Identification of Diverse Adenosine-to-Inosine RNA Editing Subtypes in Colorectal Cancer

Lee, Si-Hyun,Kim, Hwang-Phill,Kang, Jun-Kyu,Song, Sang-Hyun,Han, Sae-Won,Kim, Tae-You Korean Cancer Association 2017 Cancer Research and Treatment Vol.49 No.4

<P><B>Purpose</B></P><P>RNA editing generates protein diversity by altering RNA sequences in coding regions without changing the overall DNA sequence. Adenosine-to-inosine (A-to-I) RNA editing events have recently been reported in some types of cancer, but they are rare in human colorectal cancer (CRC). Therefore, this study was conducted to identify diverse RNA editing in CRC.</P><P><B>Materials and Methods</B></P><P>We compared transcriptome data of 39 CRC samples and paired adjacent tissues from The Cancer Genome Atlas database to identify RNA editing patterns in CRC, focusing on canonical A-to-I RNA edits in coding sequence regions. We investigated nonsynonymous RNA editing patterns by comparing tumor and normal tissue transcriptome data.</P><P><B>Results</B></P><P>The number of RNA edits varied from 12 to 42 per sample. We also observed that hypoand hyper-RNA editing patterns were distinguishable within the samples. We found 10 recurrent nonsynonymous RNA editing candidates in nine genes (<I>PDLIM, NEIL1, SRP9, GLI1, APMAP, IGFBP7, ZNF358, COPA</I>, and <I>ZNF587B</I>) and validated some by Sanger sequencing and the inosine chemical erasing assay. We further showed that editing at these positions was performed by the adenosine deaminase acting on RNA 1 enzyme. Most of these genes are hypoedited in CRC, but editing of <I>GLI1</I> was increased in cancer tissues compared with normal tissues.</P><P><B>Conclusion</B></P><P>Our results show that nonsynonymous RNA editing patterns can be used to identify CRC patients and could serve as novel biomarkers for CRC.</P>

Big Data Analytics in RNA-sequencing

WOO Sung-Hun,JUNG Byung Chul 대한임상검사과학회 2023 대한임상검사과학회지(KJCLS) Vol.55 No.4

As next-generation sequencing has been developed and used widely, RNA-sequencing (RNA-seq) has rapidly emerged as the first choice of tools to validate global transcriptome profiling. With the significant advances in RNA-seq, various types of RNA-seq have evolved in conjunction with the progress in bioinformatic tools. On the other hand, it is difficult to interpret the complex data underlying the biological meaning without a general understanding of the types of RNA-seq and bioinformatic approaches. In this regard, this paper discusses the two main sections of RNA-seq. First, two major variants of RNA-seq are described and compared with the standard RNA-seq. This provides insights into which RNA-seq method is most appropriate for their research. Second, the most widely used RNA-seq data analyses are discussed: (1) exploratory data analysis and (2) pathway enrichment analysis. This paper introduces the most widely used exploratory data analysis for RNA-seq, such as principal component analysis, heatmap, and volcano plot, which can provide the overall trends in the dataset. The pathway enrichment analysis section introduces three generations of pathway enrichment analysis and how they generate enriched pathways with the RNA-seq dataset.

