Reverse Transcription Of mRNA Research Articles

Evaluation of Expression of WNT1 and PTCH Genes in Peripheral Blood of Patients with Odontogenic Cysts and Tumours of the Jaws by Quantitative RT-PCR: A Pilot Study.

Odontogenic lesions of the maxillofacial region constitute a complex group of lesions with diverse histopathologic types and clinical behaviour. Early diagnosis is important to minimize the need for radical surgery and to improve quality of life of the patients. Tumour markers play an essential role in the molecular level understanding of Odontogenic lesions and also used for early diagnosis and target therapies which improves the quality of life of the patients. Patched, a tumour suppressor gene encodes the transmembrane protein PTCH and is a receptor for the morphogen Sonic Hedgehog. It is evident that PTCH gene mutations occur in odontogenic keratocysts and the Hedgehog signalling pathway has an important role during tooth formation. WNT 1 is a key signal molecule that controls cell growth and proliferation. WNT pathway abnormalities are reported to induce tumour occurrence. Hence, my study was to determine the presence of WNT1 and PTCH in peripheral blood of patients with Odontogenic lesions using quantitative RT-PCR. In this cross-sectional study, two groups were included: Group 1-blood samples from 8 individuals with odontogenic cysts and tumours, and Group 2-blood samples of 8 individuals without Odontogenic lesions. 2ml of blood sample was collected from radial veins into PAX gene tubes containing RNA stabilizing agent and stored at a temperature of 2 to 4 degrees and transported to Enable Biolabs India Pvt Ltd., Chennai. PAX gene tubes were subjected to centrifugation at 8000rpm to separate plasma fraction. Reverse transcription of mRNA was performed using miScript II RT Kit (Cat#218161, Qiagen, Germany) to synthesize cDNA. GAPDH house-keeping gene used as control. The study group had 3 males and 5 females (n = 8) with a mean age group of 32.6years and the control group had 2 males and 6 females (n = 8) with mean age of 35.2years. Group I (study group) showed 37.5% positive expression of WNT1 gene with a p value of 0.055 (p > 0.05) and 50% positive expression of PTCH with a p value of 0.021 (p < 0.05) (Figs. 3 and 4) which was statistically significant when compared with control group. Group II (control group) showed 100% negative expression for WNT1 and PTCH genes. WNT1 and PTCH genes were expressed in peripheral blood of patients with odontogenic lesions. WNT1 and PTCH genes may be potential predictors in individuals who would develop odontogenic lesions. Further studies on expression of WNT1 and PTCH genes with larger number of samples might give a future scope for target therapy in odontogenic lesions.

Discovery of non-reference processed pseudogenes in the Swedish population.

The vast majority of the human genome is non-coding. There is a diversity of non-coding features, some of which have functional importance. Although the non-coding regions constitute the majority of the genome, they remain understudied, and for a long time, these regions have been referred to as junk DNA. Pseudogenes are one of these features. A pseudogene is a non-functional copy of a protein-coding gene. Pseudogenes may arise through a variety of genetic mechanisms. Processed pseudogenes are formed through reverse transcription of mRNA by LINE elements, after which the cDNA is integrated into the genome. Processed pseudogenes are known to be variable across populations; however, the variability and distribution remains unknown. Herein, we apply a custom-designed processed pseudogene pipeline on the whole genome sequencing data of 3,500 individuals; 2,500 individuals from the thousand genomes dataset, as well as 1,000 Swedish individuals. Through these analyses, we discover over 3,000 pseudogenes missing from the GRCh38 reference. Utilising our pipeline, we position 74% of the detected processed pseudogenes-allowing for analyses of formation. Notably, we find that common structural variant callers, such as Delly, classify the processed pseudogenes as deletion events, which are later predicted to be truncating variants. By compiling lists of non-reference processed pseudogenes and their frequencies, we find a great variability of pseudogenes; indicating that non-reference processed pseudogenes may be useful for DNA testing and as population-specific markers. In summary, our findings highlight a great diversity of processed pseudogenes, that processed pseudogenes are actively formed in the human genome; and that our pipeline may be used to reduce false positive structural variation caused by the misalignment and subsequent misclassification of non-reference processed pseudogenes.

