Cellular responses induced by surgical procedure or ischemia-reperfusion injury (IRI) may severely alter transcriptome profiles and complicate molecular diagnostics. To investigate this effect, we characterized such pre-analytical effects in 143 non-malignant liver samples obtained from 30 patients at different time points of ischemia during surgery from two individual cohorts treated either with the Pringle manoeuvre or total vascular exclusion. Transcriptomics profiles were analyzed by Affymetrix microarrays and expression of selected mRNAs was validated by RT-PCR. We found 179 mutually deregulated genes which point to elevated cytokine signaling with NFκB as a dominant pathway in ischemia responses. In contrast to ischemia, reperfusion induced pro-apoptotic and pro-inflammatory cascades involving TNF, NFκB and MAPK pathways. FOS and JUN were down-regulated in steatosis compared to their up-regulation in normal livers. Surprisingly, molecular signatures of underlying primary and secondary cancers were present in non-tumor tissue. The reported inter-patient variability might reflect differences in individual stress responses and impact of underlying disease conditions. Furthermore, we provide a set of 230 pre-analytically highly robust genes identified from histologically normal livers (<2% covariation across both cohorts) that might serve as reference genes and could be particularly suited for future diagnostic applications.

Molecular biomarkers, like gene transcripts or enzyme activities, are potentially powerful tools for early warning assessment of pollution. However, a thorough understanding of response and baseline variation is required to distinguish actual effects from pollution. Here, we assess the freshwater mussel Anodonta anatina as a biomarker model species for freshwater ecosystems, by testing responses of six transcriptional (cat, gst, hsp70, hsp90, mt, and sod) and two biochemical (AChE and GST) biomarkers to environmentally relevant Cu water concentrations. Mussels (n = 20), collected from a stream free from point source pollution, were exposed in the laboratory, for 96 h, to Cu treatments (< 0.2 μg/L, 0.77 ± 0.87 μg/L, and 6.3 ± 5.4 μg/L). Gills and digestive glands were extracted and analyzed for transcriptional and biochemical responses. Biological and statistical effect sizes from Cu treatments were in general small (mean log2 fold-change ≤ 0.80 and Cohen’s f ≤ 0.69, respectively), and no significant treatment effects were observed. In contrast, four out of eight biomarkers (cat, gst, hsp70, and GST) showed a significant sex:tissue interaction, and additionally one (sod) showed significant overall effects from sex. Specifically, three markers in gills (cat, mt, GST) and one in digestive gland (AChE) displayed significant sex differences, independent of treatment. Results suggest that sex or tissue effects might obscure low-magnitude biomarker responses and potential early warnings. Thus, variation in biomarker baselines and response patterns needs to be further addressed for the future use of A. anatina as a biomarker model species.

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.

Interaction between organ-specific stem cells and extracellular matrix (ECM) is crucial for regeneration. We therefore, investigated novel stem cells markers in human kidney and verified the potential of human fetal kidney cells (hFKC) to repopulate decellularized porcine kidneys. Adult and fetal human kidneys were stained by immunohistochemistry for putative stem cell markers. In addition, hFKC were isolated and characterized phenotypically and by gene expression. Furthermore, whole porcine kidneys were decellularized using detergents, cut into 1 mm slices, seeded with hFKC, cultured for 14 days and characterized by histology and qPCR. We found that, decellularized porcine kidneys showed significant loss of DNA but preserved some ECM components. Human fetal kidneys including hFKC expressed stem cell markers CD133, DLK-1, EPCAM and ephrin receptor EphA6. Interestingly, EphA7 and SIX2 were markedly expressed only in fetus. Furthermore, in fetal kidneys EphA7 was co-expressed with DLK-1. Recellularized kidney pieces showed cell infiltration, growing in orchestrated fashion distributed around the scaffold. These pieces also demonstrated cells expressing CK8, CK18, DLK-1, CD133, EphA7, EphB3, PCNA, podocin and increased levels of transcriptional factors in kidney development (SIX2, EYA1, CITED1, LHX1, SALL1, DLK-1 and WT1). We conclude that decellularized porcine kidneys support the culture, proliferation of hFKC and regenerate by upregulation of transcription factors. We suggest that expanded hFKC may be the ideal cell source for whole kidney regeneration in the future. We also postulate EphA7 might be a novel stem cell marker in kidneys.

