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Experimental and Therapeutic Medicine (1)
Journal of Biomolecular Techniques : JBT (1)
BHUTANI, MANISHA (1)
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Khanna, Kanhav (1)
LEE, J. JACK (1)
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Visualizing the Transcriptome: A Comparison of Different RNA Library Preparation Methods
Journal of Biomolecular Techniques : JBT
RNA-seq is a powerful tool used to obtain in-depth information on expression profiling, gene annotation, and transcript discovery. With the growing popularity of RNA sequencing, new library preparation techniques are becoming commercially available. These techniques are improvements on the classic poly-A selection and rRNA reduction methods, and in some cases sensitive enough to analyze the transcriptome of a single cell. However, limited information is available on comparative analysis of these methods and their appropriate application for the transcriptome studies. We utilized Illumina's HiSeq technology to compare the merits of four commercial sample preparation kits: NuGen's Ovation RNA-seq system v2, Illumina's TruSeq RNA Sample Preparation kit v2, Epicentre's ScriptSeq RNA-seq kit v2 and Clontech's SMARTer Ultra Low RNA kit. We found that the quality of input RNA was critical for optimum performance of SMARTer Ultra Low RNA kit. Ovation and ScriptSeq kits, on the other hand, worked well with moderate quality input RNA as well. Based on analysis of the sequencing data, 12% of reads from ScriptSeq mapped to the mitochondrial genes as compared to 24% reads from Ovation. The library complexity and percentage of reads aligning to non-exonic region was similar between both kits. However, 28% reads aligned to the coding region for ScriptSeq versus 18% for Ovation. While TruSeq and SMARTer kits are designed for Poly-A containing RNAs only, ScriptSeq and Ovation kits provide more global analysis of the transcriptome. Analyzing the differences between these methods provides a better understanding of their specific advantage over the other. This information is especially useful for Sequencing Core Facilities, to recommend and apply appropriate methods to different transcriptome studies.
Frequent expression of MAGE1 tumor antigens in bronchial epithelium of smokers without lung cancer
PATHAK, ASHUTOSH KUMAR
FAN, YOU H.
LIU, DIANE D.
LEE, J. JACK
MORICE, RODOLFO C.
HONG, WAUN KI
Experimental and Therapeutic Medicine
Melanoma antigens (MAGE) are frequently expressed in lung cancer and are promising targets of anticancer immunotherapy. Our preliminary data suggested that MAGE may be expressed during early lung carcinogenesis, raising the possibility of targeting MAGE as a lung cancer prevention strategy. The purpose of this study was to investigate MAGE activation patterns in the airways of chronic smokers without lung cancer. MAGE-A1, -A3 and -B2 gene expression was determined in bronchial brush cells from chronic former smokers without lung cancer by reverse transcription-PCR (RT-PCR). The results were correlated with clinical parameters. The 123 subjects had a median age of 57 years, a median of 40 pack-years smoking history, and had quit smoking for at least one year prior to enrollment. Among the subjects, 31 (25%), 38 (31%), and 46 (37%) had detectable MAGE-A1, -A3 and -B2 expression, respectively, in their bronchial brush samples. Expression of MAGE-A1 and -B2 positively correlated with pack-years smoking history (P=0.03 and 0.03, respectively). The frequency of expression did not decrease despite a prolonged smoking cessation period. In conclusion, MAGE-A1, -A3 and -B2 genes are frequently expressed in the bronchial epithelial cells of chronic smokers without lung cancer, suggesting that chronic exposure to cigarette smoke activates these genes even before the malignant transformation of bronchial cells in susceptible individuals. Once activated, the expression persists despite long-term smoking cessation. These data support the targeting of MAGE as a novel lung cancer prevention strategy.
melanoma antigens; airway; smokers; lung cancer; prevention
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