464 Nanopore Cas9-targeted sequencing as a tool for identifying revertant mosaicism in the skin

Revertant mosaicism (RM) is a phenomenon in which germline mutations are spontaneously corrected in tissues. RM skin is often observed in genodermatoses, including epidermolysis bullosa (EB). However, the rigorous validation of RM in the skin has been technically challenging, especially when homologous recombination (HR) causes RM. Recently developed nanopore Cas9-targeted sequencing (nCATS) enables the enrichment and sequencing of specific regions (up to a few tens of kb) on gDNA without PCR amplification or the reverse transcription of mRNA.

Construction and identification of an immortalized rat intestinal smooth muscle cell line.

Although intestinal smooth muscle cells (ISMCs) play an important role in the remodeling of the intestinal structure, considerably less is known about the molecular mechanisms that mediate the development and growth of ISMCs. A possible reason for this lag is the lack of cell lines that recapitulate ISMCs in vivo. In this study, we separated the primary ISMCs from the rat intestinal tract and integrated the Simian Vacuolating virus 40 Large T antigen (SV40 LT) gene into the genome of the primary ISMC to construct an immortalized cell line named ISMC-Hc. ISMC-Hc proliferated persistently without any signs of senescence up to 50 passages and without neoplasticity. Analysis of the genome isolated from ISMC-Hc confirmed that the SV40LT gene recombined in the genome, and mRNA reverse transcription PCR suggested that SV40LT could be expressed normally. In addition, ISMC-Hc had few morphological differences compared with the primary ISMC. Furthermore, ISMC-Hc showed the expression of the specific protein markers (alpha-smooth muscle actin and desmin) through immunofluorescence analysis. Further studies showed that ISMC-Hc had enhanced contractility and expressed the glial-derived neurotrophic factor, nerve growth factor, ciliary neurotrophic factor, and leukemia inhibitory factor after co-stimulation with IL-1 beta and TNF-alpha. ISMC-Hc showed characteristics similar to that of primary ISMC and can be used as an in vitro model to explore the cellular and molecular mechanisms of ISMC. Additionally, the immortalized ISMCs could help investigate the basic functional mechanisms of intestinal diseases.

Internal oligo(dT) priming introduces systematic bias in bulk and single-cell RNA sequencing count data.

Significant advances in RNA sequencing have been recently made possible by using oligo(dT) primers for simultaneous mRNA enrichment and reverse transcription priming. The associated increase in efficiency has enabled more economical bulk RNA sequencing methods and the advent of high-throughput single-cell RNA sequencing, already one of the most widely adopted methods in transcriptomics. However, the effects of off-target oligo(dT) priming on gene expression quantification have not been appreciated. In the present study, we describe the extent, the possible causes, and the consequences of internal oligo(dT) priming across multiple public datasets obtained from various bulk and single-cell RNA sequencing platforms. To explore and address this issue, we developed a computational algorithm for RNA counting methods, which identifies the sequencing read alignments that likely resulted from internal oligo(dT) priming and removes them from the data. Directly comparing filtered datasets to those obtained by an alternative method reveals significant improvements in gene expression measurement. Finally, we infer a list of human genes whose expression quantification is most likely to be affected by internal oligo(dT) priming and predict that when measured using these methods, the expression of most genes may be inflated by at least 10% whereby some genes are affected more than others.

Global Identification of Co-Translational Interaction Networks by Selective Ribosome Profiling.

In recent years, it has become evident that ribosomes not only decode our mRNA but also guide the emergence of the polypeptide chain into the crowded cellular environment. Ribosomes provide the platform for spatially and kinetically controlled binding of membrane-targeting factors, modifying enzymes, and folding chaperones. Even the assembly into high-order oligomeric complexes, as well as protein-protein network formation steps, were recently discovered to be coordinated with synthesis. Here, we describe Selective Ribosome Profiling, a method developed to capture co-translational interactions in vivo. We will detail the various affinity purification steps required for capturing ribosome-nascent-chain complexes together with co-translational interactors, as well as the mRNA extraction, size exclusion, reverse transcription, deep-sequencing, and big-data analysis steps, required to decipher co-translational interactions in near-codon resolution.

EGFR transcription in non-small-cell lung cancer tumours can be revealed in ctDNA by cell-free chromatin immunoprecipitation (cfChIP).

