Transient astrocyte-like NG2 glia subpopulation emerges solely following permanent brain ischemia.

NG2 glia display wide proliferation and differentiation potential under physiological and pathological conditions. Here, we examined these two features following different types of brain disorders such as focal cerebral ischemia (FCI), cortical stab wound (SW), and demyelination (DEMY) in 3-month-old mice, in which NG2 glia are labeled by tdTomato under the Cspg4 promoter. To compare NG2 glia expression profiles following different CNS injuries, we employed single-cell RT-qPCR and self-organizing Kohonen map analysis of tdTomato-positive cells isolated from the uninjured cortex/corpus callosum and those after specific injury. Such approach enabled us to distinguish two main cell populations (NG2 glia, oligodendrocytes), each of them comprising four distinct subpopulations. The gene expression profiling revealed that a subpopulation of NG2 glia expressing GFAP, a marker of reactive astrocytes, is only present transiently after FCI. However, following less severe injuries, namely the SW and DEMY, subpopulations mirroring different stages of oligodendrocyte maturation markedly prevail. Such injury-dependent incidence of distinct subpopulations was also confirmed by immunohistochemistry. To characterize this unique subpopulation of transient astrocyte-like NG2 glia, we used single-cell RNA-sequencing analysis and to disclose their basic membrane properties, the patch-clamp technique was employed. Overall, we have proved that astrocyte-like NG2 glia are a specific subpopulation of NG2 glia emerging transiently only following FCI. These cells, located in the postischemic glial scar, are active in the cell cycle and display a current pattern similar to that identified in cortical astrocytes. Astrocyte-like NG2 glia may represent important players in glial scar formation and repair processes, following ischemia.

Performance Comparison of Reverse Transcriptases for Single-Cell Studies.

BACKGROUND: Recent advances allowing quantification of RNA from single cells are revolutionizing biology and medicine. Currently, almost all single-cell transcriptomic protocols rely on reverse transcription (RT). However, RT is recognized as a known source of variability, particularly with low amounts of RNA. Recently, several new reverse transcriptases (RTases) with the potential to decrease the loss of information have been developed, but knowledge of their performance is limited. METHODS: We compared the performance of 11 RTases in quantitative reverse transcription PCR (RT-qPCR) on single-cell and 100-cell bulk templates, using 2 priming strategies: a conventional mixture of random hexamers with oligo(dT)s and a reduced concentration of oligo(dT)s mimicking common single-cell RNA-sequencing protocols. Depending on their performance, 2 RTases were further tested in a high-throughput single-cell experiment. RESULTS: All tested RTases demonstrated high precision (R2 > 0.9445). The most pronounced differences were found in their ability to capture rare transcripts (0%-90% reaction positivity rate) and in their absolute reaction yield (7.3%-137.9%). RTase performance and reproducibility were compared with Z scores. The 2 best-performing enzymes were Maxima H- and SuperScript IV. The validity of the obtained results was confirmed in a follow-up single-cell model experiment. The better-performing enzyme (Maxima H-) increased the sensitivity of the single-cell experiment and improved resolution in the clustering analysis over the commonly used RTase (SuperScript II). CONCLUSIONS: Our comprehensive comparison of 11 RTases in low RNA input conditions identified 2 best-performing enzymes. Our results provide a point of reference for the improvement of current single-cell quantification protocols.

Multicenter Evaluation of Circulating Cell-Free DNA Extraction and Downstream Analyses for the Development of Standardized (Pre)analytical Work Flows.

BACKGROUND: In cancer patients, circulating cell-free DNA (ccfDNA) can contain tumor-derived DNA (ctDNA), which enables noninvasive diagnosis, real-time monitoring, and treatment susceptibility testing. However, ctDNA fractions are highly variable, which challenges downstream applications. Therefore, established preanalytical work flows in combination with cost-efficient and reproducible reference materials for ccfDNA analyses are crucial for analytical validity and subsequently for clinical decision-making. METHODS: We describe the efforts of the Innovative Medicines Initiative consortium CANCER-ID (http://www.cancer-id.eu) for comparing different technologies for ccfDNA purification, quantification, and characterization in a multicenter setting. To this end, in-house generated mononucleosomal DNA (mnDNA) from lung cancer cell lines carrying known TP53 mutations was spiked in pools of plasma from healthy donors generated from 2 different blood collection tubes (BCTs). ccfDNA extraction was performed at 15 partner sites according to their respective routine practice. Downstream analysis of ccfDNA with respect to recovery, integrity, and mutation analysis was performed centralized at 4 different sites. RESULTS: We demonstrate suitability of mnDNA as a surrogate for ccfDNA as a process quality control from nucleic acid extraction to mutation detection. Although automated extraction protocols and quantitative PCR-based quantification methods yielded the most consistent and precise results, some kits preferentially recovered spiked mnDNA over endogenous ccfDNA. Mutated TP53 fragments derived from mnDNA were consistently detected using both next-generation sequencing-based deep sequencing and droplet digital PCR independently of BCT. CONCLUSIONS: This comprehensive multicenter comparison of ccfDNA preanalytical and analytical work flows is an important contribution to establishing evidence-based guidelines for clinically feasible (pre)analytical work flows.

