Target mRNA Sequences Research Articles

Immune-related Gene Expression Predicts Response to Neoadjuvant Chemotherapy but not Additional Benefit from PD-L1 Inhibition in Women with Early Triple-negative Breast Cancer.

We evaluated mRNA signatures to predict response to neoadjuvant PD-L1 inhibition in combination with chemotherapy in early triple-negative breast cancer. Targeted mRNA sequencing of 2,559 transcripts was performed in formalin-fixed, paraffin-embedded samples from 162 patients of the GeparNuevo trial. We focused on validation of four predefined gene signatures and differential gene expression analyses for new predictive markers. Two signatures [GeparSixto signature (G6-Sig) and IFN signature (IFN-Sig)] were predictive for treatment response in a multivariate model including treatment arm [G6-Sig: OR, 1.558; 95% confidence interval (CI), 1.130-2.182; P = 0.008 and IFN-Sig: OR, 1.695; 95% CI, 1.234-2.376; P = 0.002), while the CYT metric predicted pathologic complete response (pCR) in the durvalumab arm, and the proliferation-associated gene signature in the placebo arm. Expression of PD-L1 mRNA was associated with better response in both arms, indicating that increased levels of PD-L1 are a general predictor of neoadjuvant therapy response. In an exploratory analysis, we identified seven genes that were higher expressed in responders in the durvalumab arm, but not the placebo arm: HLA-A, HLA-B, TAP1, GBP1, CXCL10, STAT1, and CD38. These genes were associated with cellular antigen processing and presentation and IFN signaling. Immune-associated signatures are associated with pCR after chemotherapy, but might be of limited use for the prediction of response to additional immune checkpoint blockade. Gene expressions related to antigen presentation and IFN signaling might be interesting candidates for further evaluation.

Identification of miR-802-5p and its involvement in type 2 diabetes mellitus.

MicroRNAs (miRNA) are recently discovered endogenous, small noncoding RNAs (of 22 nucleotides) that play pivotal roles in gene regulation. They are involved in post-transcriptional control of gene expression. miRNAs are emerging as important regulators of cell proliferation, development, cancer formation, stress responses, cell death and physiological conditions. Increasing evidence has demonstrated the human miRNAs bind to their target mRNA sequences with perfect or near-perfect sequence complementarily. This provides a powerful strategy for discovering potential type 2 diabetes mellitus (T2DM) targets and gives the probability to exploit them for diagnostic and therapeutic causes. About 6% of the world population is affected by T2DM, and it is recognized as a global epidemic by the World Health Organization. At present there is no valid biomarker to control or manage T2DM. Therefore, the present study applied a mature sequence of miRNAs from publicly accessible databases to identify the miRNA from T2DM expressed sequence tags, and the results are detailed and discussed below.

MIPDH: A Novel Computational Model for Predicting microRNA-mRNA Interactions by DeepWalk on a Heterogeneous Network.

Analysis of miRNA-target mRNA interaction (MTI) is of crucial significance in discovering new target candidates for miRNAs. However, the biological experiments for identifying MTIs have a high false positive rate and are high-priced, time-consuming, and arduous. It is an urgent task to develop effective computational approaches to enhance the investigation of miRNA-target mRNA relationships. In this study, a novel method called MIPDH is developed for miRNA–mRNA interaction prediction by using DeepWalk on a heterogeneous network. More specifically, MIPDH extracts two kinds of features, in which a biological behavior feature is learned using a network embedding algorithm on a constructed heterogeneous network derived from 17 kinds of associations among drug, disease, and 6 kinds of biomolecules, and the attribute feature is learned using the k-mer method on sequences of miRNAs and target mRNAs. Then, a random forest classifier is trained on the features combined with the biological behavior feature and attribute feature. When implementing a 5-fold cross-validation experiment, MIPDH achieved an average accuracy, sensitivity, specificity and AUC of 75.85, 74.37, 77.33%, and 0.8044, respectively. To further evaluate the performance of MIPDH, other classifiers and feature descriptors are conducted for comparisons. MIPDH can achieve a better performance. Additionally, case studies on hsa-miR-106b-5p, hsa-let-7d-5p, and hsa-let-7e-5p are also implemented. As a result, 14, 9, and 9 out of the top 15 targets that interacted with these miRNAs were verified using the experimental literature or other databases. All these prediction results indicate that MIPDH is an effective method for predicting miRNA-target mRNA interactions.

