Accurate Gene Expression Profiling Research Articles

Evaluation of Reference Gene Stability in Goat Skeletal Muscle Satellite Cells during Proliferation and Differentiation Phases.

The process of skeletal muscle development is intricate and involves the regulation of a diverse array of genes. Accurate gene expression profiles are crucial for studying muscle development, making it essential to choose the right reference genes for real-time quantitative PCR (RT-qPCR). In the present study, eight candidate reference genes were identified from our previous transcriptome sequencing analysis of caprine skeletal muscle satellite cells (MuSCs), and two traditional reference genes (ACTB and GAPDH) were assessed. The quantitative levels of the candidate reference genes were determined through the RT-qPCR technique, while the stability of their expression was evaluated utilizing the GeNorm, NormFinder, BestKeeper, and RefFinder programs. Furthermore, the chosen reference genes were utilized for the normalization of the gene expression levels of PCNA and Myf5. It was determined that conventional reference genes, including ACTB and GAPDH, were not appropriate for normalizing target gene expression. Conversely, RPL14 and RPS15A, identified through RNA sequencing analysis, exhibited minimal variability and were identified as the optimal reference genes for normalizing gene expression during the proliferation and differentiation of goat MuSCs. Our research offers a validated panel of optimal reference genes for the detection of differentially expressed genes in goat muscle satellite cells using RT-qPCR.

Comprehensive evaluation and validation of optimal reference genes for normalization of qPCR data in different caprine tissues.

Quantitative real-time PCR (qPCR) is a highly reliable method for validating gene expression data in molecular studies due to its sensitivity, specificity, and efficiency. To ensure accurate qPCR results, it's essential to normalize the expression data using stable reference genes. This study aimed to identify suitable reference genes for qPCR studies in goats by evaluating 18 candidate reference genes (ACTB, BACH1, B2M, GAPDH, HMBS, HPRT1, PGK1, PPIA, PPIB, RPLP0, RPL19, RPS9, RPS15, RPS28, SDHA, TBP, UXT, and YWHAZ) in 10 different caprine tissues (heart, intestine, kidney, liver, lung, muscle, rumen, skin, spleen, and testis). An integrated tool called RefFinder, which incorporates various algorithms like NormFinder, GeNorm, BestKeeper, and ΔCt, was used to assess the stability of expression among these genes. After thorough analysis, ACTB, PPIB, and B2M emerged as the most stable reference genes, while RPL19, RPS15, and RPS9 were found to be the least stable. The suitability of the selected internal control genes was further validated through target gene analysis, confirming their efficacy in ensuring accurate gene expression profiling in goats. The study determined that the geometric average of ACTB, PPIB, and B2M creates an appropriate normalization factor for gene expression studies in goat tissues.

Reference genes for gene expression profiling in mouse models of Listeria monocytogenes infection.

RT-qPCR dissects transcription-based processes but requires reference genes (RGs) for data normalization. This study prospected RGs for mouse macrophages (pMØ) and spleen infected with Listeria monocytogenes. ThepMØ were infected in vitro with L. monocytogenes or vehicle for 4h. Mice were injected with L. monocytogenes (or vehicle) and euthanized 24h post-injection. TheRGs came from a multispecies primer set, from theliterature or designed here. The RG ranking relied on GeNorm, NormFinder, BestKeeper, Delta-CTand RefFinder. B2m-H3f3a-Ppia were the most stable RGs for pMØ, albeit RG indexes fine-tuned estimations of cytokine relative expression. Actβ-Ubc-Ppia were the best RGs for spleen butmodestly impacted the cytokine relative expression. Hence, mouse models of L. monocytogenes require context-specific RGs for RT-qPCR, thus reinforcing its paramount contribution to accurate gene expression profiling.