The role of nitric oxide during embryonic wound healing.

BACKGROUND: The study of the mechanisms controlling wound healing is an attractive area within the field of biology, with it having a potentially significant impact on the health sector given the current medical burden associated with healing in the elderly population. Healing is a complex process and includes many steps that are regulated by coding and noncoding RNAs, proteins and other molecules. Nitric oxide (NO) is one of these small molecule regulators and its function has already been associated with inflammation and angiogenesis during adult healing. RESULTS: Our results showed that NO is also an essential component during embryonic scarless healing and acts via a previously unknown mechanism. NO is mainly produced during the early phase of healing and it is crucial for the expression of genes associated with healing. However, we also observed a late phase of healing, which occurs for several hours after wound closure and takes place under the epidermis and includes tissue remodelling that is dependent on NO. We also found that the NO is associated with multiple cellular metabolic pathways, in particularly the glucose metabolism pathway. This is particular noteworthy as the use of NO donors have already been found to be beneficial for the treatment of chronic healing defects (including those associated with diabetes) and it is possible that its mechanism of action follows those observed during embryonic wound healing. CONCLUSIONS: Our study describes a new role of NO during healing, which may potentially translate to improved therapeutic treatments, especially for individual suffering with problematic healing.

Multicenter Evaluation of Circulating Plasma MicroRNA Extraction Technologies for the Development of Clinically Feasible Reverse Transcription Quantitative PCR and Next-Generation Sequencing Analytical Work Flows.

BACKGROUND: In human body fluids, microRNA (miRNA) can be found as circulating cell-free miRNA (cfmiRNA), as well as secreted into extracellular vesicles (EVmiRNA). miRNAs are being intensively evaluated as minimally invasive liquid biopsy biomarkers in patients with cancer. The growing interest in developing clinical assays for circulating miRNA necessitates careful consideration of confounding effects of preanalytical and analytical parameters. METHODS: By using reverse transcription quantitative real-time PCR and next-generation sequencing (NGS), we compared extraction efficiencies of 5 different protocols for cfmiRNA and 2 protocols for EVmiRNA isolation in a multicentric manner. The efficiency of the different extraction methods was evaluated by measuring exogenously spiked cel-miR-39 and 6 targeted miRNAs in plasma from 20 healthy individuals. RESULTS: There were significant differences between the tested methods. Although column-based extraction methods were highly effective for the isolation of endogenous miRNA, phenol extraction combined with column-based miRNA purification and ultracentrifugation resulted in lower quality and quantity of isolated miRNA. Among all extraction methods, the ubiquitously expressed miR-16 was represented with high abundance when compared with other targeted miRNAs. In addition, the use of miR-16 as an endogenous control for normalization of quantification cycle values resulted in a decreased variability of column-based cfmiRNA extraction methods. Cluster analysis of normalized NGS counts clearly indicated a method-dependent bias. CONCLUSIONS: The choice of plasma miRNA extraction methods affects the selection of potential miRNA marker candidates and mechanistic interpretation of results, which should be done with caution, particularly across studies using different protocols.

Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification.

