LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

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CHAPTER 2 MATERIALS AND METHODS Array G2500A scanner (Agilent) and the expression data on human Affymetrix chip was generated. Since the numbers of samples in each treatment type were sparse, we used a bivariate simulation approach to identify differentially expressed (DE) genes for the specified threshold fold change (FC). The DE candidates across each comparison were further evaluated for their biological relevance through Gene Ontology and Pathways studies. The statistical analysis was carried out using R package and the biological analysis was carried out using Genowiz TM software. For comparative evaluation, an un-irradiated control cell was used. The expression data on Affymetrix Human 133AB chip was obtained for each sample. The primary objective of the study was to obtain differentially expressed (DE) probe sets (genes) across various comparisons. Also, the interest was to study the clustering of genes and samples and the functional relevance of the differentially expressed genes. Upon identifying the DE genes, the next interest was to study the biological relevance of selected marker genes through GO and Pathway analysis. The data analysis process involves Robust Multichip Average (RMA) normalization, quality check analysis, differential expression analysis and the gene enrichment analysis. The correlation between the expression values for samples was studied through Pearson's coefficient. A pairwise correlation analysis was performed to generate a correlation matrix indicating how the samples are related with each other. The coefficient tells about the similarity of expression trend between the two arrays. A coefficient value close to 1.0 indicates the linear 85
CHAPTER 2 MATERIALS AND METHODS expression between the arrays. The minimum correlation between the samples was 0.987, which was above the acceptable criterion of 0.8. 2.6 Semiquantitative Reverse transcriptional polymerase chain reaction (RT-PCR) Total RNA from all the groups of cells (1 x 10 6 cells) was extracted after required time periods of irradiation and eluted in 30 µl RNAase free water using RNA tissue kit (Roche). Equal amount of RNA in each group was reverse transcribed using cMaster RT kit (Eppendorf). Equal amount (2µl) of cDNA in each group was used for specific amplification of β-actin, NF-κB (p65), iNOS, p21, p53, ATM, DNA-PK, ATR, MLH1, Rad52, GADD45α, Toll-like receptor 3 (TLR3), Ferredoxin reductase (FDXR), Chk1, Chk2, Bcl-2 or Bax gene using gene specific primers (Table 2.6). The PCR condition were 94 o C, 5 min initial denaturation followed by 30 cycles of 94 o C 45 s, 55 – 62 o C 45 s (depending on the annealing temperature of specific primers given in Table 2.6), 72 o C 45 s and final extension at 72 o C for 10 min. Equal amount of each PCR product (10 µl) was run on 1.5 % agarose gel containing ethidium bromide in tris borate EDTA buffer at 60 V. The bands in the gel were visualized under UV lamp and relative intensities were quantified using Gel doc software (Syngene). 2.7 Immunofluorescence staining Cells were grown in cover slips which were kept in 35mm petri dishes and irradiated as mentioned above. Cell layer were washed at different time period after irradiation in PBS and fixed for 20 min in 4% paraformaldehyde at room temperature. Afterwards, the cells were washed twice in PBS. For immunofluorescence staining, cells were permeabilized for 3 min in 0.25% Triton X-100 in PBS, washed two times in PBS and blocked for 1 h with 5% BSA in 86
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RADIATION INDUCED SIGNALING IN MAMM
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DECLARATION I, hereby declare that
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CONTENTS Page No. SYNOPSIS………
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2.11 Measurement of NO production
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PREAMBLE SYNOPSIS Ionising radiatio
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SYNOPSIS Aims and Objectives: To St
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SYNOPSIS 2.6 Immunofluorescence sta
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SYNOPSIS ATM gene expression, which
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SYNOPSIS Percentage Survival 100 80
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SYNOPSIS Ratio of Rad52 to Actin Ra
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Relative Phosphorylation 45 40 35 3
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SYNOPSIS Fig. 3.3A Clonogenic Cell
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SYNOPSIS (A) Av No. of phospho BRCA
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SYNOPSIS Percentage Survival 120 10
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SYNOPSIS Fig.3.4B. Existance of cro
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SYNOPSIS Fig. 3.4EDNA damage in EL-
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SYNOPSIS subsequent cytotoxicity ca
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SYNOPSIS [4] Hall, E. J. Radiobiolo
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CHAPTER 1 INTRODUCTION INTRODUCTION
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CHAPTER 1 INTRODUCTION 15.8% 9.56%
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CHAPTER 1 INTRODUCTION far apart an
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Page 104 and 105: CHAPTER 3 RESULTS fractionated regi
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Page 108 and 109: CHAPTER 3 RESULTS Table 3.1.2A List
Page 110 and 111: CHAPTER 3 RESULTS 80 NM_002185 IL7R
Page 112 and 113: CHAPTER 3 RESULTS SAA2 149 AU134977
Page 114 and 115: CHAPTER 3 RESULTS 221 LOC643167 RNA
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Page 118 and 119: CHAPTER 3 RESULTS CDK4) 57 AU152107
Page 120 and 121: CHAPTER 3 RESULTS 134 AL045793 METT
Page 122 and 123: CHAPTER 3 RESULTS 58 AF237813 ABAT
Page 124 and 125: CHAPTER 3 RESULTS 127 AI809749 ORMD
Page 126 and 127: CHAPTER 3 RESULTS 31 BE615699 UNC84
Page 128 and 129: CHAPTER 3 RESULTS 47 AF026303 SULT1
Page 130 and 131: CHAPTER 3 RESULTS 117 BF062193 CYor
Page 132 and 133: CHAPTER 3 RESULTS 187 AL036662 ---
Page 134 and 135: CHAPTER 3 RESULTS 52 AV686514 EMP2
Page 136 and 137: CHAPTER 3 RESULTS exposed to 10 Gy
Page 138 and 139: CHAPTER 3 RESULTS Fig.3.1.4 Gene ex
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CHAPTER 3 RESULTS 3.1.9 Stabilizati
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CHAPTER 3 RESULTS 23±1.5% (Fig. 3.