Selection and validation of reliable reference genes for quantitative real-time PCR in Barnyard millet (Echinochloa spp.) under varied abiotic stress conditions

Quantitative real-time polymerase chain reaction (RT-qPCR) using a stable reference gene is widely used for gene expression research. Barnyard millet (Echinochloa spp.) is an ancient crop in Asia and Africa that is widely cultivated for food and fodder. It thrives well under drought, salinity, cold, and heat environmental conditions, besides adapting to any soil type. To date, there are no gene expression studies performed to identify the potential candidate gene responsible for stress response in barnyard millet, due to lack of reference gene. Here, 10 candidate reference genes, Actin (ACT), α-tubulin (α-TUB), β-tubulin (β-TUB), RNA pol II (RP II), elongation factor-1 alpha (EF-1α), adenine phosphoribosyltransferase (APRT), TATA-binding protein-like factor (TLF), ubiquitin-conjugating enzyme 2 (UBC2), ubiquitin-conjugating enzyme E2L5 (UBC5) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were selected from mRNA sequences of E. crus-galli and E. colona var frumentacea. Five statistical algorithms (geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder) were applied to determine the expression stabilities of these genes in barnyard millet grown under four different abiotic stress (drought, salinity, cold and heat) exposed at different time points. The UBC5 and ɑ-TUB in drought, GAPDH in salinity, GAPDH and APRT in cold, and EF-1α and RP II in heat were the most stable reference genes, whereas ß-TUB was the least stable irrespective of stress conditions applied. Further Vn/Vn + 1 analysis revealed two reference genes were sufficient to normalize gene expression across all sample sets. The suitability of identified reference genes was validated with Cu-ZnSOD (SOD1) in the plants exposed to different abiotic stress conditions. The results revealed that the relative quantification of the SOD1 gene varied according to reference genes and the number of reference genes used, thus highlighting the importance of the choice of a reference gene in such experiments. This study provides a foundational framework for standardizing RT-qPCR analyses, enabling accurate gene expression profiling in barnyard millet.

Reliable Reference Genes for Accurate Gene Expression Profiling across Different Tissues and Genotypes of Rice Seedlings (Oryza sativa L.) under Salt Stress

Reliable Reference Genes for Accurate Gene Expression Profiling across Different Tissues and Genotypes of Rice Seedlings (Oryza sativa L.) under Salt Stress

Diagnostic Host Gene Expression Analysis by Quantitative Reverse Transcription Loop-Mediated Isothermal Amplification to Discriminate between Bacterial and Viral Infections.

Early and accurate diagnosis of acute infections can help minimize the overprescription of antibiotics and improve patient outcomes. Discrimination between bacterial and viral etiologies in acute infection based on changes in host gene expression has been described. Unfortunately, established technologies used for gene expression profiling are typically expensive and slow, confounding integration into clinical workflows. Here we report the development of an ultra-rapid test system for host gene expression profiling from blood based on quantitative reverse transcription followed by loop-mediated isothermal amplification (qRT-LAMP). We developed 10 messenger ribonucleic acid-specific assays based on qRT-LAMP targeting 7 informative biomarkers to discriminate viral from bacterial infections and 3 housekeeping reference genes. We optimized qRT-LAMP formulations to achieve a turnaround time of 12 min without sacrificing specificity or precision. The accuracy of the test system was verified utilizing blood samples from 57 patients and comparing qRT-LAMP results to profiles obtained using an orthogonal reference technology. We observed a Pearson coefficient of 0.90 between bacterial/viral metascores generated by qRT-LAMP and the reference technology. qRT-LAMP assays can provide sufficiently accurate gene expression profiling data to enable discrimination between bacterial and viral etiologies using an established set of biomarkers and a classification algorithm.

Evaluation of Reference Genes for Transcriptional Profiling in Two Cockroach Models.

The German cockroach, Blattella germanica, and the American cockroach, Periplaneta americana are the most common and synanthropic household pests of interest to public health. While they have increasingly served as model systems in hemimetabolous insects for studying many biological issues, there is still a lack of stable reference gene evaluation for reliable quantitative real-time PCR (qPCR) outputs and functional genomics. Here, we evaluated the expression variation of common insect reference genes, including the historically used actin, across various tissues and developmental stages, and also under experimental treatment conditions in these two species by using three individual algorithms (geNorm, BestKeeper, and NormFinder) and a comprehensive program (RefFinder). RPL32 in B. germanica and EF1α in P. americana showed the overall lowest variation among all examined samples. Based on the stability rankings by RefFinder, the optimal but varied reference genes under specific conditions were selected for qPCR normalization. In addition, the combination of RPL32 and EF1α was recommended for all the tested tissues and stages in B. germanica, whereas the combination of multiple reference genes was unfavorable in P. americana. This study provides a condition-specific resource of reference gene selection for accurate gene expression profiling and facilitating functional genomics in these two important cockroaches.