Background: Current diagnosis and staging of advanced epithelial ovarian cancer (aEOC) has important limitations and better biomarkers are needed. We investigate the performance of non-haematopoietic circulating cells (CCs) at the time of disease presentation and relapse. Methods: Venous blood was collected prospectively from 37 aEOC patients and 39 volunteers. CCs were evaluated using ImageStream Technology™ and specific antibodies to differentiate epithelial cells from haematopoetic cells. qRT-PCR from whole blood of relapsed aEOC patients was carried out for biomarker discovery. Results: Significant numbers of CCs (CK+/WT1+/CD45−) were identified, quantified and characterised from aEOC patients compared to volunteers. CCs are abundant in women with newly diagnosed aEOC, prior to any treatment. Evaluation of RNA from the CCs in relapsed aEOC patients (n = 5) against a 79-gene panel revealed several differentially expressed genes compared to volunteers (n = 14). Size differentiation of CCs versus CD45+ haematopoietic cells was not reliable. Conclusion: CCs of non-haematopoetic origin are prevalent, particularly in patients with newly diagnosed aEOC. Exploiting a CC-rich population in aEOC patients offers insights into a part of the circulating microenvironment.

In May 2017, the European In Vitro Diagnostic Regulation (IVDR) entered into force and will apply to in vitro diagnostics from May 26th, 2022. This will have a major impact on the in vitro diagnostics (IVD) industry as all devices falling under the scope of the IVDR will require new or re-certification. It will also affect health institutions developing and using in-house devices. The IVDR also has implications with respect to product performance validation and verification including the pre-analytics of biological samples used by IVD developers and diagnostic service providers. In parallel to the IVDR, a series of standards on pre-analytical sample processing has been published by the International Organization for Standardization (ISO) and the European Committee for Standardization (CEN). These standards describe pre-analytical requirements for various types of analyses in various types of biospecimens. They are of relevance for IVD product developers in the context of (re)certification under the IVDR and to some extent also to devices manufactured and used only within health institutions. This review highlights the background and the rational for the pre-analytical standards. It describes the procedure that leads to these standards, the major implications of the standards and the requirements on pre-analytical workflows. In addition, it discusses the relationship between the standards and the IVDR.

Abstract MicroRNAs (miRNAs) are small noncoding RNAs that interfere with and regulate different biological pathways. Therefore, dysregulation of miRNAs could lead to disease initiation and progression, particularly in various types of cancer. In human body fluids, miRNAs can be found as circulating cell-free miRNAs (cfmiRNAs, often associated with RNA-binding proteins) as well as exosomal miRNAs (exomiRNAs), both serving as potentially valuable biomarkers to evaluate patient's disease progression, or to monitor treatment efficacy. Various methods for the extraction and analysis of miRNAs, cfmiRNAs or exomiRNAs, are commercially available. Here, we describe the efforts of the Innovative Medicines Initiative (IMI) consortium CANCER-ID (www.cancer-id.eu), a public-private partnership of experts from academia, biotech and pharmaceutical industry, in generating comparative data using different RNA/miRNA extraction technologies in a multicenter ring study. To address clinical relevance in a later stage of the project, K2EDTA plasma samples from 20 healthy donors were collected in a centralized way with respective informed consent. Five different RNA/miRNA extraction methods and two extraction methods specific for exomiRNAs were selected for comparison: mirVana (ThermoFisher Scientific), miRCURY RNA Isolation Kit (Exiqon), miRNeasy Serum/Plasma kit (QIAGEN), miRNeasy Advanced Serum/Plasma kit (QIAGEN), Plasma/Serum RNA Purification mini kit (Norgen), exoRNeasy Serum/Plasma (QIAGEN) and ultracentrifugation. The eluates were sent to one CANCER-ID partner, centrally performing RT-qPCR analysis on six miRNAs, as well as miRNA massive parallel sequencing. After extraction using the miRCURY RNA isolation kit and miRNeasy Advanced Serum/Plasma kit, highest miRNA-seq calls (mean: 2x10^6) and lowest RT-qPCR Cq-values (mean: 26.6) for six miRNAs were observed with both extraction methods. Samples from patients and healthy volunteers, respectively, were collected under signed informed consent. Based on the results of the evaluation of the different miRNA extraction technologies a single extraction technology will be selected for the next iteration of ring studies, the comparison of different analysis platforms and the integration of downstream workflows. First results of these ring studies will be presented. This work is supported by IMI JU & EFPIA (grand no. 115749). Citation Format: Martin H. Neumann, Francesca Di Pasquale, Markus Sprenger-Haussels, Jonathan M. Shaffer, Martin Schlumpberger, Fay Betsou, Wim Ammerlaan, Taija af Hallström, Evi Lianidou, Robert Sjöback, Sebastian Bender, Thomas Schlange. Multicenter evaluation of miRNA extraction technologies for the development of a clinically relevant miRNA analysis workflow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1593.