Identification and analysis of microRNAs in Candida albicans

Jin-Hyun Cho(조진현),Heon-Jin Lee(이헌진) 한국생명과학회 2017 생명과학회지 Vol.27 No.12

Candida albicans에 의한 구강 감염(캔디다증)은 구강 점막에 빈번하게 발생하며 잘 알려진 질병이다. 구강 캔디 다증은 생명을 위협하는 정도의 곰팡이 감염증은 아니나, 특정상황에서 개인에게 심각한 위험을 초래할 수도 있다. 마이크로 RNA는 세포 내에서 다른 타겟 유전자를 저해하는 작은 크기의 RNA 분자이며 단백질을 코딩하지는 않고 번역과정을 억제하는 조절자로서의 역할을 하고 있다. 본 연구는 C. albicans의 마이크로RNA를 처음으로 동정하고 그러한 마이크로RNA가 지닌 기능을 조사하기 위함이다. 이를 위하여 C. albicans의 small RNA를 차세대 염기분석법을 통하여 분석하고 그러한 RNA들의 2차 구조를 생물정보학적 방법으로 조사하였다. 분석한 small RNA들은 마이크로 RNA라고 불리울 수 있는 특징들을 가지고 있었으며, 특별히 높게 발현되고 있는 두개의 마이크로 RNA 정도 크기의 RNA가 CBP1 유전자의 3’ 말단 비번역구역(UTR)에서 반대방향으로 발현하는 것을 밝혀내었다. 우리는 이러한 C. albicans의 RNA가 CBP1 유전자를 타겟으로 하여 조절하는지 알아보기 위해 RNA를 인위적으로 합성한 후 세포 내로 주입하고, 형광형미경으로 도입 사실을 확인하였다. 하지만 4시간과 8시간 후에 CBP1의 발현 변화는 관찰되지 않았다. 따라서, 이러한 결과는 C. albicans가 마이크로RNA에 의한 RNA 간섭(RNAi) 작용이 다른 진핵세포와는 다르게 작용하는 것을 알 수 있다. Oral infection due to Candida albicans is a widely recognized and frequent cause of superficial infections of the oral mucosa (oral candidiasis). Although oral candidiasis is not a life-threatening fungemia, it can cause severe problems in individuals under certain conditions. MicroRNAs (miRNAs) are noncoding, small RNA molecules, which regulate the expression of other genes by inhibiting the translation of target mRNAs. The present study was designed to identify miRNAs in C. albicans and determine their possible roles in this organism. miRNA-sized small RNAs (msRNAs) were cloned in C. albicans by deep sequencing, and their secondary structures were analyzed. All the cloned msRNAs satisfied conditions required to qualify them as miRNAs. Bioinformatics analysis revealed that two of the most highly expressed C. albicans msRNAs, Ca-363 and Ca-2019, were located in the 3" untranslated region of the corticosteroid-binding protein 1 (CBP1) gene in a reverse orientation. miRNA mimics were transformed into C. albicans to investigate their RNA-inhibitory functions. RNA oligonucleotide-transformed C. albicans was then observed by fluorescent microscopy. Quantitative PCR analysis showed that these msRNAs did not inhibit CBP1 gene expression 4 hr and 8 hr after ectopic miRNA transformation. These results suggest that msRNAs in C. albicans possess an miRNA-triggered RNA interference gene-silencing function, which is distinct from that exhibited by other eukaryotic systems.

Identification of Alternative Splicing and Fusion Transcripts in Non-Small Cell Lung Cancer by RNA Sequencing

( Yoon Ki Hong ),( Woo Jin Kim ),( Chi Young Bang ),( Jae Cheol Lee ),( Yeon Mok Oh ) 대한결핵 및 호흡기학회 2016 Tuberculosis and Respiratory Diseases Vol.79 No.2

Background: Lung cancer is the most common cause of cancer related death. Alterations in gene sequence, structure, and expression have an important role in the pathogenesis of lung cancer. Fusion genes and alternative splicing of cancer-related genes have the potential to be oncogenic. In the current study, we performed RNA-sequencing (RNA-seq) to investigate potential fusion genes and alternative splicing in non-small cell lung cancer. Methods: RNA was isolated from lung tissues obtained from 86 subjects with lung cancer. The RNA samples from lung cancer and normal tissues were processed with RNA-seq using the HiSeq 2000 system. Fusion genes were evaluated using Defuse and ChimeraScan. Candidate fusion transcripts were validated by Sanger sequencing. Alternative splicing was analyzed using multivariate analysis of transcript sequencing and validated using quantitative real time polymerase chain reaction. Results: RNA-seq data identified oncogenic fusion genes EML4-ALK and SLC34A2-ROS1 in three of 86 normal-cancer paired samples. Nine distinct fusion transcripts were selected using DeFuse and ChimeraScan; of which, four fusion transcripts were validated by Sanger sequencing. In 33 squamous cell carcinoma, 29 tumor specific skipped exon events and six mutually exclusive exon events were identified. ITGB4 and PYCR1 were top genes that showed significant tumor specific splice variants. Conclusion: In conclusion, RNA-seq data identified novel potential fusion transcripts and splice variants. Further evaluation of their functional significance in the pathogenesis of lung cancer is required.