Identification and selection of reference genes for gene expression analysis by quantitative real-time PCR in Suaeda glauca\u2019s response to salinity

Quantitative real-time polymerase chain reaction (qPCR) using a stable reference gene is widely used for gene expression research. Suaeda glauca L. is a succulent halophyte and medicinal plant that is extensively used for phytoremediation and extraction of medicinal compounds. It thrives under high-salt conditions, which promote the accumulation of high-value secondary metabolites. However, a suitable reference gene has not been identified for gene expression standardization in S. glauca under saline conditions. Here, 10 candidate reference genes, ACT7, ACT11, CCD1, TUA5, UPL1, PP2A, DREB1D, V-H+-ATPase, MPK6, and PHT4;5, were selected from S. glauca transcriptome data. Five statistical algorithms (ΔCq, geNorm, NormFinder, BestKeeper, and RefFinder) were applied to determine the expression stabilities of these genes in 72 samples at different salt concentrations in different tissues. PP2A and TUA5 were the most stable reference genes in different tissues and salt treatments, whereas DREB1D was the least stable. The two reference genes were sufficient to normalize gene expression across all sample sets. The suitability of identified reference genes was validated with MYB and AP2 in germinating seeds of S. glauca exposed to different NaCl concentrations. Our study provides a foundational framework for standardizing qPCR analyses, enabling accurate gene expression profiling in S. glauca.

MethCORR infers gene expression from DNA methylation and allows molecular analysis of ten common cancer types using fresh-frozen and formalin-fixed paraffin-embedded tumor samples

BackgroundTranscriptional analysis is widely used to study the molecular biology of cancer and hold great biomarker potential for clinical patient stratification. Yet, accurate transcriptional profiling requires RNA of a high quality, which often cannot be retrieved from formalin-fixed, paraffin-embedded (FFPE) tumor tissue that is routinely collected and archived in clinical departments. To overcome this roadblock to clinical testing, we previously developed MethCORR, a method that infers gene expression from DNA methylation data, which is robustly retrieved from FFPE tissue. MethCORR was originally developed for colorectal cancer and with this study, we aim to: (1) extend the MethCORR method to 10 additional cancer types and (2) to illustrate that the inferred gene expression is accurate and clinically informative.ResultsRegression models to infer gene expression information from DNA methylation were developed for ten common cancer types using matched RNA sequencing and DNA methylation profiles (HumanMethylation450 BeadChip) from The Cancer Genome Atlas Project. Robust and accurate gene expression profiles were inferred for all cancer types: on average, the expression of 11,000 genes was modeled with good accuracy and an intra-sample correlation of R2 = 0.90 between inferred and measured gene expression was observed. Molecular pathway analysis and transcriptional subtyping were performed for breast, prostate, and lung cancer samples to illustrate the general usability of the inferred gene expression profiles: overall, a high correlation of r = 0.96 (Pearson) in pathway enrichment scores and a 76% correspondence in molecular subtype calls were observed when using measured and inferred gene expression as input. Finally, inferred expression from FFPE tissue correlated better with RNA sequencing data from matched fresh-frozen tissue than did RNA sequencing data from FFPE tissue (P < 0.0001; Wilcoxon rank-sum test).ConclusionsIn all cancers investigated, MethCORR enabled DNA methylation-based transcriptional analysis, thus enabling future analysis of cancer in situations where high-quality DNA, but not RNA, is available. Here, we provide the framework and resources for MethCORR modeling of ten common cancer types, thereby widely expanding the possibilities for transcriptional studies of archival FFPE material.

Genomic Landscape of Liquid Biopsy for Hepatocellular Carcinoma Personalized Medicine.

Hepatocellular carcinoma (HCC) is the sixth most frequently diagnosed cancer and the third leading cause of cancer-related deaths worldwide. Advanced-stage HCC patients have poor survival rates and this requires the discovery of novel clear biomarkers for HCC early diagnosis and prognosis, identifying risk factors, distinguishing HCC from non-HCC liver diseases, and assessment of treatment response. Liquid biopsy has emerged as a novel minimally invasive approach to enable monitoring tumor progression, metastasis, and recurrence. Since the liquid biopsy analysis has relatively high specificity and low sensitivity in cancer early detection, there is a risk of bias. Next-generation sequencing (NGS) technologies provide accurate and comprehensive gene expression and mutational profiling of liquid biopsies including cell-free circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), and genomic components of extracellular vesicles (EVs) including micro-RNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs). Since HCC is a highly heterogeneous cancer, HCC patients can display various genomic, epigenomic, and transcriptomic patterns and exhibit varying sensitivity to treatment options. Identification of individual variabilities in genomic signatures in liquid biopsy has the potential to greatly enhance precision oncology capabilities. In this review, we highlight and critically discuss the latest progress in characterizing the genomic landscape of liquid biopsy, which can advance HCC personalized medicine.