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.

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.

Quantitative Real-Time Polymerase Chain Reaction, better known as qPCR, is the most sensitive and specific technique we have for the detection of nucleic acids. Even though it has been around for more than 30 years and is preferred in research applications, it has yet to win broad acceptance in routine practice. This requires a means to unambiguously assess the performance of specific qPCR analyses. Here we present methods to determine the limit of detection (LoD) and the limit of quantification (LoQ) as applicable to qPCR. These are based on standard statistical methods as recommended by regulatory bodies adapted to qPCR and complemented with a novel approach to estimate the precision of LoD.

Here we report the fabrication of a novel composite gel from decellularized gal-gal-knockout porcine skin and human peripheral blood mononuclear cells (hPBMCs) for full-thickness skin wound healing. Decellularized skin extracellular matrix (ECM) powder was prepared via chemical treatment, freeze drying, and homogenization. The powder was mixed with culture medium containing hyaluronic acid to generate a pig skin gel (PSG). The effect of the gel in regeneration of full-thickness wounds was studied in nude mice. We found significantly accelerated wound closure already on day 15 in animals treated with PSG only or PSG + hPBMCs compared to untreated and hyaluronic acid-treated controls (p < 0.05). Addition of the hPBMCs to the gel resulted in marked increase of host blood vessels as well as the presence of human blood vessels. At day 25, histologically, the wounds in animals treated with PSG only or PSG + hPBMCs were completely closed compared to those of controls. Thus, the gel facilitated generation of new skin with well-arranged epidermal cells and restored bilayer structure of the epidermis and dermis. These results suggest that porcine skin ECM gel together with human cells may be a novel and promising biomaterial for medical applications especially for patients with acute and chronic skin wounds.

Analysis of multiple analytes from biological samples can be challenging as different analytes require different preservation measures. Heat induced enzymatic inactivation is an efficient way to preserve proteins and their modifications in biological samples but RNA quality, as measured by RIN value, has been a concern in such samples. Here, we investigate the effect of heat stabilization compared with standard snap freezing on RNA quality using two RNA extraction protocols, QiaZol with and without urea pre-solubilization, and two RNA quality measurements: RIN value, as defined by the Agilent Bioanalyzer, and an alternative qPCR based method. DNA extraction from heat stabilized brain samples was also examined. The snap frozen samples had RIN values about 1 unit higher than heat stabilized samples for the direct QiaZol extraction but equal with stabilized samples using urea pre-solubilization. qPCR based RNA quality measurement showed no difference in quality between snap frozen and heat inactivated samples. The probable explanation for this discrepancy is that the RIN value is an indirect measure based on rRNA, while the qPCR score is based on actual measurement of mRNA quality. The DNA yield from heat stabilized brain tissue samples was significantly increased, compared to the snap frozen tissue, without any effects on purity or quality. Hence, heat stabilization of tissues opens up the possibility for a two step preservation protocol, where proteins and their modifications can be preserved in the first heat based step, while in a second step, using standard RNA preservation strategies, mRNA be preserved. This collection strategy will enable biobanking of samples where the ultimate analysis is not determined without loss of sample quality.

