Detection and Characterization of Apoptosis-Related Proteins in Hippocampal Neurodegeneration: From mRNA Expression to Protein Quantification.

The involvement of apoptosis in neurodegeneration can be detected by quantifying the apoptotic proteins in hippocampal lysate. Apoptosis can occur due to the overproduction of apoptotic proteins under the influence of external trigger or due to the overexpression of the apoptotic genes. Thus, the imbalance in the production of apoptotic proteins can be quantified using the Western blotting technique and the overexpression of apoptotic genes in hippocampal DNA can be quantified using the real-time quantification of mRNA expression of the apoptotic proteins. Here we provide the methodology of detecting the apoptosis-related proteins like Bax and Bcl-2 and their mRNA expression in hippocampal neurodegeneration. In this chapter, we have described the methodology for quantification of mRNA expression of these apoptosis-related proteins in the hippocampal lysate using the real-time quantitative polymerase chain reaction (qPCR) technique and the methodology of detection and characterization of respective protein expression in the hippocampal lysate using the Western blotting technique.

Purification of Circulating Tumor Cells Based on Multiantibody-Modified Magnetic Nanoparticles and Molecular Analysis toward Epithelial Ovarian Cancer Detection.

Circulating tumor cells (CTCs) are valuable circulating biomarkers of cancer, which carry primary tumor information and may provide real-time assessment of tumor status as well as treatment response in cancer patients. Herein, we developed a novel assay for accurate diagnosis and dynamic monitoring of epithelial ovarian cancer (EOC) using CTC RNA analysis. Multiantibody-modified magnetic nanoparticles were prepared for purification of EOC CTCs from whole blood samples of clinical patients. Subsequently, nine EOC-specific mRNAs of purified CTCs were quantified using droplet digital PCR. The EOC CTC Score was generated using a multivariate logistic regression model for each sample based on the transcripts of the nine genes. This assay exhibited a distinguishing diagnostic performance for the detection of EOC (n = 17) from benign ovarian tumors (n = 30), with an area under the receiver operating characteristic curve (AUC) of 0.96 (95% CI = 0.91-1.00). Moreover, dynamic changes of the EOC CTC Score were observed in patients undergoing treatment, demonstrating the potential of the assay for monitoring EOC. In conclusion, we present an accurate assay for the diagnosis and monitoring of EOC via CTC RNA analysis, and the results suggest that it may provide a promising solution for the detection and treatment response assessment of EOC.

Hydrogel-Based Biosensors for Effective Therapeutics.

Nanotechnology and polymer engineering are navigating toward new developments to control and overcome complex problems. In the last few decades, polymer engineering has received researchers' attention and similarly, polymeric network-engineered structures have been vastly studied. Prior to therapeutic application, early and rapid detection analyses are critical. Therefore, developing hydrogel-based sensors to manage the acute expression of diseases and malignancies to devise therapeutic approaches demands advanced nanoengineering. However, nano-therapeutics have emerged as an alternative approach to tackling strenuous diseases. Similarly, sensing applications for multiple kinds of analytes in water-based environments and other media are gaining wide interest. It has also been observed that these functional roles can be used as alternative approaches to the detection of a wide range of biomolecules and pathogenic proteins. Moreover, hydrogels have emerged as a three-dimensional (3D) polymeric network that consists of hydrophilic natural or synthetic polymers with multidimensional dynamics. The resemblance of hydrogels to tissue structure makes them more unique to study inquisitively. Preceding studies have shown a vast spectrum of synthetic and natural polymer applications in the field of biotechnology and molecular diagnostics. This review explores recent studies on synthetic and natural polymers engineered hydrogel-based biosensors and their applications in multipurpose diagnostics and therapeutics. We review the latest studies on hydrogel-engineered biosensors, exclusively DNA-based and DNA hydrogel-fabricated biosensors.

Prandial state and biological sex modulate clinically relevant efflux transporters to different extents in Wistar and Sprague Dawley rats.