A smart deoxyribozyme-based fluorescent sensor for in vitro detection of androgen receptor mRNA.

Nowadays a variety of biosensors are widely used in different fields, including biomedical diagnostics and self-testing. Nucleic acid-based biosensors are typically applied to detect another nucleic acid, proteins, ions, and several other types of compounds. It is most promising to develop simple and effective biosensors for the use in situations where traditional methods are not available due to their complexity and laboriousness. In this project, a novel smart deoxyribozyme-based fluorescent sensor for the detection of androgen receptor mRNA was developed. It consists of several functional modules including two deoxyribozymes 10–23, an RNA-dependent split malachite green aptamer, and an oligonucleotide platform. Deoxyribozymes specifically release a 27-nucleotide RNA fragment that is readily available for the interaction with the aptamer module. This solves a problem of secondary structure in hybridization with the target sequence of full-length mRNA. It was shown that within 24 hours the proposed sensor specifically recognized both a synthetic 60-nucleotide RNA fragment (LOD is 1.4 nM of RNA fragment at 37 °C) and a full-sized mRNA molecule of the androgen receptor. The constructed sensor is easy to use, has high efficiency and selectivity for the RNA target, and can be reconstructed for the detection of various nucleic acid sequences due to its modular structure. Thus, similar biosensors may be useful for the differential diagnosis.

Silencing Immunity: miR159 Suppresses Pathogen Responses in Tobacco.

To ensure optimal growth and development, plants must precisely control gene expression networks in a tissue-specific manner. MicroRNAs (miRNAs) are a class of small noncoding RNA molecules that posttranscriptionally silence genes by binding to complementary sequences in target mRNAs. Many land

Role of MicroRNAs in Establishing Latency of Human Immunodeficiency Virus.

Acquired immunodeficiency syndrome (AIDS) emerged as an epidemic in Africa in 1981, and now it has become a most destructive global pandemic. Human immunodeficiency virus (HIV) is responsible for the pathogenesis of AIDS, and it is usually transmitted through unsafe sexual activities. HIV is a lentivirus that can remain latent in the host cells for a long period, and it has various mechanisms to establish latency. The HIV genome encodes several microRNAs (miRNA-TAR, miRNA-H1, miRNA-H3, and miRNA-Nef-367) that act as posttranscriptional control by targeting mRNA sequences. The miRNA-TAR, miRNA-Nef-367, and miRNA-H1 have established roles in HIV latency, whereas miRNA-H3 can activate the latent reservoirs of HIV. The human genome also encodes several miRNAs that have defensive roles against infections. Cellular miRNAs (miRNA-29a, miRNA-146a, miRNA-34c-5'p, miRNA-186, miRNA-210 and miRNA-222) also contribute to viral latency. The most challenging hurdle in the development of effective HIV therapeutics is viral latency. A complete understanding of latency will enable us to develop efficient therapeutics and to eradicate HIV from the globe.

Hepatitis B virus X protein upregulates alpha-fetoprotein to promote hepatocellular carcinoma by targeting miR-1236 and miR-329.