Abstract Background: Circulating tumor cells (CTC) are discussed to be an ideal surrogate marker to monitor disease progression in metastatic breast cancer (MBC). Besides CTC characterization for targeted therapies, it would also be desirable to know where these cells derive from or to which organ site they are going to. Here we investigated whether it is possible to predict the origin of metastatic lesion based on the expression of 46 genes in CTC of MBC patients (pts). Materials and Methods: 2×5 ml blood of 45 MBC pts and 20 healthy controls was collected at the time of disease progression (T0) and at two consecutive clinical staging (T1 and T2) for the detection of CTC applying immunomagnetic enrichment using the AdnaTest EMT-2/Stem Cell Select (QIAGEN Hannover GmbH, Germany). Pts were grouped a) into overall responders (OR) and overall non-responders (ONR), thus responding or not responding at T1 and T2 and b) according to sites of metastasis. PCR assays, targeting 46 transcripts and reference markers were used for a workflow based on pre-amplification and high throughput profiling (each samples in duplicates) with the full set of markers including also ValidPrime to correct for genomic background and InterPlate Calibrator to even out variations between runs. Data were analyzed using GenEx (MultiD, Sweden) and SAS. qPCR as well as technical reads were normalized using several normalization strategies. Results: The multidrug resistant protein gene MRP1 was significantly differently expressed if OR and ONR groups were compared. In the following order of significance, VEGFR1, Keratin (KRT) 19, EGFR, MET1, ALDH, progesterone receptor (PR), UPA, Cathepsin D, KIT1 and Ki67 were differentially expressed in CTC of pts who had already developed liver metastasis as compared to pts without liver metastasis. Interestingly, a small group of pts, developing liver metastases in the course of disease, showed the estrogen receptor (ER), PR, HER2, mammaglobin, KRT19 on a significantly lower level as compared to the other pts. Similarly, once CTC were ER and PR positive, the probability of bone metastasis development decreased. Conclusion: Our preliminary results indicate that the development of metastatic lesions is associated with site-specific CTC. Thus, besides using CTC as a monitoring tool to guide therapy, they might also indicate the site of metastasis which will allow a more precise decision concerning treatment strategy. Citation Format: Maren Bredemeier, Philippos Edimiris, Pawel Mach, Mikael Kubista, Robert Sjoback, Marie Jindrichova, Eva Rohlova, Vendula Novosadova, Katarina Kolostova, Siegfried Hauch, Bahriye Aktas, Mitra Tewes, Rainer Kimmig, Sabine Kasimir-Bauer. Gene expression signatures in circulating tumor cells are prognostic for metastatic lesions in breast cancer patients and correlate with response to therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 502.

Abstract Background: Circulating tumor cells (CTCs) are discussed to be an ideal surrogate marker to monitor disease progression in metastatic breast cancer (MBC) since response to therapy can only be assessed retrospectively after a therapy strategy has already failed. Here we established a new profiling method to characterize the heterogeneous CTC population and investigated if it is possible to predict treatment response based on expression of 46 genes in CTCs of MBC patients. Materials and Methods: 2×5 ml blood of 45 MBC patients was collected at the time of disease progression (T0) and at two consecutive clinical staging (T1 and T2) after 8-12 weeks of chemo-, hormone or antibody therapy for the detection of CTCs applying positive immunomagnetic selection targeting EpCAM, EGFR and HER2 using the AdnaTest EMT-2/Stem Cell Select (AdnaGen GmbH, Germany). Patients were classified into responders and non-responders at the time of clinical staging according to RECIST criteria. PCR assays targeting 46 selected transcript comprising breast cancer, stem cell, EMT, and references markers were designed and extensively optimized for a workflow based on pre-amplification and high throughput profiling. Each sample was profiled in duplicates with the full set of markers including also ValidPrime to correct for genomic background and InterPlate Calibrator to even out variations between runs. The entire workflow was validated to establish excellent technical reproducibility from sample collection through extraction, pre-amplification and analysis. Measured data were analyzed using parametric as well as non-parametric statistics with GenEx and SAS. qPCR as well as technical reads were normalized using the average expression of the reference genes B2M and ActB. Results: A number of genes, including ADAM17, CD24L4, EPCAM, KRT19, MTOR, HER2, TOP2A, and CD44 were differentially expressed in MBC patients (n = 45) as compared to healthy controls (n = 20). A group of non-responders could be identified based on gene expression. Interestingly, expression of ADAM17 (tumor necrosis factor-α-converting enzyme) differed significantly when responders were compared with non-responders at T1 (p = 0.000567). Conclusion: It is possible to distinguish MBC patients from healthy controls based on the expression of the genes investigated. Preliminary results indicate that ADAM17 is a key marker, distinguishing responders from non-responders. For more detailed analysis, it is desirable to build up a larger patient cohort in order to correlate gene expression profiles of CTC enriched samples to a given therapy for individualized treatment. Citation Format: Maren Bredemeier, Mikael Kubista, Robert Sjöback, Marie Jendrichova, Eva Rohlova, Vednula Novosadova, Katarina Kolostova, Siegfried Hauch, Bahriye Aktas, Mitra Tewes, Rainer Kimmig, Sabine Kasimir-Bauer. Expression profiling of circulating tumor cells: A prognostic and predictive biomarker in metastatic breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 372. doi:10.1158/1538-7445.AM2015-372