In Vivo Gene Expression Profile of Human Intestinal Epithelial Cells: From the Viewpoint of Drug Metabolism and Pharmacokinetics.

Orally administered drugs are absorbed and metabolized in the intestine. To accurately predict pharmacokinetics in the intestine, it is essential to understand the intestinal expression profiles of the genes related to drug absorption, distribution, metabolism, and excretion (ADME). However, in many previous studies, gene expression analysis in the intestine has been carried out using specimens from patients with cancer. In this study, to obtain more accurate gene expression profiles, biopsy samples were collected under endoscopic observation from the noninflammatory regions of 14 patients with inflammatory bowel disease, and RNA-seq analysis was performed. Gene expression analysis of drug-metabolizing enzymes (cytochromes P450), non-cytochrome P450 enzymes, nuclear receptors, drug-conjugating enzymes (UDP-glucuronosyltransferases and sulfotransferases), and apical and basolateral drug transporters was performed in biopsy samples from the duodenum, ileum, colon, and rectum. The proportions of the cytochromes P450 expressed in the ileum were 25% (CYP3A4), 19% (CYP2C18), and 14% (CYP3A5). CYP3A4 and CYP2C19 were highly expressed in the duodenum and ileum, but not in the colon and rectum. In the ileum, apical transporters such as P-gp, peptide transporter 1, breast cancer resistance protein, MRP2, and ASBT were strongly expressed, and the expression levels of P-gp and ASBT in the ileum were higher than those in other regions. In the ileum, basolateral transporters such as OSTα, OSTβ, and MRP3 were strongly expressed. We succeeded in obtaining gene expression profiles of ADME-related genes in human intestinal epithelial cells in vivo. We expect that this information would be useful for accurate prediction of the pharmacokinetics of oral drugs. SIGNIFICANCE STATEMENT: To obtain gene expression profiles of ADME-related genes in human intestinal epithelial cells in vivo, biopsy samples were collected under endoscopic observation from the noninflammatory regions of 14 patients with inflammatory bowel disease, and RNA-seq analysis was performed. Gene expression profiles of drug-metabolizing enzymes (cytochromes P450), non-cytochrome P450 enzymes, nuclear receptors, drug-conjugating enzymes (UDP-glucuronosyltransferases and sulfotransferases), and apical and basolateral drug transporters in biopsy samples from the duodenum, ileum, colon, and rectum were obtained in this study.

Gene Expression Analysis and Metabolite Profiling of Silymarin Biosynthesis during Milk Thistle (Silybum marianum (L.) Gaertn.) Fruit Ripening.

Mature fruits (i.e., achenes) of milk thistle (Silybum marianum (L.) Gaertn., Asteraceae) accumulate high amounts of silymarin (SILM), a complex mixture of bioactive flavonolignans deriving from taxifolin. Their biological activities in relation with human health promotion and disease prevention are well described. However, the conditions of their biosynthesis in planta are still obscure. To fill this gap, fruit development stages were first precisely defined to study the accumulation kinetics of SILM constituents during fruit ripening. The accumulation profiles of the SILM components during fruit maturation were determined using the LC-MS analysis of these defined developmental phases. The kinetics of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) and peroxidase (POX) activities suggest in situ biosynthesis of SILM from l-Phenylalanine during fruit maturation rather than a transport of precursors to the achene. In particular, in contrast to laccase activity, POX activity was associated with the accumulation of silymarin, thus indicating a possible preferential involvement of peroxidase(s) in the oxidative coupling step leading to flavonolignans. Reference genes have been identified, selected and validated to allow accurate gene expression profiling of candidate biosynthetic genes (PAL, CAD, CHS, F3H, F3’H and POX) related to SILM accumulation. Gene expression profiles were correlated with SILM accumulation kinetic and preferential location in pericarp during S. marianum fruit maturation, reaching maximum biosynthesis when desiccation occurs, thus reinforcing the hypothesis of an in situ biosynthesis. This observation led us to consider the involvement of abscisic acid (ABA), a key phytohormone in the control of fruit ripening process. ABA accumulation timing and location during milk thistle fruit ripening appeared in line with a potential regulation of the SLIM accumulation. A possible transcriptional regulation of SILM biosynthesis by ABA was supported by the presence of ABA-responsive cis-acting elements in the promoter regions of the SILM biosynthetic genes studied. These results pave the way for a better understanding of the biosynthetic regulation of SILM during the maturation of S. marianum fruit and offer important insights to better control the production of these medicinally important compounds.