MicroRNAs are a class of small non-coding RNAs that serve as important regulators of gene expression at the posttranscriptional level. They are stable in body fluids and pose great potential to serve as biomarkers. Here, we present a highly specific, sensitive and cost-effective system to quantify miRNA expression based on two-step RT-qPCR with SYBR-green detection chemistry called Two-tailed RT-qPCR. It takes advantage of novel, target-specific primers for reverse transcription composed of two hemiprobes complementary to two different parts of the targeted miRNA, connected by a hairpin structure. The introduction of a second probe ensures high sensitivity and enables discrimination of highly homologous miRNAs irrespectively of the position of the mismatched nucleotide. Two-tailed RT-qPCR has a dynamic range of seven logs and a sensitivity sufficient to detect down to ten target miRNA molecules. It is capable to capture the full isomiR repertoire, leading to accurate representation of the complete miRNA content in a sample. The reverse transcription step can be multiplexed and the miRNA profiles measured with Two-tailed RT-qPCR show excellent correlation with the industry standard TaqMan miRNA assays (r2 = 0.985). Moreover, Two-tailed RT-qPCR allows for rapid testing with a total analysis time of less than 2.5 hours.

A single-cell analysis reveals multiple roles of oligodendroglial lineage cells during post-ischemic regeneration.

NG2 cells represent precursors of oligodendrocytes under physiological conditions; however, following cerebral ischemia they play an important role in glial scar formation. Here, we compared the expression profiles of oligodendroglial lineage cells, after focal cerebral ischemia (FCI) and in Alzheimer's-like pathology using transgenic mice, which enables genetic fate-mapping of Cspg4-positive NG2 cells and their progeny, based on the expression of red fluorescent protein tdTomato. tdTomato-positive cells possessed the expression profile of NG2 cells and oligodendrocytes; however, based on the expression of cell type-specific genes, we were able to distinguish between them. To shed light on the changes in the expression patterns caused by FCI, we employed self-organizing Kohonen maps, enabling the division of NG2 cells and oligodendrocytes into subpopulations based on similarities in the expression profiles of individual cells. We identified three subpopulations of NG2 cells emerging after FCI: proliferative; astrocyte-like and oligodendrocyte-like NG2 cells; such phenotypes were further confirmed by immunohistochemistry. Oligodendrocytes themselves formed four subpopulations, which reflected the process of oligodendrocytes maturation. Finally, we used 5-ethynyl-2' deoxyuridine (EdU) labeling to reveal that NG2 cells can differentiate directly into reactive astrocytes without preceding proliferation. In contrast, in Alzheimer's-like pathology we failed to identify these subpopulations. Collectively, here we identified several yet unknown differences between the expression profiles of NG2 cells and oligodendrocytes, and characterized specific genes contributing to oligodendrocyte maturation and phenotypical changes of NG2 cells after FCI. Moreover, our results suggest that, unlike in Alzheimer's-like pathology, NG2 cells acquire a multipotent phenotype following FCI.

Human oocyte maturation in vitro is improved by co-culture with cumulus cells from mature oocytes.

The conventional method of human oocyte maturation in vitro in the presence of gonadotrophins continues to be a relatively low-success procedure in the assisted conception programme owing to suboptimal maturation conditions in the absence of an ovarian 'niche' and poor understanding of this procedure at the molecular level in oocytes. In this study, the gene expression profiles of human oocytes were analysed according to their manner of maturation: in vivo (in the ovaries) or in vitro (matured either by the conventional method or by a new approach - co-cultured with cumulus cells of mature oocytes from the same patient). Our results show that the in-vitro maturation procedure strongly affects the gene expression profile of human oocytes, including several genes involved in transcriptional regulation, embryogenesis, epigenetics, development, and the cell cycle. The in-vitro maturation of oocytes co-cultured with cumulus cells from mature oocytes provides an ovarian 'niche' to some degree, which improves oocyte maturation rates and their gene expression profile to the extent that they are more comparable to oocytes that naturally mature in the ovarian follicle.

Platforms for Single-Cell Collection and Analysis.

Single-cell analysis has become an established method to study cell heterogeneity and for rare cell characterization. Despite the high cost and technical constraints, applications are increasing every year in all fields of biology. Following the trend, there is a tremendous development of tools for single-cell analysis, especially in the RNA sequencing field. Every improvement increases sensitivity and throughput. Collecting a large amount of data also stimulates the development of new approaches for bioinformatic analysis and interpretation. However, the essential requirement for any analysis is the collection of single cells of high quality. The single-cell isolation must be fast, effective, and gentle to maintain the native expression profiles. Classical methods for single-cell isolation are micromanipulation, microdissection, and fluorescence-activated cell sorting (FACS). In the last decade several new and highly efficient approaches have been developed, which not just supplement but may fully replace the traditional ones. These new techniques are based on microfluidic chips, droplets, micro-well plates, and automatic collection of cells using capillaries, magnets, an electric field, or a punching probe. In this review we summarize the current methods and developments in this field. We discuss the advantages of the different commercially available platforms and their applicability, and also provide remarks on future developments.