Determination of tumour‐specific transcription based on liquid biopsies possesses a large diagnostic and prognostic potential in non‐small cell lung cancer (NSCLC). Cell‐free DNA (cfDNA) packed in nucleosomes mirrors the histone modification profiles present in the cells of origin. H3 lysine 36 trimethylation (H3K36me3)‐modified nucleosomes are associated with active genes, and therefore, cell‐free chromatin immunoprecipitation (cfChIP) of H3K36me3‐associated cfDNA has the potential to delineate whether transcription of a particular gene is occurring in the cells from which its cfDNA originates. We hypothesized that cfChIP can delineate transcriptional status of genes harbouring somatic cancer mutations and analysed the recurrently observed EGFR‐L858R mutation as an example. In representative NSCLC cell lines, the relationship between wild‐type (WT) and mutated EGFR transcriptional activity and mRNA expression levels was analysed using H3K36me3 ChIP and EGFR mRNA reverse transcription quantitative PCR (RT‐qPCR), respectively. The ChIP analysis showed that both WT and mutated EGFR are transcribed and that mRNA is similarly expressed per EGFR copy. Based on this observation, we proceeded with EGFR cfChIP using blood plasma from NSCLC patients harbouring the EGFR‐L858R mutation. EGFR‐WT fragments can originate from both nontumour cells with no or low EGFR transcription and tumour cells with active EGFR transcription, whereas EGFR‐L858R fragments must specifically originate from tumour cells. H3K36me3 cfChIP followed by droplet digital PCR (ddPCR) revealed significantly higher enrichment of EGFR‐L858R compared to EGFR‐WT fragments. This is in alignment with EGFR‐L858R being actively transcribed in the NSCLC tumour cells. This study is proof‐of‐principle that cfChIP can be used to identify tumour‐specific transcriptional activity of mutated alleles, which can expand the utility of liquid biopsy‐based cfDNA analyses to enhance tumour diagnostics and therapeutics.

Genome-wide analysis of European sea bass provides insights into the evolution and functions of single-exon genes.

Several studies have attempted to understand the origin and evolution of single‐exon genes (SEGs) in eukaryotic organisms, including fishes, but few have examined the functional and evolutionary relationships between SEGs and multiple‐exon gene (MEG) paralogs, in particular the conservation of promoter regions. Given that SEGs originate via the reverse transcription of mRNA from a “parental” MEGs, such comparisons may enable identifying evolutionarily‐related SEG/MEG paralogs, which might fulfill equivalent physiological functions. Here, the relationship of SEG proportion with MEG count, gene density, intron count, and chromosome size was assessed for the genome of the European sea bass, Dicentrarchus labrax. Then, SEGs with an MEG parent were identified, and promoter sequences of SEG/MEG paralogs were compared, to identify highly conserved functional motifs. The results revealed a total count of 1,585 (8.3% of total genes) SEGs in the European sea bass genome, which was correlated with MEG count but not with gene density. The significant correlation of SEG content with the number of MEGs suggests that SEGs were continuously and independently generated over evolutionary time following species divergence through retrotranscription events, followed by tandem duplications. Functional annotation showed that the majority of SEGs are functional, as is evident from their expression in RNA‐seq data used to support homology‐based genome annotation. Differences in 5′UTR and 3′UTR lengths between SEG/MEG paralogs observed in this study may contribute to gene expression divergence between them and therefore lead to the emergence of new SEG functions. The comparison of nonsynonymous to synonymous changes (Ka/Ks) between SEG/MEG parents showed that 74 of them are under positive selection (Ka/Ks > 1; p = .0447). An additional fifteen SEGs with an MEG parent have a common promoter, which implies that they are under the influence of common regulatory networks.

Semi-Quantitative RT-PCR: An Effective Method to Explore the Regulation of Gene Transcription Level Affected by Environmental Pollutants.

Semi-quantitative reverse transcription and polymerase chain reaction (sqRT-PCR) is a simple and specific method for quantitative RNA in recent years. The relative quantity of a specific mRNA in the samples can be inferred by reverse transcription of mRNA into cDNA, and PCR amplification and determination of the quantity of PCR products. The semi-quantitative analysis is carried out under a fixed number of PCR cycles, and the total RNA concentration is kept in the exponential phase of the PCR. The method is to use a housekeeping gene (usually actin, GAPDH, and EF1α) as a reference standard in treated and control organisms to observe the expression of the interested genes (upregulated or downregulated) in toxicology. In this chapter, we describe a step-by-step method for determining the differential regulation of target genes in organisms exposed to environmental pollutants.