P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein 2 (MRP2) are clinically relevant efflux transporters implicated in the oral absorption of many food and drug substrates. Here, we hypothesised that food intake could influence protein and mRNA intestinal expression of P-gp/abcb1a, BCRP/abcg2, and MRP2/abcc2 differently in male and female Wistar and Sprague Dawley rats. To test this hypothesis, we used enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR) to quantify the protein and mRNA intestinal expression of these transporters, respectively. Our study found food and sex differences in P-gp expression, whereby in the fed state P-gp expression decreased in male Wistar rats, but P-gp expression increased in females. In the fed state, BCRP expression increased in both male and female Wistar rats, compared with the fasted state. In contrast, no sex differences or food effect differences were seen in Sprague Dawley rats for P-gp and BCRP expression. On the other hand, in the fed state, MRP2 expression was higher in male and female Wistar and Sprague Dawley rats when compared with the fasted state. Sex differences were also observed in the fasted state. Overall, significant strain differences were reported for P-gp, BCRP and MRP2 expression. Strong to moderate positive linear correlations were found between ELISA and PCR quantification methods. ELISA may be more useful than PCR as it reports protein expression as opposed to transcript expression. Researchers must consider the influence of sex, strain and feeding status in preclinical studies of P-gp, BCRP and MRP2 drug substrates.

Elevated atmospheric CO2 concentrations caused a shift of the metabolically active microbiome in vineyard soil.

BACKGROUND: Elevated carbon dioxide concentrations (eCO2), one of the main causes of climate change, have several consequences for both vine and cover crops in vineyards and potentially also for the soil microbiome. Hence soil samples were taken from a vineyard free-air CO2 enrichment (VineyardFACE) study in Geisenheim and examined for possible changes in the soil active bacterial composition (cDNA of 16S rRNA) using a metabarcoding approach. Soil samples were taken from the areas between the rows of vines with and without cover cropping from plots exposed to either eCO2 or ambient CO2 (aCO2). RESULTS: Diversity indices and redundancy analysis (RDA) demonstrated that eCO2 changed the active soil bacterial diversity in grapevine soil with cover crops (p-value 0.007). In contrast, the bacterial composition in bare soil was unaffected. In addition, the microbial soil respiration (p-values 0.04-0.003) and the ammonium concentration (p-value 0.003) were significantly different in the samples where cover crops were present and exposed to eCO2. Moreover, under eCO2 conditions, qPCR results showed a significant decrease in 16S rRNA copy numbers and transcripts for enzymes involved in N2 fixation and NO2- reduction were observed using qPCR. Co-occurrence analysis revealed a shift in the number, strength, and patterns of microbial interactions under eCO2 conditions, mainly represented by a reduction in the number of interacting ASVs and the number of interactions. CONCLUSIONS: The results of this study demonstrate that eCO2 concentrations changed the active soil bacterial composition, which could have future influence on both soil properties and wine quality.

Reference genes for quantitative Arabidopsis single molecule RNA fluorescence in situ hybridization.

Subcellular mRNA quantities and spatial distributions are fundamental for driving gene regulatory programmes. Single molecule RNA fluorescence in situ hybridization (smFISH) uses fluorescent probes to label individual mRNA molecules, thereby facilitating both localization and quantitative studies. Validated reference mRNAs function as positive controls and are required for calibration. Here we present selection criteria for the first set of Arabidopsis smFISH reference genes. Following sequence and transcript data assessments, four mRNA probe sets were selected for imaging. Transcript counts per cell, correlations with cell size, and corrected fluorescence intensities were all calculated for comparison. In addition to validating reference probe sets, we present sample preparation steps that can retain green fluorescent protein fluorescence, thereby providing a method for simultaneous RNA and protein detection. In summary, our reference gene analyses, modified protocol, and simplified quantification method together provide a firm foundation for future quantitative single molecule RNA studies in Arabidopsis root apical meristem cells.

Quantitative Real-Time PCR for Evaluating Transcriptional Changes in T-Lymphocytes.