Hepatitis B virus (HBV) infection is the most common cause of hepatocellular carcinoma (HCC) worldwide, wherein the expression of alpha-fetoprotein (AFP) is reactivated to promote tumorgenesis. Hepatitis B virus X protein (HBx) protein encoded by the HBV virus X gene has been considered to be oncogenic and implicated in hepatocarcinogenesis. However, the relationship between HBx and abnormal AFP expression in HCC is yet to be fully understood. To explore the potential regulation of HBx on AFP re-expression in HCC, 97 HCC samples of different etiologies were analyzed, and extremely higher serum AFP levels were found in patients with HBsAg+ . Analyses of HBV-related HCC specimens showed that the expression of AFP was negatively correlated with the levels of miR-1236 and miR-329. Further analyses indicated that HBx promotes the expression of AFP by orchestrating the levels of miR-1236 and miR-329 both in vitro and in vivo. Specifically, miR-1236 and miR-329 bind to the potential target sequences in AFP mRNA 3'-untranslated region to suppress its expression. HBx transfection resulted in the significant decrement of these microRNAs and increment of AFP expression. Moreover, AFP promotes the proliferation of hepatoma cells and attenuates the proapoptotic effect of chemotherapy agents. These findings revealed a novel regulatory mechanism of HBx on the abnormal AFP expression in HCC, which may provide a therapeutic approach for combating HBV-related HCC by targeting the regulation of AFP expression.

Regulatory roles of small RNAs in prokaryotes: parallels and contrast with eukaryotic miRNA

Small non-coding RNAs continue to be identified that regulate the processes of translation and transcription in prokaryotes. A variety of regulatory mechanisms have been characterized by these regulatory RNAs that occur by complementary base pairing between the small regulatory RNA (sreRNA) and a target mRNA, including transcription attenuation, translation inhibition, translation activation, and mRNA protection. Here, we discuss the description of these mechanisms, and the key components that contribute to the interactions between the sreRNA and the target mRNA. Additionally, we classify sreRNAs into categories based on their origins. Antisense RNA (asRNA) is defined strictly as cis-encoded, trans-acting regulatory RNA, while small RNA (sRNA) is strictly defined as trans-encoded, trans-acting regulatory RNA. Although both RNAs bind the target mRNA by Watson-Crick base pairing to the complementary sequence of mRNA, sRNA binding typically requires the presence of a chaperone protein, is only partially complementary to the target mRNA, and often targets multiple mRNAs. Therefore, we characterize the mechanism of sRNA as similar to the well characterized eukaryotic miRNA and discuss the parallels and differences between the two. The binding of asRNA to its target mRNA, by contrast, is typically independent of a chaperone protein, is completely or almost completely complementary to the mRNA target sequence and targets only a single mRNA. While this categorization is likely to evolve as the research identifies more relevant and distinguishing characteristics to classify sreRNAs, the classification used here of prokaryotic sreRNAs should lead to a more refined approach to the discussion and investigation of regulatory RNAs until then.

The Regulatory Role of MicroRNAs in Breast Cancer.

MicroRNAs (miRNAs) are small non-coding RNA molecules which function as critical post-transcriptional gene regulators of various biological functions. Generally, miRNAs negatively regulate gene expression by binding to their selective messenger RNAs (mRNAs), thereby leading to either mRNA degradation or translational repression, depending on the degree of complementarity with target mRNA sequences. Aberrant expression of these miRNAs has been linked etiologically with various human diseases including breast cancer. Different cellular pathways of breast cancer development such as cell proliferation, apoptotic response, metastasis, cancer recurrence and chemoresistance are regulated by either the oncogenic miRNA (oncomiR) or tumor suppressor miRNA (tsmiR). In this review, we highlight the current state of research into miRNA involved in breast cancer, with particular attention to articles published between the years 2000 to 2019, using detailed searches of the databases PubMed, Google Scholar, and Scopus. The post-transcriptional gene regulatory roles of various dysregulated miRNAs in breast cancer and their potential as therapeutic targets are also discussed.