Follow-up Study on the Outcomes of Recovered Pregnant Women with a History of COVID-19 in the First and Second Trimesters: A Case Series from China.

Objective:To determine the pregnancy and neonatal outcomes of women who recovered from coronavirus disease 2019 (COVID-19) that developed in early pregnancy.Methods:This case series analyzed five pregnant women (26–33 years) whom recovered from COVID-19 which were developed in early pregnancy (6–27 weeks) and admitted at the Wuhan Union Hospital from January 15, 2020 to April 30, 2020. The clinical manifestation, laboratory examinations, treatment, pregnancy outcomes, maternal and neonatal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) throat swab reverse transcription polymerase chain reaction test results, and SARS-CoV-2 antibody test results in neonates were reviewed. The placental pathology, placental angiotensin-converting enzyme 2 expression were studied by hematoxylin-eosin and immunohistochemistry staining, SARS-CoV-2 presence was examined by QT-PCR. We also followed up the infants at 3–6 months.Results:Three pregnant women were diagnosed with COVID-19 in early pregnancy (Cases 1–3), and two were serum immunoglobulin G positive asymptomatic cases (Cases 4 and 5). Cases 1–3 showed complete recovery after severe COVID-19. Case 3 was infected at 6 weeks of gestation during the first trimester and had induced medical abortion at 12 weeks of gestation. All neonates had no pneumonia, SARS-CoV-2 mRNA reverse transcription polymerase chain reaction and serum immunoglobulin M were negative, and immunoglobulin G were positive. All placental samples were negative for SARS-CoV-2 in the nucleic acid test. Placental pathology showed chronic ischemia changes. ACE-2 expressed in both placenta and decidua. The follow-up showed that the infants were healthy and asymptomatic at 3–6 months.Conclusion:No adverse outcomes was observed in our case series. However, systemic inflammatory responses to SARS-CoV-2 infection may cause placental injury. At the time of delivery after recovery from COVID-19, no SARS-CoV-2 positive results was found in the placenta in this case series.

Abstract B44: An optimized method of multimodal mRNA and gDNA isolation from low biomass input

Abstract Background: In cell-based applications in liquid biopsy research (circulating tumor cells [CTC], fetal cells, endothelial cells), often transcriptomes get analyzed. Here and in single-cell research there is a requirement to also investigate genomic variants. Therefore, a multimodal sample preparation for both gDNA and RNA (preferably mRNA) is increasingly needed in the market. There are several combined RNA/DNA preparation solutions already available with the following caveats: Optimizing DNA yield and quality is often achieved under the compromise of accepting losses in RNA yield and/or quality or vice versa. This may be acceptable for larger sample inputs but is critical for low-biomass samples. The goal of this project, therefore, was to develop an optimized AllPrep solution for low-biomass samples that provides both highest mRNA and gDNA quality and yield without any compromises. Material and Methods: As starting material for the mRNA purification from low-biomass input samples, the AdnaTest EMT 2/StemCell Select/Detect procedure was used. The procedure has already been tested widely to get highest yields of mRNA from as few as 2 cells spiked into 5 mL of blood. Cells captured with immunomagnetic beads as provided in the AdnaTest CTC-Select procedure were lysed using the AdnaTest Lysis/Binding Buffer and mRNA was purified using Oligo(dT)25 beads. After mRNA depletion cell lysates still contained gDNA, which can be isolated using magnetic affinity binding beads in an optimized workflow. mRNA recovery was demonstrated by reverse transcription and subsequent endpoint PCR. gDNA was analyzed using PCR and pyrosequencing as downstream applications. Results: It has been shown that the optimized workflow combination allowed the isolation of mRNA and gDNA from as few as 5 tumor cells spiked into 5 mL of blood. Tumor cell-related gene expression profiles can be analyzed after reverse transcription of mRNA and multiplex PCR. In parallel, the recovered gDNA was successfully used in PCR assays as well as for detection of known gene variants by means of pyrosequencing. Furthermore, gel electrophoretic analysis of the gDNA eluates showed good gDNA integrity with mean molecular weight values of &amp;gt;60 kbp. Citation Format: Jens van de Flierdt, Markus Sprenger-Haussels, Michael Otte, Siegfried Hauch. An optimized method of multimodal mRNA and gDNA isolation from low biomass input [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B44.