The real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) is an indispensable technology that enables reliable transcriptional analysis routinely used in molecular biology studies. The qRT-PCR technique quantifies mRNA by taking advantage of the reverse transcriptase-dependent conversion of RNA into cDNA and subsequent amplification of the cDNA using PCR. The amount of PCR product is directly proportional to the initial starting quantity of mRNA. The straightforward but complex methodologies used in this technique involve multiple sequential steps that include isolation of mRNA, conversion of mRNA into cDNA, amplification of the cDNA, and quantification of amplicons. In this chapter, we describe an optimized protocol for performing qRT-PCR in human T-lymphocytes.

Protein and RNA quantification of multiple genes in single cells.

Single-cell analysis overcomes the problems of cellular heterogeneity by revealing the individual differences between cells in tissue. The current tools used to profile gene expression at the single-cell level are arduous and often require specialized equipment. We have previously developed a technique to quantify protein expression levels in single living cells. Here, we combine quantification of protein expression with absolute measurement of mRNA amounts of the same gene in the same cell, to profile the expression of genes at the transcriptional and translational levels. We show that high heterogeneity exists at both the mRNA and protein levels for multiple genes, even among monoclonal cells. We demonstrate a rapid, straightforward approach to single-cell profiling of RNA and protein production.

Simultaneous Quantification of Multiple Alternatively Spliced mRNA Transcripts Using Droplet Digital PCR.

Currently there is no sensitive, precise, and reproducible method to quantitate alternative splicing of mRNA transcripts. Droplet digital™ PCR (ddPCR™) analysis allows for accurate digital counting for quantification of gene expression. Human telomerase reverse transcriptase (hTERT) is one of the essential components required for telomerase activity and for the maintenance of telomeres. Several alternatively spliced forms of hTERT mRNA in human primary and tumor cells have been reported in the literature. Using one pair of primers and two probes for hTERT, four alternatively spliced forms of hTERT (α-/ß+, α+/ß- single deletions, α-/ß- double deletion, and nondeletion α+/ß+) were accurately quantified through a novel analysis method via data collected from a single ddPCR reaction. In this chapter, we describe this ddPCR method that enables direct quantitative comparison of four alternatively spliced forms of the hTERT messenger RNA without the need for internal standards or multiple pairs of primers specific for each variant, eliminating the technical variation due to differential PCR amplification efficiency for different amplicons and the challenges of quantification using standard curves. This simple and straightforward method should have general utility for quantifying alternatively spliced gene transcripts.

Quantification of classical HLA class I mRNA by allele-specific, real-time polymerase chain reaction for most Han individuals.

Recent studies have shown that expression levels of different alleles at the same HLA class I locus can vary dramatically, which might have a broad influence on human disease. However, precise quantification of the relative expression level of each HLA allele is challenging, because distinguishing different alleles on the same locus is difficult. Here, we developed a series of allele-specific, real-time polymerase chain reaction assays for quantifying HLA class I allele mRNA in most Han individuals. The alleles of almost all heterozygous genotypes with a frequency higher than 0.5% in our population (78 alleles on HLA-A locus, 124 alleles on HLA-B locus, and 74 alleles on HLA-C locus) were specifically amplified. The specificity of the amplification was strictly validated by setting the corresponding negative control for each allele of each genotype. The amplification efficiency of each reaction was determined, and the slopes of the reactions were compared. This study provides a tool for detecting the comprehensive expression profile of HLA class I alleles and will be useful not only for the investigation of the molecular mechanism underlying HLA allele expression regulation but also for exploration of immunological mechanisms involving HLA expression in the fields of tumour immune evasion, viral infection, auto-immune disorders, and graft vs host disease after haematopoietic stem cell transplantation.

Gaining insight into plant gene transcription using smFISH.

Single molecule RNA fluorescent in situ hybridization (smFISH) enables gene transcription to be assessed at the cellular level. In this point of view article, we describe our recent smFISH research in the model plant Arabidopsis thaliana and discuss how this technique could further knowledge of plant gene transcription in the future.

Efficient in situ detection of mRNAs using the Chlorella virus DNA ligase for padlock probe ligation.

