Evaluation and Optimization of Lipofectamine 3000 Reagents for Transient Gene Expression in KYSE-30 Esophagus Cancer Cell Line

Background and Aim: Transfection of DNA/RNA sequence into eukaryotic cells has a major effect on scientific studies. Various methods are used to transfer the DNA/RNA sequence into cells, such as lipid-based carriers as the available and easy procedure. Transfection with cationic lipid liposome is introduced as a simple and efficient procedure for monitoring the DNA/RNA sequence through gene function analysis, including fluorescence imaging RNA and protein expression. This study aimed to investigate the transfection efficiency and cell death through GFP expression in human esophageal squamous cell carcinoma (ESCC) cell line KYSE-30 using Lipofectamine 3000 reagent. Methods: The pCDH-513b plasmid DNA was transfected into KYSE-30 cells using Lipofectamine 3000 in different concentrations of the plasmid DNA and reagent. The transfection efficiency was evaluated by fluorescence microscope and flow cytometry analysis to determine the percentage of GFP-expressing cells. Moreover, the viability and death of transfected KYSE-30 cells were evaluated using a trypan blue exclusion assay. Results: The transfection efficiency of KYSE-30 with Lipofectamine 3000 was increased with higher plasmid DNA concentration and a lower amount of Lipofectamine 3000 reagent. The Optimized concentration of 1.5 µg plasmid DNA and volume of one µl of lipofectamine 3000 reagents were identified for 95% transfection efficiency in the KYSE-30 cell line. The viability and death of transfected cells were 43% and 58% after transfection, respectively. Conclusion: The results indicated that Lipofectamine 3000 might not be suitable for transfection in KYSE-30 cells due to increased cell death. *Corresponding Author: Mohammad Reza Abbaszadegan; Email: [email protected] Please cite this article as: Mahmoudian RA, Farshchian M, Abbaszadegan MR. Evaluation and Optimization of Lipofectamine 3000 Reagents for Transient Gene Expression in KYSE-30 Esophagus Cancer Cell Line. Arch Med Lab Sci. 2019;5(4):1-9. https://doi.org/10.22037/amls.v5i4.3108

Fully automatic classification of breast cancer microarray images

AbstractA microarray image is used as an accurate method for diagnosis of cancerous diseases. The aim of this research is to provide an approach for detection of breast cancer type. First, raw data is extracted from microarray images. Determining the exact location of each gene is carried out using image processing techniques. Then, by the sum of the pixels associated with each gene, the amount of “genes expression” is extracted as raw data. To identify more effective genes, information gain method on the set of raw data is used. Finally, the type of cancer can be recognized via analyzing the obtained data using a decision tree. The proposed approach has an accuracy of 95.23% in diagnosing the breast cancer types

Amplification of Tumor Transcripts from Limited Quantity of Esophageal Squamous Cell Carcinoma Tissue Samples

To examine the template-switching technology accompanied by in vitro transcription (the Switch Mechanism At the 5ʹ end of Reverse Transcript) to amplify enough amount of mRNA as input for gene expression experiments. We amplified limited quantity of esophageal squamous cell carcinoma (ESCC) transcripts samples using generated ds cDNA as template and in vitro transcription (IVT) reaction. In addition, the quality and quantity of amplified mRNA were assessed by comparative real-time PCR of genes such as stem cell markers CD44, OCT4 and SNAIL as well as MAGE-A4 as a cancer-testis antigens, and XRCC5 as an underexpressed gene in ESCC.The results obtained from this study demonstrated that optimal amounts of mRNA are generated by template-switching and IVT reaction. Integrity and purity of all RNA samples were assessed. By using this approach, over 10 micrograms of amplified mRNA were generated from 100 ng of starting total RNA. The results of comparative real-time PCR of five genes with different levels of expression illustrated that the expression level of amplified sense RNA was almost similar when compared with non-amplified RNA. Our results clearly showed the usefulness of the T7-based IVT technique for amplification of limited quantity of input total RNA