RNA sequencing of the nephron transcriptome: a technical note

( Jae Wook Lee ) 대한신장학회 2015 Kidney Research and Clinical Practice Vol.34 No.4

To understand the functions of the kidney, the transcriptome of each part of the nephron needs to be profiled using a highly sensitive and unbiased tool. RNA sequencing (RNA-seq) has revolutionized transcriptomic research, enabling researchers to define transcription activity and functions of genomic elements with unprecedented sensitivity and precision. Recently, RNA-seq for polyadenylated messenger RNAs [poly(A)0-mRNAs] and classical microdissection were successfully combined to investigate the transcriptome of glomeruli and 14 different renal tubule segments. A rat kidney is perfused with and incubated in collagenase solution, and the digested kidney was manually dissected under a stereomicroscope. Individual glomeruli and renal tubule segments are identified by their anatomical and morphological characteristics and collected in phosphate-buffered saline. Poly(A)0- tailed mRNAs are released from cell lysate, captured by oligo-dT primers, and made into complementary DNAs (cDNAs) using a highly sensitive reverse transcription method. These cDNAs are sheared by sonication and prepared into adapter-ligated cDNA libraries for Illumina sequencing. Nucleotide sequences reported from the sequencing reaction are mapped to the rat reference genome for gene expression analysis. These RNA-seq transcriptomic data were highly consistent with prior knowledge of gene expression along the nephron. The gene expression data obtained in this work are available as a public Web page (https://helixweb.nih.gov/ ESBL/Database/NephronRNAseq/) and can be used to explore the transcriptomic landscape of the nephron.

유리비드를 포함한 PDMS 마이크로칩을 이용한 고감도 감염성 병원균 측정에 관한 연구

원지영(Ji Yeong Won),민준홍(Junhong Min) 한국생물공학회 2009 KSBB Journal Vol.24 No.5

본 연구는 환경샘플 중 병원균을 진단하기 위한 목적을 가진다. 최소 챔버 칩에서 환경 샘플 중 병원균을 농축하고 mRNA를 증폭하여 효과적이고 간단한 진단방법을 고안 하였다. PDMS로 면적 1.5 cm x 1.5 cm, 높이 100 μL의 칩을 제작하여 유리에 부착시켰다. RNase에 의한 진단 오류 또는 실패를 막고자 RNase away 처리를 하고, RNA와 PDMS의 결합을 막기 위해 BSA 처리를 하였다. 수질에 있는 병원균은 매우 적은 농도로 존재하므로 농축의 과정이 필요하다. 농축의 방법에는 여러 가지가 있으나 본 연구에서는 유리 비드를 칩 내에 삽입하고 저농도의 시료를 주입 함으로서 고농도로 농축을 하는 방법을 사용하였다. 그러나 부피가 작은 칩 내에서 수행하기에는 내부 압력이 작용하여 문제가 발생하여 100 μm의 유리 비드를 사용하고 유리 비드의 칩 내부 이탈을 방지하기 위하여 댐을 만들어 농축에 가장 적합한 칩의 형태를 잡았다. 시료의 주입속도에 따라 내부 압력이 상승하여 댐의 기능이 상실하여 유리 비드가 이탈하게 되므로 그것을 방지하기 위하여 칩 내에 댐을 강화하여 만들고 내부압력 증가가 방지되는 최적의 댐을 개발하여 시료의 주입 속도 5 mL/min까지 유리 비드의 이탈을 막았다. 유리 비드에서의 RNA 농축은 pH 5에서 효과적이고 pH가 증가할수록 유리 비드와 RNA의 결합이 끊어지는 현상을 보였으므로 시료에 pH 5의 버퍼를 첨가하여 농축을 진행하고 중성의 NASBA 용액을 주입하여 유리비드에서 탈착된 농축된 고농도의 RNA를 증폭하였다. NASBA는 항온 수조에서 온도에 변화 없이 41℃에서 1시간 30분 동안 진행하며 증폭된 mRNA는 직접 확인하였다. 이 방법은 LOC 기술을 적용하여 저농도의 시료를 효과적으로 측정할 수 있도록 편리한 바이오 칩을 개발함으로써 대용량의 샘플 중 극 저농도의 대장균을 효과적으로 검출 할 수 있는 장점을 가지고 있다. Here, we demonstrated simple nucleic acid, RNA, concentration method using polymer micro chip containing glass bead (100 μm). Polymer micro chip was fabricated by PDMS (1.5 cm x 1.5 cm, 100 μm in the height) including pillar structure (160 μm (l) x 80 μm (w) x 100 μm (h), gap size 50 μm) for blocking micro bead. RNA could be adsorbed on micro glass bead at low pH by hydrogen bonding whereas RNA was released at high pH by electrostatic force between silica surface and RNA. Amount of glass beads and flow rate were optimized in aspects of adsorption and desorption of RNA. Adsorption and desorption rate was measured with real time PCR. This concentrated RNA was applied to amplification micro chip in which NASBA (Nucleic Acid Sequence B ased Amplification) was performed. As a result, E.coli O157:H7 in the concentration of 10 c.f.u./10 mL was successfully detected by these serial processes (concentration and amplification) with polymer micro chips. It implies this simple concentration method using polymer micro chip can be directly applied to ultra sensitive method to measure viable bacteria and virus in clinical samples as well as environmental samples.