Gene Expression Signatures in Circulating Tumor Cells Correlate with Response to Therapy in Metastatic Breast Cancer.

BACKGROUND: Circulating tumor cells (CTCs) are thought to be an ideal surrogate marker to monitor disease progression in metastatic breast cancer (MBC). We investigated the prediction of treatment response in CTCs of MBC patients on the basis of the expression of 46 genes. METHODS: From 45 MBC patients and 20 healthy donors (HD), 2 × 5 mL of blood was collected at the time of disease progression (TP0) and at 2 consecutive clinical staging time points (TP1 and TP2) to proceed with the AdnaTest EMT-2/StemCellSelectTM (QIAGEN). Patients were grouped into (a) responder (R) and non-responder (NR) at TP1 and (b) overall responder (OR) and overall non-responder (ONR) at TP2. A 46-gene PCR assay was used for preamplification and high-throughput gene expression profiling. Data were analyzed by use of GenEx (MultiD) and SAS. RESULTS: The CTC positivity was defined by the four-gene signature (EPCAM, KRT19, MUC1, ERBB2 positivity). Fourteen genes were identified as significantly differentially expressed between CTC+ and CTC- patients (KRT19, FLT1, EGFR, EPCAM, GZMM, PGR, CD24, KIT, PLAU, ALDH1A1, CTSD, MKI67, TWIST1, and ERBB2). KRT19 was highly expressed in CTC+ patients and ADAM17 in the NR at TP1. A significant differential expression of 4 genes (KRT19, EPCAM, CDH1, and SCGB2A2) was observed between OR and ONR when stratifying the samples into CTC+ or CTC-. CONCLUSIONS: ADAM17 could be a key marker in distinguishing R from NR, and KRT19 was powerful in identifying CTCs.

Generation of reactive astrocytes from NG2 cells is regulated by sonic hedgehog.

NG2 cells, a fourth glial cell type in the adult mammalian central nervous system, produce oligodendrocytes in the healthy nervous tissue, and display wide differentiation potential under pathological conditions, where they could give rise to reactive astrocytes. The factors that control the differentiation of NG2 cells after focal cerebral ischemia (FCI) are largely unknown. Here, we used transgenic Cspg4-cre/Esr1/ROSA26Sortm14(CAG-tdTomato) mice, in which tamoxifen administration triggers the expression of red fluorescent protein (tomato) specifically in NG2 cells and cells derived therefrom. Differentiation potential (in vitro and in vivo) of tomato-positive NG2 cells from control or postischemic brains was determined using the immunohistochemistry, single cell RT-qPCR and patch-clamp method. The ischemic injury was induced by middle cerebral artery occlusion, a model of FCI. Using genetic fate-mapping method, we identified sonic hedgehog (Shh) as an important factor that influences differentiation of NG2 cells into astrocytes in vitro. We also manipulated Shh signaling in the adult mouse brain after FCI. Shh signaling activation significantly increased the number of astrocytes derived from NG2 cells in the glial scar around the ischemic lesion, while Shh signaling inhibition caused the opposite effect. Since Shh signaling modifications did not change the proliferation rate of NG2 cells, we can conclude that Shh has a direct influence on the differentiation of NG2 cells and therefore, on the formation and composition of a glial scar, which consequently affects the degree of the brain damage. GLIA 2016;64:1518-1531.

The need for transparency and good practices in the qPCR literature.

Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.

Accession of Tumor Heterogeneity by Multiplex Transcriptome Profiling of Single Circulating Tumor Cells.