Background: With the introduction of the first high-throughput qPCR instrument on the market it became possible to perform thousands of reactions in a single run compared to the previous hundreds. In the high-throughput reaction, only limited volumes of highly concentrated cDNA or DNA samples can be added. This necessity can be solved by pre-amplification, which became a part of the high-throughput experimental workflow. Here, we focused our attention on the limits of the specific target pre-amplification reaction and propose the optimal, general setup for gene expression experiment using BioMark instrument (Fluidigm).Results: For evaluating different pre-amplification factors following conditions were combined: four human blood samples from healthy donors and five transcripts having high to low expression levels; each cDNA sample was pre-amplified at four cycles (15, 18, 21, and 24) and five concentrations (equivalent to 0.078 ng, 0.32 ng, 1.25 ng, 5 ng, and 20 ng of total RNA). Factors identified as critical for a success of cDNA pre-amplification were cycle of pre-amplification, total RNA concentration, and type of gene. The selected pre-amplification reactions were further tested for optimal Cq distribution in a BioMark Array. The following concentrations combined with pre-amplification cycles were optimal for good quality samples: 20 ng of total RNA with 15 cycles of pre-amplification, 20x and 40x diluted; and 5 ng and 20 ng of total RNA with 18 cycles of pre-amplification, both 20x and 40x diluted.Conclusions: We set up upper limits for the bulk gene expression experiment using gene expression Dynamic Array and provided an easy-to-obtain tool for measuring of pre-amplification success. We also showed that variability of the pre-amplification, introduced into the experimental workflow of reverse transcription-qPCR, is lower than variability caused by the reverse transcription step.

Integrity of the mRNA in clinical samples has major impact on the quality of measured expression levels. This is independent of the measurement technique being next generation sequencing (NGS), Quantitative real-time PCR (qPCR) or microarray profiling. If mRNA is highly degraded or damaged, measured data will be very unreliable and the whole study is likely a waste of time and money. It is therefore common strategy to test the quality of RNA in samples before conducting large and costly studies. Most methods today to assess the quality of RNA are ignorant to the nature of the RNA and, therefore, reflect the integrity of ribosomal RNA, which is the dominant species, rather than of mRNAs, microRNAs and long non-coding RNAs, which usually are the species of interest. Here, we present a novel molecular approach to assess the quality of the targeted RNA species by measuring the differential amplification (ΔAmp) of an Endogenous RNase Resistant (ERR) marker relative to a reference gene, optionally combined with the measurement of two amplicons of different lengths. The combination reveals any mRNA degradation caused by ribonucleases as well as physical, chemical or UV damage. ΔAmp has superior sensitivity to common microfluidic electrophoretic methods, senses the integrity of the actual targeted RNA species, and allows for a smoother and more cost efficient workflow.

There is an increasing need for proper quality control tools in the pre-analytical phase of the molecular diagnostic workflow. The aim of the present study was to identify biomarkers for monitoring pre-analytical mRNA quality variations in two different types of blood collection tubes, K2EDTA (EDTA) tubes and PAXgene Blood RNA Tubes (PAXgene tubes). These tubes are extensively used both in the diagnostic setting as well as for research biobank samples. Blood specimens collected in the two different blood collection tubes were stored for varying times at different temperatures, and microarray analysis was performed on resultant extracted RNA. A large set of potential mRNA quality biomarkers for monitoring post-phlebotomy gene expression changes and mRNA degradation in blood was identified. qPCR assays for the potential biomarkers and a set of relevant reference genes were generated and used to pre-validate a sub-set of the selected biomarkers. The assay precision of the potential qPCR based biomarkers was determined, and a final validation of the selected quality biomarkers using the developed qPCR assays and blood samples from 60 healthy additional subjects was performed. In total, four mRNA quality biomarkers (USP32, LMNA, FOSB, TNRFSF10C) were successfully validated. We suggest here the use of these blood mRNA quality biomarkers for validating an experimental pre-analytical workflow. These biomarkers were further evaluated in the 2nd ring trial of the SPIDIA-RNA Program which demonstrated that these biomarkers can be used as quality control tools for mRNA analyses from blood samples.