Non-invasive Potential Circulating mRNA Markers for Colorectal Adenoma Using Targeted Sequencing

We have developed an optimized protocol for plasma targeted mRNA sequencing in our previous study. Here, we performed plasma targeted mRNA sequencing for 40 colorectal adenoma patients and 39 colonoscopy-proven normal controls in order to find potential circulating mRNA markers for colorectal adenoma. Results showed that GSK3A and RHOA were differential expressed genes identified by a cut-off of fold change >2 and adjusted P value < 0.05. More detailed analysis showed that the expression of both GSK3A (0.01-fold with adjusted P < 1 × 10−6) and RHOA (0.35-fold with adjusted P < 0.01) in adenoma patients was significantly lower than those in normal healthy subjects. Based on the enrichment analysis of biological process for potential markers, we found that the regulation of programmed cell death (GO: 0043067; GO: 0043069), regulation of cell death (GO: 0010941; GO: 0060548) and cell differentiation (GO: 0021861) were the main processes involved in adenoma formation. In summary, this study is a cutting-edge research on the detection of plasma mRNA in colorectal adenoma patients and normal healthy subjects.

Abstract 2795: Dissemination score of CD8+ TILs by automated image analysis is a potential marker of immune activity in human cancers

Abstract The overall density of CD8+ tumor-infiltrating lymphocytes (TILs) is important for characterizing the level of immune activity in the tumor microenvironment (TME). Beyond the densities of CD8+ TILs, both their location and distributional patterns may also have relevance to immune activity. We evaluated 645 resected tumors encompassing seven cancer types, and correlate location and spatial patterns of CD8+ TILs to immune pathway activity. We integrated image analysis results from digitized immunohistochemistry (IHC) slides with gene expression data from a targeted Ion Torrent Panel. Overall density of CD8+ TILs and the exact position of individual CD8+ lymphocytes were determined from IHC slides. A dissemination score was defined as ratio of global density and average local density of CD8+ TILs. This score is the inverse of the Ripley’s K statistic and becomes high for disseminated spatial patterns. We used this quotient as a continuous metric to identify tumors with a disseminated TIL pattern and to distinguish them from tumors with focal distribution of CD8+ TILs. Within a subset of tumors, the continuous dissemination metric was correlated with biological pathways using targeted mRNA sequencing and gene set enrichment analysis. In addition, association of the dissemination score with overall survival was tested on a subset of cases. CD8+ TIL distributional patterns differed significantly between tumor types. Breast and pancreatic cancers more frequently showed a focal distribution of CD8+ TILs, while lung tumors comparatively exhibited a disseminated pattern. Transcriptional profiling data revealed differences between both image analysis phenotypes. On average, cases with more disseminated patterns of CD8+ T cells were associated with mRNA expression of genes that fall in pathways related to motility, migration and activation status of tumor infiltrating T cells. We also found a trend to better overall survival in patients whose tumors had a disseminated TIL score compared to those with a focal pattern. This trend was significant in non-small cell adenocarcinoma of the lung (log rank p = 0.018). We demonstrate the value of spatial image analysis to automatically score CD8+ TIL dissemination as a marker of immune activity in the TME. Jointly analyzing transcriptional profiles appears to identify a biologically meaningful activation phenotype in tumors with high dissemination scores. Our data further suggests that this phenotype is associated with improved overall survival in some cancer patients. Citation Format: Stefan Bentink, Andreas Spitzmueller, Tze Heng Tan, Hadassah Sade, Song Wu, Brandon W. Higgs, Keith E. Steele. Dissemination score of CD8+ TILs by automated image analysis is a potential marker of immune activity in human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2795.

MiR-148a-3p inhibits alpaca melanocyte pigmentation by targeting MITF

microRNAs (miRNAs) are well established as post-transcriptional regulators of gene expression, and function by binding regulatory regions, such as the 3′ untranslated region (UTR) of their target mRNA sequences. They have been implicated in the regulation of pigmentation, including the determination of alpaca hair-fiber color; however, the mechanisms underlying their effects are not yet well understood. Thus, the present study investigated whether the miRNA miR-148a-3p regulates the expression of the micropthalmia-associated transcription factor (MITF), since MITF is known to control the expression of key genes linked to coat color. The results of the conducted analyses showed that overexpressing miR-148a-3p in alpaca melanocytes caused the expression of MITF, pigmentation-associated protein tyrosinase (TYR), and both tyrosinase-related proteins 1 and 2 (TYRP1 and TYRP2, respectively) to be reduced, and thereby led to a decrease in the overall melanin content of the analyzed cells. Thus, the present study supports that miR-148a-3p is likely an important regulator of MITF expression.