Application of FFDNET for Image Denoising On Microarray Images

Microarray technology allows the simultaneous profiling of thousands of genes. Denoising is an important pre-processing step in microarray image analysis for accurate gene expression profiling. In this paper, as FFDNet provides model independent denoising technique, it is been applied on microarray images. FFDNet is validated on AWGN based images and real noisy images trained network. The application is compared with the standard denoising methods. The results revealed that optimal sigma value to efficiently remove noise while preserving details for AWGN based images and real noisy trained methods were 15 and 20 respectively. Overall, the performance of the FFDNet is better compared to other metrics considered in the study as it is flexible, effective and fast. It is also capable to maintain the trade-off between denoising and feature preservation.

Systematic expression analysis of genes related to generation of action potentials in human iPS cell-derived cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a valuable tool to characterize the pharmacology and toxic effects of drugs on heart cells. In particular, hiPSC-CMs can be used to identify drugs that generate arrhythmias. However, it is unclear whether the expression of genes related to generation of CM action potentials differs between hiPSC-CM cell lines and the mature human heart. To address this, we obtained accurate gene expression profiles of commercially available hiPSC-CM cell lines with quantitative real time RT-PCR analysis. Expression analysis of ten cardiac proteins important for generation of action potentials and three cardiac proteins important for muscle contractility was performed using GAPDH for normalization. Comparison revealed large variations in expression levels among hiPSC-CM cell lines and between hiPSC-CMs and normal human heart. In general, gene expression in hiPSC-CM cell lines was more similar to an immature, stem-like cell than a mature cardiomyocyte from human heart samples. These results provide quantitative information about differences in gene expression between hiPSC-CM cell lines, essential for interpreting pharmacology experiments. Our approach can be used as an experimental guideline for future research on gene expression in hiPSC-CMs.

Clinical Features and Cell of Origin Subtyping Using Gene Expression Profiling in HIV-Negative Patients with Primary Central Nervous System Lymphoma