Identification of Alternative Splicing and Fusion Transcripts in Non-Small Cell Lung Cancer by RNA Sequencing

홍윤기,김우진,방지영,이재철,오연목 대한결핵및호흡기학회 2016 Tuberculosis and Respiratory Diseases Vol.79 No.2

Background: Lung cancer is the most common cause of cancer related death. Alterations in gene sequence, structure, and expression have an important role in the pathogenesis of lung cancer. Fusion genes and alternative splicing of cancer-related genes have the potential to be oncogenic. In the current study, we performed RNA-sequencing (RNA-seq) to investigate potential fusion genes and alternative splicing in non-small cell lung cancer. Methods: RNA was isolated from lung tissues obtained from 86 subjects with lung cancer. The RNA samples from lung cancer and normal tissues were processed with RNA-seq using the HiSeq 2000 system. Fusion genes were evaluated using Defuse and ChimeraScan. Candidate fusion transcripts were validated by Sanger sequencing. Alternative splicing was analyzed using multivariate analysis of transcript sequencing and validated using quantitative real time polymerase chain reaction. Results: RNA-seq data identified oncogenic fusion genes EML4-ALK and SLC34A2-ROS1 in three of 86 normal-cancer paired samples. Nine distinct fusion transcripts were selected using DeFuse and ChimeraScan; of which, four fusion transcripts were validated by Sanger sequencing. In 33 squamous cell carcinoma, 29 tumor specific skipped exon events and six mutually exclusive exon events were identified. ITGB4 and PYCR1 were top genes that showed significant tumor specific splice variants. Conclusion: In conclusion, RNA-seq data identified novel potential fusion transcripts and splice variants. Further evaluation of their functional significance in the pathogenesis of lung cancer is required.

Identification of Alternative Splicing and Fusion Transcripts in Non-Small Cell Lung Cancer by RNA Sequencing

Hong, Yoonki,Kim, Woo Jin,Bang, Chi Young,Lee, Jae Cheol,Oh, Yeon-Mok The Korean Academy of Tuberculosis and Respiratory 2016 Tuberculosis and Respiratory Diseases Vol.79 No.2

Background: Lung cancer is the most common cause of cancer related death. Alterations in gene sequence, structure, and expression have an important role in the pathogenesis of lung cancer. Fusion genes and alternative splicing of cancer-related genes have the potential to be oncogenic. In the current study, we performed RNA-sequencing (RNA-seq) to investigate potential fusion genes and alternative splicing in non-small cell lung cancer. Methods: RNA was isolated from lung tissues obtained from 86 subjects with lung cancer. The RNA samples from lung cancer and normal tissues were processed with RNA-seq using the HiSeq 2000 system. Fusion genes were evaluated using Defuse and ChimeraScan. Candidate fusion transcripts were validated by Sanger sequencing. Alternative splicing was analyzed using multivariate analysis of transcript sequencing and validated using quantitative real time polymerase chain reaction. Results: RNA-seq data identified oncogenic fusion genes EML4-ALK and SLC34A2-ROS1 in three of 86 normal-cancer paired samples. Nine distinct fusion transcripts were selected using DeFuse and ChimeraScan; of which, four fusion transcripts were validated by Sanger sequencing. In 33 squamous cell carcinoma, 29 tumor specific skipped exon events and six mutually exclusive exon events were identified. ITGB4 and PYCR1 were top genes that showed significant tumor specific splice variants. Conclusion: In conclusion, RNA-seq data identified novel potential fusion transcripts and splice variants. Further evaluation of their functional significance in the pathogenesis of lung cancer is required.