MicroRNA-143/Musashi-2/KRAS cascade contributes positively to carcinogenesis in human bladder cancer.

It has been well established that microRNA (miR)‐143 is downregulated in human bladder cancer (BC). Recent precision medicine has shown that mutations in BC are frequently observed in FGFR3, RAS and PIK3CA genes, all of which correlate with RAS signaling networks. We have previously shown that miR‐143 suppresses cell growth by inhibiting RAS signaling networks in several cancers including BC. In the present study, we showed that synthetic miR‐143 negatively regulated the RNA‐binding protein Musashi‐2 (MSI2) in BC cell lines. MSI2 is an RNA‐binding protein that regulates the stability of certain mRNAs and their translation by binding to the target sequences of the mRNAs. Of note, the present study clarified that MSI2 positively regulated KRAS expression through directly binding to the target sequence of KRAS mRNA and promoting its translation, thus contributing to the maintenance of KRAS expression. Thus, miR‐143 silenced KRAS and MSI2, which further downregulated KRAS expression through perturbation of the MSI2/KRAS cascade.

In silico identification of microRNAs and their target genes in watermelon (Citrullus lanatus)

Watermelon (Citrullus lanatus) is an annual herbaceous crop, mainly grown for its fleshy fruit, which is an excellent source of vitamins and therefore fetches a huge market demand all over the world. Yield of watermelon can be improved by increasing the productivity that relies on plant growth, development and stress response. MicroRNAs (miRNAs), a group of short (∼22 nt), noncoding, endogenous and evolutionary conserved post-transcriptional regulatory RNAs, play a major role in various biological and metabolic processes associated with growth, development and stress response. In this study, we deployed a comparative genomics-based approach using expressed sequence tags (ESTs) to identify and characterize potential miRNAs and their target genes in watermelon. Three potential miRNAs (cla-miR-2515, cla-miR482d and cla-miR-8461) were predicted from 17,594 publicly available watermelon EST sequences, according to a series of filtering criteria used in the protocol. Moreover, 21 potential mRNA targets encoding for 19 different proteins linked to various metabolic processes such as inositol-3-phosphate synthase activity, metal ion binding and oxidoreductase activity were identified in watermelon genome, based on perfect or nearly perfect complementarity of miRNAs with their targeted mRNA sequences. Phylogenetic analysis of these miRNAs revealed that cla-miR8461 and cla-miR2515 are unique miRNAs closely related to Schistosoma mansoni and Drosophila pseudoobscura, respectively.

Factors Regulating microRNA Expression and Function in Multiple Myeloma.

Intensive research has been undertaken during the last decade to identify the implication of microRNAs (miRNAs) in the pathogenesis of multiple myeloma (MM). The expression profiling of miRNAs in MM has provided relevant information, demonstrating different patterns of miRNA expression depending on the genetic abnormalities of MM and a key role of some miRNAs regulating critical genes associated with MM pathogenesis. However, the underlying causes of abnormal expression of miRNAs in myeloma cells remain mainly elusive. The final expression of the mature miRNAs is subject to multiple regulation mechanisms, such as copy number alterations, CpG methylation or transcription factors, together with impairment in miRNA biogenesis and differences in availability of the mRNA target sequence. In this review, we summarize the available knowledge about the factors involved in the regulation of miRNA expression and functionality in MM.