Objectives: Primary central nervous system lymphoma (PCNSL) is a rare aggressive B-cell lymphoma, particularly in HIV-negative individuals, that represents a clinical challenge due to its location and lack of comprehensive molecular and biologic description. Histopathologic features are that of diffuse large B-cell lymphoma with expression of pan-B-cell markers as well as cell of origin (COO) germinal center B cell (GCB) and post-germinal center B cell (non-GCB) markers. Previous studies using immunohistochemistry (IHC) suggest that the majority of PCNSL cases are non-GCB. However, gene expression profiling has revealed non-GCB to be comprised of two distinct subtypes, namely activated B-cell (ABC) and unclassified (UNC) subtypes that are indistinguishable by IHC. To date COO testing using the highly accurate Lymph2Cx gene expression profiling assay has not been reported in PCNSL.Methods: IRB approval was obtained and HIV negative patients diagnosed with PCNSL, who had given informed consent and for whom archived tumor tissue was available for testing were identified. COO testing was performed using the Lymph2Cx NanoString assay on RNA extracted from formalin-fixed, paraffin embed tissues using our established laboratory protocols. Clinical data including patient demographics, lines of treatment and survival outcomes were collected and correlated with each other and Lymph2Cx COO results.Results: Thirty-two HIV-negative patients diagnosed between January 2005 and June 2015 were included. Median age was 61 years (30-82) and 53% were male. Radiographic information was available for 18/32 patients. Eleven (61%) had a single brain lesion at diagnosis, while 7 (39%) had >1 brain lesion. Lines of systemic therapy were 1 (91%) and 2 (9%). All patients received methotrexate-based induction therapy (44% received methotrexate, rituximab and temozolomide, 16% received single-agent methotrexate, 31% received methotrexate and rituximab, and 9% received the modified Bonn regimen; methotrexate and cytarabine-based induction). A total of 10 patients (31%) received high-dose chemotherapy and autologous stem cell transplant (ASCT) for consolidation. Of the 10 patients that underwent consolidation therapy, 9 underwent ASCT after first line induction, and 1 underwent ASCT after second line therapy. None of the patients received whole-brain radiation therapy. At a median follow-up of 29 months (range of 2-107) median event-free survival (EFS) was 16.3 months (95% CI, 8.8-45.7), and median overall survival (OS) was 41.2 months (95% CI, 28.5-NE).COO testing using the Lymph2Cx assay revealed that 91% (29/32) were ABC, 9% (3/32) were GCB, and none were UNC. Histopathology reports described COO using the Hans algorithm in 11 of the 32 cases. Of the 3 determined to be GCB on Lymph2Cx, 1 was denoted GCB by the Hans algorithm and 2 were not stained to determine COO. Of the 29 determined to be ABC by Lymph2Cx, 9 were denoted non-GCB and 1 was denoted GCB by the Hans algorithm, and 19 were not stained to determine COO.Conclusions: This series of HIV-negative patients with PCNSL showed median survival consistent with previous studies. In this first series using the Lymph2Cx assay, we confirmed that over 90% of PCNSLs are of ABC subtype, which concurs with previous reports that PCNSL tumors are predominately non-GCB by the Hans algorithm. These findings provide biological rationale forthat pharmacologic interventions targeting B-cell receptor signaling to be explored in clinical trials in the majority of patients with PCNSL. [Display omitted] DisclosuresRimsza:NanoString: Other: Inventor on the patent for the Lymph2Cx assay.

The thanatotranscriptome: Gene expression of male reproductive organs after death

The prostate gland is one of the last internal organs to deteriorate during human decomposition; however, this phenomenon is still mysterious. Gene expression in antemortem cases has been widely studied and a majority of the analyses concentrate on discovering basic physiological processes. The question of “What happens to gene expression after a human dies?” is a novel and emerging topic. Thanatotranscriptome (thanatos-, Greek for death) involves research on mRNA transcript abundances and gene expression in human tissues after death. Our previous studies have shown that RNA is a suitable and stable molecule in postmortem liver samples up to two days. Consequently, we hypothesized that there are also measurable and significant differences in mRNA transcript abundances in prostate tissues from human remains. In the current study, the goal was to identify apoptotic molecular markers (i.e., pro- and/or anti-apoptosis genes) that provide accurate gene expression profiles regarding the time of death.Tissue samples were removed by a medical examiner from the prostate of five cadavers during autopsy. After RNA extraction, cDNA was synthesized and the concentration was determined. The cDNA was reacted in apoptosis-related gene expression profiling by human PCR Array. The PCR Array results showed that at 38 h after death, a majority of the genes for apoptosis induction and positive regulation (i.e., caspases) were over-expressed more than at five days. The expression of anti-apoptotic genes such as BAG1, BCL2, and negative regulator of apoptosis, XIAP, was significantly elevated in a time-dependent manner. However, pro-apoptotic gene expression such as TP53 and TNFSF10 was not significantly upregulated.Therefore, postmortem prostate cells counteract programmed cell death with its anti-apoptotic machinery; yet as time progresses, pro-apoptotic mechanisms dominate. In conclusion, our study implies that over-expression of genes in male reproductive organs still occurs during decomposition, which may play substantial roles in forensic research and clinical application. These findings demonstrate that there is still active postmortem gene expression; however, our future research question will be, “When does gene expression terminate after death?”

Challenges in using liquid biopsies for gene expression profiling.