Two distinct extracellular RNA signatures released by a single cell type identified by microarray and next-generation sequencing

Lä,sser, Cecilia,Shelke, Ganesh Vilas,Yeri, Ashish,Kim, Dae-Kyum,Crescitelli, Rossella,Raimondo, Stefania,Sjö,strand, Margareta,Gho, Yong Song,Van Keuren Jensen, Kendall,Lö,tvall, Jan TaylorFrancis 2017 RNA BIOLOGY Vol.14 No.1

<P><B>ABSTRACT</B></P><P>Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in many body fluids such as blood, breast milk and cerebrospinal fluid. However, there are conflicting results regarding the nature of exRNA. Here, we have separated 2 distinct exRNA profiles released by mast cells, here termed high-density (HD) and low-density (LD) exRNA. The exRNA in both fractions was characterized by microarray and next-generation sequencing. Both exRNA fractions contained mRNA and miRNA, and the mRNAs in the LD exRNA correlated closely with the cellular mRNA, whereas the HD mRNA did not. Furthermore, the HD exRNA was enriched in lincRNA, antisense RNA, vault RNA, snoRNA, and snRNA with little or no evidence of full-length 18S and 28S rRNA. The LD exRNA was enriched in mitochondrial rRNA, mitochondrial tRNA, tRNA, piRNA, Y RNA, and full-length 18S and 28S rRNA. The proteomes of the HD and LD exRNA-containing fractions were determined with LC-MS/MS and analyzed with Gene Ontology term finder, which showed that both proteomes were associated with the term extracellular vesicles and electron microscopy suggests that at least a part of the exRNA is associated with exosome-like extracellular vesicles. Additionally, the proteins in the HD fractions tended to be associated with the nucleus and ribosomes, whereas the LD fraction proteome tended to be associated with the mitochondrion.</P><P>We show that the 2 exRNA signatures released by a single cell type can be separated by floatation on a density gradient. These results show that cells can release multiple types of exRNA with substantial differences in RNA species content. This is important for any future studies determining the nature and function of exRNA released from different cells under different conditions.</P>

단세포 RNA 시퀀싱 데이터를 위한 가중변수 스펙트럼 군집화 기법

박민영,박세영 한국통계학회 2020 응용통계연구 Vol.33 No.4

Single-cell RNA-sequencing (scRNA-seq) data consists of each cell’s RNA expression extracted from large populations of cells. One main purpose of using scRNA-seq data is to identify inter-cellular heterogeneity. However, scRNA-seq data pose statistical challenges when applying traditional clustering methods because they have many missing values and high level of noise due to technical and sampling issues. In this paper, motivated by analyzing scRNA-seq data, we propose a novel spectral-based clustering method by imposing different weights on genes when computing a similarity between cells. Assigning weights on genes and clustering cells are performed simultaneously in the proposed clustering framework. We solve the proposed non-convex optimization using an iterative algorithm. Both real data application and simulation study suggest that the proposed clustering method better identifies underlying clusters compared with existing clustering methods. 단세포 RNA 시퀀싱 데이터(single-cell RNA-sequencing data, 이하 단세포 RNA 데이터)는 세포 조직으로부터 추출한 각 단세포 별 유전자의 신호를 기록한 데이터로, 세포 간의 이질성을 파악하는 것을 주요 목적으로 한다. 그러나 단세포 RNA 데이터는 샘플링 및 기술적인 한계로 인해 결측비율이 높고, 노이즈가 크다. 이러한 이유 때문에 기존의 군집화 방법을 적용하는 데에 한계가 존재한다. 본 논문에서는 단세포 RNA 데이터 분석에서 모티브를 얻어 스펙트럼 군집화(spectral clustering) 기반의 방법을 제안한다. 특히 유사도 행렬(similarity matrix) 계산에서 유전자 별로 가중치를 부여하여 기존의 단세포 데이터 분석 방법과 차별화하였다. 제안하는 군집화 방법은 유전자 별 가중치를 부여함과 동시에 세포를 군집화한다. 군집화는 반복 알고리즘을 통해 제안하는 비볼록식(non-convex optimization)을 풀어 진행한다. 또한 실데이터 적용과 시뮬레이션을 통해 제안하는 군집화 방법이 기존의 방법보다 군집을 잘 구분하는 것을 보인다.