Cancer Stem Cell Markers in Esophageal Cancer

Esophageal carcinoma is one of the most malignant of all tumors, and affected patients have low survival rates. The lack of good prognostic and therapeutic targets indicate the mysterious biology of this cancer. Recently, studies with cancer stem cells (CSCs) revealed some clues for better understanding of cancer biology and development. CSCs are derived from normal stem cells and have essential roles in tumor initiation and development of malignancies, such as esophageal carcinoma. Self-renewal studies in CSCs have improved our understanding of the factors that regulate CSCs behaviour and may result in improved prognostic markers and new therapeutic targets. Abnormal activity of major cell signaling pathways such as Shh, Notch, and Wnt play important roles in converting stem cells from normal to cancerous. This manuscript reviews the importance of several processes in the maintenance of esophageal CSCs and introduces probable useful markers for CSC based ESCC therapy

Role of SIZN1 in Esophageal Squamous Cell Carcinoma

Background: Bone morphogenetic proteins are a family of cytokines and growth factors that are involved in tumorigenesis. ZCCHC12 (SIZN1), as a transcriptional coactivator of bone morphogenetic protein signaling, is identified as a positive regulator of central nervous system development during embryogenesis. It positively regulates the CREB and AP1 transcription factors that cooperate with the bone morphogenetic protein signaling pathway. In the present study, SIZN1 mRNA expression was assessed in esophageal squamous cell carcinoma patients. Methods: The levels of SIZN1 mRNA expression in tumor tissues from 50 patients with esophageal squamous cell carcinoma were compared with their corresponding normal margins by using real-time polymerase chain reaction. Results: We observed that 10 out of 50 (20%) cases overexpressed SIZN1, whereas 40 out of 50 (80%) cases showed either normal or under expression of SIZN1. There was a significant correlation between the levels of SIZN1 mRNA expression and tumor depth of invasion (P=0.040). Furthermore, a significant correlation between lymph node metastasis and SIZN1 mRNA expression was observed in esophageal squamous cell carcinoma patients (P=0.036). Conclusion: This study is the first report that has assessed SIZN1 expression in esophageal squamous cell carcinoma patients. SIZN1 can be a potential therapeutic target for primary esophageal squamous cell carcinoma because of its role in the early stages of tumor progression and metastasis

Structural Biology: Modeling applications and techniques at a glance

As recent advancements in biology shows, the molecular machines specially proteins, RNA and complex molecules play the main role of the so called cell functionality. It means a very big part of the system biology is concerned with the interactions of such molecular components. Drug industries and research institutes are trying hard to better understand the concepts underlying these interactions and are highly dependent on the issues regarding these molecular elements. However the costs for such projects are so high and in many cases these projects will be funded by governments or profit making companies. With this in mind it has to be said that the techniques like stimulation are always a very good candidate to decrease such costs and to provide scientists with a bright future of the project results before undergoing costly experiments. However the costs involved projects that determine an approximation for the problem is not that much high but they are also costly. So it is of utmost importance to invent special techniques for the concept of stimulation that can also decrease the project costs and also predict much accurately. Since the system biology and proteomics as the study of the proteins and their functions are in the center of consideration for the purpose of drug discovery, understanding the cell functionalities and the underlying causes behind diseases; so we need advance software and algorithms that can predict the structure of the molecular components and to provide researchers with the computational tools to analyze such models. In this paper we make review of the importance of molecular modeling, its limitations and applications

Rapid DNA Extraction Protocol from Stool, Suitable for Molecular Genetic Diagnosis of Colon Cancer