Circulating tumor cells (CTCs) have potential utility as a surrogate biomarker of tumor biology via a liquid biopsy. The aim of this study was to evaluate if the nCounter NanoString assay could be used for accurate gene expression profiling of CTCs using the PAM50 research-use-only CodeSet. Analysis was performed on CTCs isolated by the ANGLE Parsortix system from healthy blood spiked with the breast cancer cell lines Hs578T, SkBr3, MDA-MB-231 or MCF7. Using cell lines as gold standard positive controls and Parsortix processed blood without spiking (unspiked) as negative controls, we found an average of 12 significantly differentially expressed genes among spiked samples versus unspiked controls. We validated our findings with the NanoStringDiff differential expression statistical method. The NanoString recommended targeted pre-amplification introduced false positive results due to pre-amplification bias, and the amplification of non-cancer genes from normal leukocytes confounded gene expression profiling of CTCs. Pre-amplification bias is a concern for other similar assays that may be used as discovery tools or target validation of transcripts of interest in gene expression profiling of CTCs. We recommend the use of an unspiked negative control when evaluating CTC technologies regarding gene expression profiling. Given that the molecular profiling of CTCs as a liquid biopsy may have clinical ramifications for potential treatment selection in future clinical trials, our study emphasizes cautious consideration of pre-analytical variables such as amplification bias in the context of liquid biopsy studies.

SPOTs fill a major gap in RNA quantification.

Direct RNA quantification using barcoded fluorescence hybridization enables accurate and scalable gene expression profiling.

Abstract P1-01-11: Something from nothing? The case for quality control in liquid biopsy studies

Abstract Background: Circulating tumor cells (CTCs) as a liquid biopsy strategy are currently being studied as a surrogate biomarker that may reflect metastatic tumor biology. Given the rarity of CTCs, target enrichment is commonly used to profile the gene signatures of valuable clinical samples to evaluate for multiplexed gene panels of interest. The aim of our study was to evaluate if the NanoString PAM50 could be used for accurate gene expression profiling of CTCs and controls using the research-use-only probeset. Methods: We collected two 7.5mL EDTA tubes of blood from 12 healthy female volunteers. CTC assays were performed using the ANGLE Parsortix system as a microfluidics filter that separates cells based on size and deformability. The cell lines Hs578T (basal-like) and SK-BR-3 (HER2 amplified) were used to spike 20 cells into n=6 blood tubes per cell line (termed spiked samples). N=12 7.5mL tubes of blood (termed unspiked samples) and n=12 spiked samples were processed using Parsortix for CTC harvesting and lysis using a 10 micron cassette. From each lysate, 5uL was taken for cDNA amplification, multiple target enrichment for 14 cycles, followed by NanoString PAM50 assays. From each of the 12 peripheral blood (PB) samples, we extracted RNA and used 100ng for NanoString PAM50 assays. For cell line controls, 100ng of Hs578T or SK-BR-3 were subjected to NanoString Assays. Results: Low PAM50 gene expression was observed in all 12 PB samples. Unspiked PB harvested from the CTC assay showed a higher level of PAM50 gene expression compared to PB, suggesting that the target enrichment amplification produces false positive detection of expected breast cancer related transcripts. On ANOVA testing, 10/12 (83%) of unspiked, sorted, target enriched samples had significant differential expression (p&amp;lt;0.0001) of the mean log normalized counts for the PAM50 genes compared to PB. In spiked experiments using n=20 cells in 7.5mL of PB, sorted Hs578T were found to be triple negative in only 3/6 (50%) while sorted SK-BR-3 were found to be HER2 positive in only 3/6 (50%). On ANOVA testing, the spiked/sorted and bulk were found to have a difference among the mean log normalized counts for the PAM50 genes across all samples for both cell lines (p&amp;lt;0.0001). However, 3/6 (50%) samples had a difference in mean PAM50 gene expression when compared to bulk Hs578t on multiple comparison testing while 2/6 (33%) were statistically significantly different when comparing spiked, sorted SK-BR-3 versus bulk cell line. Conclusions: Unspiked blood processed via a CTC assay and subjected to target enrichment showed high expression of genes in the NanoString PAM50 assay, likely due to amplification bias. When working with enriched but not ultra-pure CTC samples, amplified gene expression of background leukocytes may influence read counts. This is important to consider in assays that enrich for CTCs but retain a leukocyte background. Further studies will address the effect of the CTC assay procedure and number of leukocytes on accuracy of gene expression of rare CTC mimics. This study emphasizes the importance of selecting genes that are not expressed in PB or performing background subtraction or normalization as strategies for accurate gene expression profiling of CTCs. Citation Format: Porras TB, Bains PK, Ring A, Carrasco S, Forte V, Punj V, Lang JE. Something from nothing? The case for quality control in liquid biopsy studies [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-11.

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis.

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.