Y-shaped DNA-Mediated hybrid nanoflowers as efficient gene carriers for fluorescence imaging of tumor-related mRNA in living cells

Organic-inorganic hybrid nanomaterial has gained much attention due to its excellent performances in bioanalysis and biomedicine. However, the preparation of DNA-inorganic hybrid nanomaterial with suitable size for cell uptake remains a huge challenge. Herein, a moderate biomineralization strategy for synthesis of Y-DNA@Cu3(PO4)2 (Y-DNA@CuP) hybrid nanoflowers is reported. Y-DNA with a loop structure is used as both the biomineralization template and the recognition unit for thymidine kinase 1 (TK1) mRNA. The Y-DNA probe can linearly response to TK1 mRNA target sequence in a range from 2 nM to 150 nM with the limit of detection as low as 0.56 nM. Interestingly, the presence of Y-DNA significantly decreases the size of Cu3(PO4)2 (CuP) particles, which allows them suitable for intracellular applications as gene nanocarriers. Once inside the cells, the hybrid nanoflowers dissolve and release the Y-DNA probes. Then, the intracellular TK1 mRNA hybridizes with the loop region of Y-DNA, which dissociates the Cy3-labeled loop strand and turns on the red fluorescence. Through the real-time imaging of the intracellular TK1 mRNA, the assessment of tumor cells before and after the treatment of drugs including β-estradiol and tamoxifen is achieved.

Comparing Agent-Based Delivery of DNA and PNA Forced Intercalation (FIT) Probes for Multicolor mRNA Imaging.

Fluorogenic oligonucleotide probes allow mRNA imaging in living cells. A key challenge is the cellular delivery of probes. Most delivery agents, such as cell‐penetrating peptides (CPPs) and pore‐forming proteins, require interactions with the membrane. Charges play an important role. To explore the influence of charge on fluorogenic properties and delivery efficiency, we compared peptide nucleic acid (PNA)‐ with DNA‐based forced intercalation (FIT) probes. Perhaps counterintuitively, fluorescence signaling by charged DNA FIT probes proved tolerant to CPP conjugation, whereas CPP–FIT PNA conjugates were affected. Live‐cell imaging was performed with a genetically engineered HEK293 cell line to allow the inducible expression of a specific mRNA target. Blob‐like features and high background were recurring nuisances of the tested CPP and lipid conjugates. By contrast, delivery by streptolysin‐O provided high enhancements of the fluorescence of the FIT probe upon target induction. Notably, DNA‐based FIT probes were brighter and more responsive than PNA‐based FIT probes. Optimized conditions enabled live‐cell multicolor imaging of three different mRNA target sequences.

DICER1 mutation and pituitary prolactinoma

SummaryA young woman carrying germline DICER1 mutation was discovered to have a pituitary microprolactinoma when she became amenorrhoic. The mutation was identified as a result of family screening following the early death of the patient’s daughter with ovarian cancer. The patient was in follow-up screening for thyroid disease, and investigations were initiated when she became amenorrhoic. MR scan revealed a 6 mm diameter pituitary microadenoma and raised prolactin. The prolactin was efficiently suppressed with low-dose cabergoline, and her menstrual cycles resumed. Dicer is an RNase enzyme, which is essential for processing small non-coding RNAs. These molecules play pleiotropic roles in regulating gene expression, by targeting mRNA sequences for degradation. DICER1 plays different roles depending on cell context, but is thought to be a functional tumour suppressor gene. Accordingly, germline mutation in one DICER1 allele is insufficient for oncogenesis, and a second hit on the other allele is required, as a result of postnatal somatic mutation. Loss of DICER1 is linked to multiple tumours, with prominent endocrine representation. Multinodular goitre is frequent, with increased risk of differentiated thyroid cancer. Rare, developmental pituitary tumours are reported, including pituitary blastoma, but not reports of functional pituitary adenomas. As DICER1 mutations are rare, case reports are the only means to identify new manifestations and to inform appropriate screening protocols.Learning points:DICER1 mutations lead to endocrine tumours.DICER1 is required for small non-coding RNA expression.DICER1 carriage and microprolactinoma are both rare, but here are reported in the same individual, suggesting association.Endocrine follow-up of patients carrying DICER1 mutations should consider pituitary disease.