Padlock probes are single-stranded DNA molecules that are circularized upon hybridization to their target sequence by a DNA ligase. In the following, the circulated padlock probes are amplified and detected with fluorescently labeled probes complementary to the amplification product. The hallmark of padlock probe assays is a high detection specificity gained by the ligation reaction. Concomitantly, the ligation reaction is the largest drawback for a quantitative in situ detection of mRNAs due to the low affinities of common DNA or RNA ligases to RNA-DNA duplex strands. Therefore, current protocols require that mRNAs be reverse transcribed to DNA before detection with padlock probes. Recently, it was found that the DNA ligase from Paramecium bursaria Chlorella virus 1 (PBCV-1) is able to efficiently ligate RNA-splinted DNA. Hence, we designed a padlock probe assay for direct in situ detection of mRNAs using the PBCV-1 DNA ligase. Experimental single-cell data were used to optimize and characterize the efficiency of mRNA detection with padlock probes. Our results demonstrate that the PBCV-1 DNA ligase overcomes the efficiency limitation of current protocols for direct in situ mRNA detection, making the PBCV-1 DNA ligase an attractive tool to simplify in situ ligation sequencing applications.

Tracing the expression of circular RNAs in human pre-implantation embryos.

BACKGROUND: PolyA- RNAs have not been widely analyzed in human pre-implantation embryos due to the scarcity of materials. In particular, circular RNA (circRNA), a novel type of polyA- RNA, has not been characterized during human pre-implantation development. RESULTS: We systematically analyze polyA+ messenger RNAs (mRNAs) and polyA- RNAs in individual human oocytes and pre-implantation embryos using SUPeR-seq. We de novo identify 10,032 circRNAs from 2974 hosting genes. Most of these circRNAs are developmentally stage-specific and dynamically regulated. Many of them are maternally expressed, implying their potentially important regulatory functions in oogenesis and the formation of totipotent zygotes. Comparison between human and mouse embryos reveals both high conservation and clear distinction between these two species. Human pre-implantation embryos generate more types of circRNA compared with mouse embryos and this is associated with a striking increase of the length of the circRNA flanking introns in humans. We also perform RNA de novo assembly and identify novel transcript units, many of which are potentially novel long non-coding RNAs. CONCLUSIONS: This study reports the first analysis of the whole transcriptome comprising both polyA+ mRNAs and polyA- RNAs including circRNAs during human pre-implantation development. It provides an invaluable resource for analyzing the unique function and complex regulatory mechanisms of circRNAs during this process.

Gene Expression of Col11A1 Is a Marker Not only for Pancreas Carcinoma But also for Adenocarcinoma of the Papilla of Vater, Discriminating Between Carcinoma and Chronic Pancreatitis.

AIM: For tumors of the periampullary region clinical differentiation between primary and tumor-associated pancreatitis might be difficult. Early diagnosis of these malignancies is essential, as they present with early invasion of surrounding tissue thus limiting therapeutic options. Using molecular markers, the preoperative diagnosis (EUS-guided needle biopsy, brush biopsy pancreatic duct) could be optimized and surgical therapy potentially adapted. Alpha1 (XI) collagen Col11A1 is essential for the extracellular matrix and normal skeletal development and has been associated with carcinogenesis. MATERIALS AND METHODS: Forty-three patients with adenocarcinoma of the pancreas, 11 with adenocarcinoma of the papilla of Vater and 23 patients with chronic pancreatitis were included in the study. For all patients mRNA expression of Col11A1 was quantified by TaqMan RT-PCR in tumor or pancreatitis specimen, as well as in the corresponding normal uninvolved tissue and correlated with diagnosis of cancer and chronic pancreatitis. RESULTS: Col11A1 mRNA expression was 5.25-fold higher in adenocarcinoma of the pancreas (p=0.006) and 8.25-fold in the papilla of Vater (p=0.002) compared to that of chronic pancreatitis specimen. CONCLUSION: Differential mRNA expression of Col11A1 may be applied to preoperatively differentiate between tumors of the periampullary region and chronic pancreatitis and this may potentially have a positive effect on patient survival.

Tissue-dependent VEGF and GLUT1 induction in a rat hemorrhage model: With regard to diagnostic application of mRNA quantification in forensic pathology.