BACKGROUND: Transcriptome analysis of circulating tumor cells (CTCs) holds great promise to unravel the biology of cancer cell dissemination and identify expressed genes and signaling pathways relevant to therapeutic interventions. METHODS: CTCs were enriched based on their EpCAM expression (CellSearch®) or by size and deformability (ParsortixTM), identified by EpCAM and/or pan-keratin-specific antibodies, and isolated for single cell multiplex RNA profiling. RESULTS: Distinct breast and prostate CTC expression signatures could be discriminated from RNA profiles of leukocytes. Some CTCs positive for epithelial transcripts (EpCAM and KRT19) also coexpressed leukocyte/mesenchymal associated markers (PTPRC and VIM). Additional subsets of CTCs within individual patients were characterized by divergent expression of genes involved in epithelial-mesenchymal transition (e.g., CDH2, MMPs, VIM, or ZEB1 and 2), DNA repair (RAD51), resistance to cancer therapy (e.g., AR, AR-V7, ERBB2, EGFR), cancer stemness (e.g., CD24 and CD44), activated signaling pathways involved in tumor progression (e.g., PIK3CA and MTOR) or cross talks between tumors and immune cells (e.g., CCL4, CXCL2, CXCL9, IL15, IL1B, or IL8). CONCLUSIONS: Multimarker RNA profiling of single CTCs reveals distinct CTC subsets and provides important insights into gene regulatory networks relevant for cancer progression and therapy.

Post-treatment recovery of suboptimal DNA repair capacity and gene expression levels in colorectal cancer patients.

DNA repair in blood cells was observed to be suboptimal in cancer patients at diagnosis, including colorectal cancer (CRC). To explore the causality of this phenomenon, we studied the dynamics of DNA repair from diagnosis to 1 yr follow-up, and with respect to CRC treatment. Systemic CRC therapy is targeted to DNA damage induction and DNA repair is thus of interest. CRC patients were blood-sampled three times in 6-mo intervals, starting at the diagnosis, and compared to healthy controls. DNA repair was characterized by mRNA levels of 40 repair genes, by capacity of nucleotide excision repair (NER), and by levels of DNA strand breaks (SBs). NER and base excision repair genes were significantly under-expressed (P < 0.016) in patients at diagnosis compared to controls, in accordance with reduced NER function (P = 0.008) and increased SBs (P = 0.015). Six months later, there was an increase of NER capacity, but not of gene expression levels, in treated patients only. A year from diagnosis, gene expression profiles and NER capacity were significantly modified in all patients and were no longer different from those measured in controls. All patients were free of relapse at the last sampling, so we were unable to clarify the impact of DNA repair parameters on treatment response. However, we identified a panel of blood DNA repair-related markers discerning acute stage of the disease from the remission period. In conclusion, our results support a model in which DNA repair is altered as a result of cancer.

Quantitative Analysis of Glutamate Receptors in Glial Cells from the Cortex of GFAP/EGFP Mice Following Ischemic Injury: Focus on NMDA Receptors.

Cortical glial cells contain both ionotropic and metabotropic glutamate receptors. Despite several efforts, a comprehensive analysis of the entire family of glutamate receptors and their subunits present in glial cells is still missing. Here, we provide an overall picture of the gene expression of ionotropic (AMPA, kainate, NMDA) and the main metabotropic glutamate receptors in cortical glial cells isolated from GFAP/EGFP mice before and after focal cerebral ischemia. Employing single-cell RT-qPCR, we detected the expression of genes encoding subunits of glutamate receptors in GFAP/EGFP-positive (GFAP/EGFP(+)) glial cells in the cortex of young adult mice. Most of the analyzed cells expressed mRNA for glutamate receptor subunits, the expression of which, in most cases, even increased after ischemic injury. Data analyses disclosed several classes of GFAP/EGFP(+) glial cells with respect to glutamate receptors and revealed in what manner their expression correlates with the expression of glial markers prior to and after ischemia. Furthermore, we also examined the protein expression and functional significance of NMDA receptors in glial cells. Immunohistochemical analyses of all seven NMDA receptor subunits provided direct evidence that the GluN3A subunit is present in GFAP/EGFP(+) glial cells and that its expression is increased after ischemia. In situ and in vitro Ca(2+) imaging revealed that Ca(2+) elevations evoked by the application of NMDA were diminished in GFAP/EGFP(+) glial cells following ischemia. Our results provide a comprehensive description of glutamate receptors in cortical GFAP/EGFP(+) glial cells and may serve as a basis for further research on glial cell physiology and pathophysiology.

Increased expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in reactive astrocytes following ischemia.

