed significant changes in gene expression between control and the treatment groups and represent potential biomarkers for androgen receptor ligands in whole blood.