Systemic hypoxia is inevitably involved in the death process to a varying extent. Hypoxia-response factors proved useful in forensic pathology in previous studies; however, fundamental investigations using animal models are expected to reinforce the findings from autopsy practice. An animal experiment using a rat model of fixed-volume hemorrhage was performed to apply basic insight into quantitative mRNA analyses in forensic pathology. Male Sprague-Dawley rats (n=5) were anesthetized, bled from the femoral artery (24ml/kg; about 30% of total circulating blood volume), and decapitated after 1 or 2h. Tissue samples of the heart, brain (hippocampus), kidney, liver, lung and skeletal muscle were collected for RNA and protein analyses. Quantitative analyses of VEGF, GLUT1 and GAPDH mRNAs were performed with TaqMan real-time RT-PCR assay. In the sham control without bleeding, mRNA quantification revealed the tissue-dependent mRNA levels in physiological condition. Relative quantification of VEGF and GLUT1 showed significant inductions under hemorrhage at the mRNA level, using GAPDH as endogenous reference. In conclusion, tissue-dependent induction patterns of VEGF and GLUT1 were revealed in the volume-fixed hemorrhage rat model. This study could practically guide the selection of mRNA markers and tissue samples in forensic pathology related to tissue ischemia and cellular hypoxia for autopsy cases.

The expression of nitrate transporter genes reveals different nitrogen statuses of dominant diatom groups in the southern East China Sea.

In this study, the mRNA levels of the Nrt2 nitrate transporter gene were used as a molecular indicator of nitrogen status in two dominant diatom groups, Skeletonema and Chaetoceros, which inhabit the southern East China Sea (ECS). To accurately interpret the abundance of Nrt2 transcripts in situ, maximum and minimum expression levels were determined under conditions of nitrogen deprivation and ammonium addition, respectively. In August 2010, Nrt2 transcript levels in Skeletonema at the inner shelf region exhibited a mean of 111 mmole/(mole EFL); at the mid-shelf region, the mean Nrt2 mRNA levels were 298 mmole/(mole EFL), which was very close to the maximum levels observed under nitrogen starvation. By contrast, the Nrt2 transcript levels in Chaetoceros were low at all of the shelf locations, except at one station in the mid-shelf region. The cross-shelf mean was 2.86 mmole/(mole EFL), which was similar to the expression levels observed in cultured Chaetoceros under conditions of sufficient ammonium. Similar expression patterns were observed in diatoms in the southern ECS in June 2011, but the Nrt2 transcript levels in Skeletonema at the inner shelf region were reduced to a mean of 28.6 mmole/(mole EFL). Regression analysis indicated that cell abundance and Nrt2 expression were closely related to the nutricline depth in the coastward half of the southern ECS for Skeletonema but not for Chaetoceros. These results indicate that the evaluated species differ in nitrogen status, which may reflect their evolutionary strategies to survive in a fluctuating marine environment.

Reliable quantification of mRNA in archived formalin-fixed tissue with or without paraffin embedding.

INTRODUCTION: Formalin fixation and paraffin embedding (FFPE) is a standard method for tissue sample storage and preservation in pathology archives. The Reverse Transcriptase Quantitative Polymerase Chain Reaction (RT-qPCR) is a useful method for gene expression analysis, but its sensitivity is significantly decreased in FFPE tissue due to the fixation process. This process results in chemical modifications of RNA, cross-links proteins to RNA, and degrades RNA in these archived samples, hindering the reverse transcription step of the conventional RT-pPCR method and preventing generation of a cDNA that is long enough for the subsequent quantitative PCR step. METHODS: In this study, we used a multi-species RT-qPCR method originally developed to detect mRNA in tissue homogenate samples (Wang et al., 2011) and applied it to effectively detect a specific mRNA in formalin-fixed tissues with or without paraffin-embedding by targeting mRNA sequences as short as 24 nucleotides. RESULTS: Target sizes ranging from 24 to 91 nucleotides were evaluated using this multi-species RT-qPCR assay. Data generated with FFPE tissues demonstrated that use of short target sequences relieved the dependence on RNA quality and could reliably quantify mRNA. This method was highly sensitive, reproducible, and had a dynamic range of five orders of magnitude. Importantly, this method could quantify mRNA in prolonged formalin-fixed and FFPE tissue, where conventional RT-qPCR assays failed. Moreover, a similar result for small interfering RNA (siRNA)-mediated Apob mRNA knockdown was obtained from tissues fixed in formalin solution for 3months to 4years, and was found to be comparable to results obtained with frozen liver tissues. DISCUSSION: Therefore, the method presented here allows for preclinical and clinical retrospective and prospective studies on mRNA derived from archived FFPE and prolonged formalin-fixed tissue.