Astrocytes respond to ischemic brain injury by proliferation, the increased expression of intermediate filaments and hypertrophy, which results in glial scar formation. In addition, they alter the expression of ion channels, receptors and transporters that maintain ionic/neurotransmitter homeostasis. Here, we aimed to demonstrate the expression of Hcn1-4 genes encoding hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in reactive astrocytes following focal cerebral ischemia (FCI) or global cerebral ischemia (GCI) and to characterize their functional properties. A permanent occlusion of the middle cerebral artery (MCAo) was employed to induce FCI in adult GFAP/EGFP mice, while GCI was induced by transient bilateral common carotid artery occlusion combined with hypoxia in adult rats. Using FACS, we isolated astrocytes from non-injured or ischemic brains and performed gene expression profiling using single-cell RT-qPCR. We showed that 2 weeks after ischemia reactive astrocytes express high levels of Hcn1-4 transcripts, while immunohistochemical analyses confirmed the presence of HCN1-3 channels in reactive astrocytes 5 weeks after ischemia. Electrophysiological recordings revealed that post-ischemic astrocytes are significantly depolarized, and compared with astrocytes from non-injured brains, they display large hyperpolarization-activated inward currents, the density of which increased 2-3-fold in response to ischemia. Their activation was facilitated by cAMP and their amplitudes were decreased by ZD7288 or low extracellular Na(+) concentration, suggesting that they may belong to the family of HCN channels. Collectively, our results demonstrate that regardless of the type of ischemic injury, reactive astrocytes express HCN channels, which could therefore be an important therapeutic target in poststroke therapy.

RT-qPCR work-flow for single-cell data analysis.

Individual cells represent the basic unit in tissues and organisms and are in many aspects unique in their properties. The introduction of new and sensitive techniques to study single-cells opens up new avenues to understand fundamental biological processes. Well established statistical tools and recommendations exist for gene expression data based on traditional cell population measurements. However, these workflows are not suitable, and some steps are even inappropriate, to apply on single-cell data. Here, we present a simple and practical workflow for preprocessing of single-cell data generated by reverse transcription quantitative real-time PCR. The approach is demonstrated on a data set based on profiling of 41 genes in 303 single-cells. For some pre-processing steps we present options and also recommendations. In particular, we demonstrate and discuss different strategies for handling missing data and scaling data for downstream multivariate analysis. The aim of this workflow is provide guide to the rapidly growing community studying single-cells by means of reverse transcription quantitative real-time PCR profiling.

Validation of kinetics similarity in qPCR.

Quantitative real-time PCR (qPCR) is the method of choice for specific and sensitive quantification of nucleic acids. However, data validation is still a major issue, partially due to the complex effect of PCR inhibition on the results. If undetected PCR inhibition may severely impair the accuracy and sensitivity of results. PCR inhibition is addressed by prevention, detection and correction of PCR results. Recently, a new family of computational methods for the detection of PCR inhibition called kinetics outlier detection (KOD) emerged. KOD methods are based on comparison of one or a few kinetic parameters describing a test reaction to those describing a set of reference reactions. Modern KOD can detect PCR inhibition reflected by shift of the amplification curve by merely half a cycle with specificity and sensitivity >90%. Based solely on data analysis, these tools complement measures to improve and control pre-analytics. KOD methods do not require labor and materials, do not affect the reaction accuracy and sensitivity and they can be automated for fast and reliable quantification. This review describes the background of KOD methods, their principles, assumptions, strengths and limitations. Finally, the review provides recommendations how to use KOD and how to evaluate its performance.

Correction of RT-qPCR data for genomic DNA-derived signals with ValidPrime.

Genomic DNA (gDNA) contamination is an inherent problem during RNA purification that can lead to non-specific amplification and aberrant results in reverse transcription quantitative PCR (RT-qPCR). Currently, there is no alternative to RT(-) controls to evaluate the impact of the gDNA background on RT-PCR data. We propose a novel method (ValidPrime) that is more accurate than traditional RT(-) controls to test qPCR assays with respect to their sensitivity toward gDNA. ValidPrime measures the gDNA contribution using an optimized gDNA-specific ValidPrime assay (VPA) and gDNA reference sample(s). The VPA, targeting a non-transcribed locus, is used to measure the gDNA contents in RT(+) samples and the gDNA reference is used to normalize for GOI-specific differences in gDNA sensitivity. We demonstrate that the RNA-derived component of the signal can be accurately estimated and deduced from the total signal. ValidPrime corrects with high precision for both exogenous (spiked) and endogenous gDNA, contributing ∼60% of the total signal, whereas substantially reducing the number of required qPCR control reactions. In conclusion, ValidPrime offers a cost-efficient alternative to RT(-) controls and accurately corrects for signals derived from gDNA in RT-qPCR.