Abstract 3251: TaqMan Advanced miRNA cDNA Synthesis Kit to simultaneously study expression of miRNA and mRNA and to detect somatic mutations by qPCR

Abstract MicroRNAs (miRNA) are a class of small non-coding RNAs (approximately 21 nt long) that bind complementary sequences in target mRNAs to specifically regulate gene expression. Aberrant regulation of miRNAs and their targets has been associated with several diseases including cancer. The relationship between miRNA and mRNA has been found to be important in cancer development and progression. Simultaneous expression studies of miRNA and mRNA and detection of mutations in mRNA transcripts can be valuable in understanding molecular mechanisms that have an underlying role in various diseases. We demonstrate the technical verification of a novel method to reverse-transcribe and pre-amplify miRNA and mRNA from sample-limiting serum research samples using the TaqMan® Advanced miRNA cDNA Synthesis Kit. Based on results from previous studies, a signature of 49 mRNA and 37 miRNA targets has been identified that may help distinguish between benign and malignant pancreatic tissues. In this study, these targets and an additional set of transcript mutations were analyzed in serum from normal and test samples. TaqMan assays for miRNA and mRNA targets and custom TaqMan Mutation Detection Assays were placed on TaqMan Array Cards to facilitate investigation of several samples in a single experiment. Results demonstrate that transcript mutations can be detected and miRNA and mRNA targets can be reliably quantified from a single reverse transcription reaction. (For research use only. Not for use in diagnostic purposes.) Citation Format: Kathleen Hayashibara, Harita Veereshlingam, Malte Buchholz. TaqMan Advanced miRNA cDNA Synthesis Kit to simultaneously study expression of miRNA and mRNA and to detect somatic mutations by qPCR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3251.

The Effect of Centrifugal Force in Quantification of Colorectal Cancer-Related mRNA in Plasma Using Targeted Sequencing.

In our previous study, we detected the effects of centrifugal forces on plasma RNA quantification by quantitative reverse transcription PCR. The aims of this study were to perform targeted mRNA sequencing and data analysis in healthy donors' plasma prepared by two centrifugation protocols and to investigate the effects of centrifugal forces on plasma mRNA quality and quantity. Targeted mRNA sequencing was performed using a custom panel with 108 colorectal cancer-related genes in 18 healthy donors' plasma that prepared by (1) 3,500 g for 10 min at 4°C and (2) 1,600 g for 10 min at 4°C followed by 16,000 g for 10 min at 4°C. Results showed that plasma ribosomal RNA was detected in 16/18 (88.9%) 3,500 g and 6/18 (33.3%) 1,600 g followed by 16,000 g centrifuged plasma. For targeted sequencing, 75/108 (69.4%) and 86/108 (79.6%) genes were detected in 3,500 and 1,600 g followed by 16,000 g, respectively, while 16/108 (14.8%) genes were not detected in both centrifugations. Detailed analysis showed that 2 of 108 (1.85%) genes showed lower expressions in 3,500 g than in 1,600 g followed by 16,000 g. The median expressions of genes in 3,500 g were positively correlated with the expressions in 1,600 g followed by 16,000 g (R2 = 0.9471, P < 0.0001, Spearman rank correlation). Meanwhile, plasma samples were not distinctively clustered based on centrifugal forces according to hierarchical clustering. Targeted mRNA sequencing and subsequent data analysis were performed in this study to investigate the effects of two different centrifugal forces that are commonly used in plasma collection. Our targeted sequencing results help to understand the centrifugal force effects on plasma mRNA, and these findings show that the centrifugation protocol for plasma mRNA research using targeted sequencing can be standardized which facilitates multicenter studies for comparison and quality assurance in the future.