Involvement of transposable elements in neurogenesis.

The article is about the role of transposons in the regulation of functioning of neuronal stem cells and mature neurons of the human brain. Starting from the first division of the zygote, embryonic development is governed by regular activations of transposable elements, which are necessary for the sequential regulation of the expression of genes specific for each cell type. These processes include differentiation of neuronal stem cells, which requires the finest tuning of expression of neuron genes in various regions of the brain. Therefore, in the hippocampus, the center of human neurogenesis, the highest transposon activity has been identified, which causes somatic mosaicism of cells during the formation of specific brain structures. Similar data were obtained in studies on experimental animals. Mobile genetic elements are the most important sources of long non-coding RNAs that are coexpressed with important brain protein-coding genes. Significant activity of long non-coding RNA was detected in the hippocampus, which confirms the role of transposons in the regulation of brain function. MicroRNAs, many of which arise from transposon transcripts, also play an important role in regulating the differentiation of neuronal stem cells. Therefore, transposons, through their own processed transcripts, take an active part in the epigenetic regulation of differentiation of neurons. The global regulatory role of transposons in the human brain is due to the emergence of protein-coding genes in evolution by their exonization, duplication and domestication. These genes are involved in an epigenetic regulatory network with the participation of transposons, since they contain nucleotide sequences complementary to miRNA and long non-coding RNA formed from transposons. In the memory formation, the role of the exchange of virus-like mRNA with the help of the Arc protein of endogenous retroviruses HERV between neurons has been revealed. A possible mechanism for the implementation of this mechanism may be reverse transcription of mRNA and site-specific insertion into the genome with a regulatory effect on the genes involved in the memory.

In Situ Sequencing: A High-Throughput, Multi-Targeted Gene Expression Profiling Technique for Cell Typing in Tissue Sections.

Recent advances of image-based in situ mRNA quantification methods allow to visualize where in a tissue section a set of genes is expressed. It enables to map large numbers of genes in parallel and by capturing cellular boundaries allows to assign genes to cells. Here, we present a high-throughput, multi-targeted gene expression profiling technique called in situ sequencing that is capable of localizing hundreds of genes simultaneously and supports cell type classifications that follow transcriptome-based taxonomy. In situ sequencing is a targeted, amplified, and barcoded approach using padlock probes (PLPs) and rolling circle amplification (RCA). The current protocol relies on mRNA fixation, mRNA reverse transcription, residual mRNA degradation, and PLP hybridization. PLPs are amplified by RCA and labeled with fluorophore-conjugated probes, allowing their detection under conventional fluorescence microscopes.

Amplification of cDNA Generated by Reverse Transcription of mRNA: Two-Step Reverse Transcription-Polymerase Chain Reaction (RT-PCR).

Reverse transcription-polymerase chain reaction (RT-PCR) is a powerful method to detect and synthesize cDNA copies of low-copy-number mRNAs. Two enzymes are used: reverse transcriptase to produce single-stranded cDNA copies, which are then used as templates in an amplification reaction catalyzed by a thermostable DNA polymerase. For this reason, the method is known as "two-step RT-PCR." This protocol describes the traditional method of RT-PCR in which the two synthetic reactions are performed separately and sequentially.

Taking the ‘Secret’ out of secretions: evaluation of the ParaDNA body fluid ID system at NSWHP FASS

ABSTRACTThe ParaDNA Body Fluid ID (BFID) system has been developed for use as either a screening or confirmatory test for the simultaneous identification of six biological fluids: saliva, vaginal fluid, seminal fluid, spermatozoa, peripheral blood and menstrual blood. The ParaDNA BFID kit consists of a closed, single-tube assay, incorporating multiplexed reverse transcription of mRNA and cDNA amplification, followed by fluorescent melt curve analysis with LGC’s proprietary HyBeacon DNA Probe technology. The ParaDNA instrument and early access version of the BFID kit were evaluated in this study. The evaluation included an assessment of the system’s useability, sensitivity, substrate specificity, and performance with mixtures and mock forensic samples. The ParaDNA BFID system was user-friendly, and capable of detecting most body fluids at neat concentrations. However, the system was unable to detect diluted fluids. The system’s capacity to detect saliva was also limited. Performance of the assay with different substrates was most influenced by the effectiveness of the ParaDNA sample collection tool. The tool sampled well from dried stains on fabrics, but was less effective at sampling from rayon swabs and other absorbent substrates. The system is capable of detecting fluids in mixtures, although detection appeared dependent on the combination of fluids tested.