ABSTRACT Background: Colorectal cancer (CRC) is one of the most common forms of cancers in the world and is curable if diagnosed at the early stage. Analysis of DNA extracted from stool specimens is a recent advantage to cancer diagnostics. Many protocols have been recommended for DNA extraction from stool, and almost all of them are difficult and time consuming, dealing with high amount of toxic materials like phenol. Their results vary due to sample collection method and further purification treatment. In this study, an easy and rapid method was optimized for isolating the human DNA with reduced PCR inhibitors present in stool. Methods: Fecal samples were collected from 10 colonoscopy-negative adult volunteers and 10 patients with CRC. Stool (1 g) was extracted using phenol/chloroform based protocol. The amplification of P53 exon 9 was examined to evaluate the extraction efficiency for human genomic targets and also compared its efficiency with Machiels et al. and Ito et al. protocols. Results: The amplification of exon 9 of P53 from isolated fecal DNA was possible in most cases in 35 rounds of PCR using no additional purification procedure for elimination of the remaining inhibitors. Conclusion: A useful, rapid and easy protocol for routine extraction of DNA from stool was introduced and compared with two previous protocols. Iran. Biomed. J. 1

Inherited deletion of 9p22.3-p24.3 and duplication of 18p11.31-p11.32 associated with neurodevelopmental delay: Phenotypic matching of involved genes.

We describe a 3.5-year-old Iranian female child and her affected 10-month-old brother with a maternally inherited derivative chromosome 9 [der(9)]. The postnatally detected rearrangement was finely characterized by aCGH analysis, which revealed a 15.056 Mb deletion of 9p22.3-p24.3p22.3 encompassing 14 OMIM morbid genes such as DOCK8, KANK1, DMRT1 and SMARCA2, and a gain of 3.309 Mb on 18p11.31-p11.32 encompassing USP14, THOC1, COLEC12, SMCHD1 and LPIN2. We aligned the genes affected by detected CNVs to clinical and functional phenotypic features using PhenogramViz. In this regard, the patient\u27s phenotype and CNVs data were entered into PhenogramViz. For the 9p deletion CNV, 53 affected genes were identified and 17 of them were matched to 24 HPO terms describing the patient\u27s phenotypes. Also, for CNV of 18p duplication, 22 affected genes were identified and six of them were matched to 13 phenotypes. Moreover, we used DECIPHER for in-depth characterization of involved genes in detected CNVs and also comparison of patient phenotypes with 9p and 18p genomic imbalances. Based on our filtration strategy, in the 9p22.3-p24.3 region, approximately 80 pathogenic/likely pathogenic/uncertain overlapping CNVs were in DECIPHER. The size of these CNVs ranged from 12.01 kb to 18.45 Mb and 52 CNVs were smaller than 1 Mb in size affecting 10 OMIM morbid genes. The 18p11.31-p11.32 region overlapped 19 CNVs in the DECIPHER database with the size ranging from 23.42 kb to 1.82 Mb. These CNVs affect eight haploinsufficient genes

Primary Angle Closure Glaucoma-associated Genetic Polymorphisms in Northeast Iran

Purpose: To evaluate the association of five different polymorphisms from a genomewide- associated study with susceptibility to glaucoma in the northeast Iranian population. Methods: Hundred and thirty patients with primary angle closure glaucoma (PACG) and 130 healthy controls were genotyped for the polymorphic regions with the aid of tetraamplification refractory mutation system-polymerase chain reaction. The association of these variants with the disease susceptibility was measured statistically with the logistic regression method. Results: Hundred and thirty patients with PACG (53 males, 77 females) with a mean age of 64.5 ± 6.2 years and 130 healthy control subjects (51 males, 79 females) with a mean age of 64.0 ± 5.7 years were selected for evaluation. There was a significant association between rs3816415 (P = 0.005), rs736893 (P < 0.001), rs7494379 (P < 0.001), and rs1258267 (P = 0.02) with PACG susceptibility. This association could not be shown for rs3739821. Conclusion: It was revealed that studied variants in GLIS3, EPDR1, FERMT2, and CHAT genes can contribute to the incidence of PACG. Additional studies in other populations are needed to evaluate DPM2-FAM102A