Second harmonic super-resolution microscopy for quantification of mRNA at single copy sensitivity.

Cell-specific information on the quantity and localization of key mRNAs at single copy sensitivity in single cells is critical for evaluating basic cellular process, disease risk, and efficacy of therapy. Quantification of overexpressed mRNAs beyond the diffraction limit is constrained by the optical property of the probes and microscopy techniques. In this report, nanosized barium titanium oxide (BaTiO3, BTO) crystals were utilized as probes for mRNA quantification by a second harmonic super-resolution microscopy (SHaSM). The SHaSM was able to detect a single copy of the human epidermal growth factor receptor 2 (Her2) mRNA at a resolution of 55.6 nm with the ability to resolve multiple mRNA copies in a diffraction-limited spot. Her2 mRNA per cell was counted in SK-BR-3, MCF-7, and HeLa cell lines as 595±79.1, 38.9±8.26, and 1.5±2.8, respectively. Our single-cell quantification results were validated with the fluorescence in situ hybridization studies and quantitative PCR, showing better specificity and selectivity over current single-molecule approaches for transcript detection. The SHaSM is expected to have an upper limit of resolving ∼10(4) transcripts in a single cell with the ability to monitor intracellular transcriptional dynamics at video rate. The developed approach has strong potential in clinical research and in the early diagnosis of life-threatening diseases such as cancer.

Early embryonic expression of a putative ecdysteroid-phosphate phosphatase in the water flea, Daphnia magna (Cladocera: Daphniidae).

Ecdysteroids, known as molting hormones, play central roles in the onset of molting, metamorphosis, and reproduction in arthropods. The ecdysteroids stored in eggs also play an important role in embryogenesis. In insects, ecdysteroids are stored as phosphate esters, which are converted to an active form by ecdysteroid-phosphate phosphatase (EPPase). Although EPPase is believed to be widely conserved in the Ecdysozoa, little is known about its expression in clades other than Insecta. In this study, we cloned a putative EPPase gene from a small fresh water crustacean known as a water flea, Daphnia magna Straus (Cladocera: Daphniidae), and examined its expression during embryogenesis. The amino acid sequence of the putative crustacean EPPase cDNA showed high similarity to insect EPPase and human suppressor of T-cell receptor signaling-1. We also found that the D. magna EPPase was highly expressed during early embryogenesis; its expression rapidly decreased 6 h after oviposition. This timing corresponds to the onset of organogenesis in D. magna. The expression of EPPase could not be detected in diapaused eggs. This is the first report of an EPPase from crustaceans, and the results suggest that the function of EPPase is conserved between insects and crustaceans.

Comparison between Real-Time PCR and Agarose Gel Electrophoresis for DNA Quantification / 대한진단검사의학회지

BACKGROUND: Real-time polymerase chain reaction (PCR) is generally regarded as a very accurate and time-saving method, but it is expensive to run. We evaluated the reliability of an inexpensive and a researcher-friendly gel electrophoresis-based PCR method for the quantification of mRNA, and the results were compared with those obtained by real-time PCR. METHODS: We compared the results of relative quantification for MMP-1 measured by real-time PCR and by ethidium bromide stained-agarose gel electrophoresis after end-point PCR. RESULTS: There was significant but very weak correlation between real-time PCR and end-point PCR for relative quantification of MMP-1 (r=0.16, P<0.01). CONCLUSIONS: Our results suggest that the use of the gel electrophoresis-based end-point PCR is inappropriate for quantifying mRNA. Therefore, in order to confirm the result of relative quantification by end-point PCR, the newly established real-time PCR method or northern hybridization should be applied.