Altered Expression Profiling of Spinal Genes Modulated by Compound 48/80 in A Mouse Itch Model

Background: The molecular mechanisms underlying itch are constantly being studied to create new opportunities to prevent or alleviate itch. The aim of our study was to determine the spinal gene expression changes induced by compound 48/80 in a mouse itch model.Methods: Mice were divided into saline group (control group, n=12) and compound 48/80 group (48/80 group, n=12). 100 µl saline or compound 48/80 was microinjected intradermally in the nape of the neck. After injection, pruritic behavior was immediately measured every 5 minutes. 30 minutes after injection, tissue was prepared to carry out mRNA profiling microarray and reverse transcription polymerase chain reaction (RT-qPCR) analyses.Results: The total numbers of scratching bout after compound 48/80 injection (100 µg/100 µl) were significantly increased in 48/80 group (190±11.33) as compared with control group (6.83±1.17). We screened the dorsal part of the cervical spinal cord of the mouse itch model for differentially expressed genes. Out of 45037 studied transcripts, the abundance levels of 15 transcripts were altered following compound 48/80 injection. 9 and 6 genes were up- and down-regulated in 48/80 group, respectively. We validated the reliability of the microarray results by RT-qPCR, and found 6 up-regulated mRNA, including Sgk1, Bag4, Fos, Ehd2, Edn3 and Wdfy, were significantly increased, whereas 3 down-regulated mRNA, including Corin, 4921511E18Rik and 4930423020Rik, were significantly decreased.Conclusions: These findings indicate that the alterations of spinal gene expression are involved in acute itch, and provide a translational bridge for spinal drugs targeting their signaling pathway to prevent or alleviate itch. Citation: Zhi-Gang He, Bao-Wen Liu, Zhi-Xiao Li, Cheng Liu, Hong-Bing Xiang. Altered expression profiling of spinal genes modulated by compound 48/80 in a mouse itch model. J Anesth Perioper Med 2017; 4: 220-4. doi:10.24015/JAPM.2017.0020This is an open-access article, published by Evidence Based Communications (EBC). This work is licensed under the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium or format for any lawful purpose. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Lab-on-a-chip mRNA purification and reverse transcription via a solid-phase gene extraction technique.

Extraction and purification of high quality RNA is a crucial initial step required for a variety of genomic assays. We report a solid phase gene extraction (SPGE) method for automated extraction, purification and reverse transcription of mRNA in a microfluidic device. This is performed using a 130 μm diameter stainless steel needle that is amino-linked to dT(15) oligonucleotides for selective hybridization of mRNA. By inserting this probe into the biological sample for only 30 seconds, mRNA is captured with high selectivity and a yield greater than 10 pg per mm of probe length. The probe is then inserted into a lab-on-a-chip device, where the bound poly-adenylated RNA is thermally released and immediately reverse transcribed for subsequent PCR amplification. The insertion of the probe into the microfluidic device is straightforward: the microchannel is formed with an elastomer (PDMS) that, when punctured, will seal around the probe. The specificity and RNA loading capacity of the probes were evaluated using conventional qPCR. This procedure was successfully used to extract, purify, and transcribe mRNA from rat glioblastoma cell spheroids in less than seven minutes. Analysis of the product confirmed that the SPGE technique selectively captures and inherently purifies high-quality mRNA directly from biological material with no need for additional pre-processing steps. Integrating this elegant sample preparation method into a complete lab-on-a-chip system will substantially enhance the speed and automation of mRNA assays for research and clinical diagnostics.

P3.02a-010 Evaluation of Aberrant ALK Expression in Lung Cancer by RT-PCR and Comparison with FISH and Immunohistochemistry: Topic: ALK Biomarkers

In advanced lung cancer patients the gold standard for detecting ALK gene rearrangements is fluorescence in situ hybridization (FISH), and ALK protein expression can be also evaluated by immunohistochemistry (IHC). A single analysis performed alone may not detect all the ALK-positive cases and some patients with discordant FISH and IHC respond to tyrosine kinase inhibitors (TKIs). In this study we evaluated ALK aberrant expression in lung cancer patients by a reverse-transcription (RT)-PCR, to investigate its clinical utility and its concordance with FISH and IHC. ALK aberrant expression was retrospectively investigated on RNA from formalin-fixed paraffin-embedded tissue (FFPEt) of 24 advanced lung adenocarcinoma patients, previously evaluated by FISH and IHC. We used a one-step Scorpion RT-PCR that allowed in a single reaction either the mRNA reverse transcription and the cDNA amplification for ALK kinase-domain, normally not expressed, and a control gene, to assess RNA quality. Results are reported in Table 1. Despite the instability of mRNA from FFPEt, only 2 samples resulted inadequate for RT-PCR. RT-PCR was in disagreement with both FISH and IHC in one case, which is likely to be a RT-PCR false positive. RT-PCR did not detect ALK aberrant expression in a FISH positive case, which was negative also by IHC; unfortunately, this patient died after a cycle of pemetrexed therapy, before undergoing a second line TKI treatment. The presence of ALK rearrangements does not necessarily imply increased protein levels, because of the complex transcriptional and post-transcriptional regulations, so further analysis at RNA levels may clarify discrepancy between FISH and IHC allowing a better stratification of patients who could benefit from TKIs. Therefore, according to our results the RT-PCR evaluating ALK aberrant expression regardless of the fusion partners should be considered for introduction into routine ALK testing in lung cancer.

A microfluidic multiwell chip for enzyme-free detection of mRNA from few cells

Isogenic cell populations possess heterogeneous gene expression patterns. Most methods for mRNA expression analysis start with the reverse transcription of mRNA into cDNA, a process that can introduce strong signal variations not related to the actual mRNA levels. Miniaturized lab-on-a-chip systems offer properties – e.g. low sample dilution, low contamination – that enable new reaction schemes for molecular analyses. To enable transcription-free mRNA expression analysis of few single cells, a one-step cell lysis, target labelling and hybridisation approach as well as a corresponding passive multiwell chip with a volume of 25.5 nL/well were developed. The method enabled the parallel analysis of up to 96 samples and 6 target genes per sample. Preceding light microscopy of the living cells allowed correlating mRNA levels and cell number. As a proof-of-principle, the pancreatic cancer cell line Panc-1 was investigated for expression heterogeneity of a reference gene plus 5 genes reported to be overexpressed in cancer stem cells (CSCs). A good correlation (r(51)=0.739, p<0.001; rs(51)=0.744, p<0.001) between the cell number per well and the number of detected reference gene mRNA confirmed the proper function of the device. Moreover, a heterogeneous expression of the CSC-associated target genes was found which matched well with reports on the presence of CSCs in the Panc-1 cell line.

The YY1/MMP2 axis promotes trophoblast invasion at the maternal-fetal interface.

YY1 is a sequence‐specific DNA‐binding transcription factor that has many important biological roles. However, its function in trophoblasts at the maternal–fetal interface remains to be elucidated. In this study, we used an mRNA microarray and reverse transcription qPCR and compared the YY1 mRNA expression level in trophoblasts between patients with recurrent miscarriage (RM) and healthy control subjects. Our results revealed that YY1 mRNA expression was significantly lower in the trophoblasts of the RM group compared with the healthy control group. Furthermore, immunofluorescence and immunohistochemical data showed that YY1 was highly expressed in human placental villi during early pregnancy, especially in cytotrophoblast cells and invasive extravillous trophoblasts, and it was expressed at a much lower level in the placental villi of term pregnancy. YY1 overexpression enhanced, and knockdown repressed, the invasion and proliferation of trophoblasts. Antibody array screening revealed that YY1 significantly promoted MMP2 expression in trophoblasts. Bioinformatics analysis identified three YY1‐binding sites in the MMP2 promoter region, and chromatin immunoprecipitation analysis verified that YY1 binds directly to its promoter region. Importantly, inhibition of YY1 by siRNA clearly decreased trophoblast invasion in an ex vivo explant culture model. Overall, our findings revealed a new regulatory pathway of YY1/MMP2 in trophoblast cell invasion during early pregnancy and indicated that YY1 may be involved in the pathogenesis of RM. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.