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CHAPTER 3 RESULTS whereas only the
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CHAPTER 3 RESULTS Fig.3.2.2 Gene ex
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CHAPTER 3 RESULTS Fig.3.2.4 Gene ex
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CHAPTER 3 RESULTS with equal doses
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CHAPTER 3 RESULTS Fig.3.3.1.2 Forma
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CHAPTER 3 RESULTS Fig.3.3.1.3 Phosp
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CHAPTER 3 RESULTS (7.5±0.64) (Fig.
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CHAPTER 3 RESULTS Fig.3.3.1.7b Phos
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CHAPTER 3 RESULTS 3.3.2 Oxygen beam
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CHAPTER 3 RESULTS in all the cells.
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CHAPTER 3 RESULTS 3.3.2.3 Radiation
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CHAPTER 3 RESULTS Fig.3.3.2.7 Apopt
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CHAPTER 3 RESULTS PMA stimulated U9
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CHAPTER 3 RESULTS Fig.3.4.2. Exista
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CHAPTER 3 RESULTS up-regulation of
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CHAPTER 3 RESULTS Fig.3.4.3.2 NO pr
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CHAPTER 3 RESULTS 3.4.3.4 Apoptotic
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CHAPTER 3 RESULTS 3.4.4 Bystander e
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CHAPTER 3 RESULTS Fig.3.4.4b DNA da
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CHAPTER 3 RESULTS Fig.3.4.5a Gene e
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CHAPTER 4 DISCUSSION DISCUSSION 185
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CHAPTER 4 DISCUSSION directly expos
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CHAPTER 4 DISCUSSION dose is delive
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CHAPTER 4 DISCUSSION A clear cause
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CHAPTER 4 DISCUSSION 4.2 Proton bea
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CHAPTER 4 DISCUSSION Although the e
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CHAPTER 4 DISCUSSION Similar number
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CHAPTER 4 DISCUSSION the mechanism
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CHAPTER 4 DISCUSSION Thus unlike th
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CHAPTER 4 DISCUSSION different from
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CHAPTER 4 DISCUSSION upregulates ma
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CHAPTER 4 DISCUSSION Numerous studi
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CHAPTER 4 DISCUSSION inhibition of
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CHAPTER 4 DISCUSSION The survival o
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CHAPTER 5 REFERENCES [1] Khan, F. T
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CHAPTER 5 REFERENCES [19] Woo, R. A
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CHAPTER 5 REFERENCES [35] Nghiem, P
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CHAPTER 5 REFERENCES [52] McKay, B.
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CHAPTER 5 REFERENCES [69] Cary, R.
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CHAPTER 5 REFERENCES [84] Uziel, T.
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CHAPTER 5 REFERENCES [98] Liu, Y.;
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CHAPTER 5 REFERENCES [111] Fei, P.;
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CHAPTER 5 REFERENCES [127] Frank, K
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CHAPTER 5 REFERENCES [141] Pierce,
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CHAPTER 5 REFERENCES [156] Roots, R
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CHAPTER 5 REFERENCES [171] Xia, Z.;
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CHAPTER 5 REFERENCES (MAP) kinase c
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CHAPTER 5 REFERENCES phosphorylatio
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CHAPTER 5 REFERENCES [211] Anderson
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CHAPTER 5 REFERENCES [229] Konca, K
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CHAPTER 5 REFERENCES [245] Kastan,
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CHAPTER 5 REFERENCES [261] Miralbel
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CHAPTER 5 REFERENCES [278] Weizman,
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CHAPTER 5 REFERENCES [294] Zhou, H.
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PUBLICATIONS AND PRESENTATIONS Publ
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Mutation Research 729 (2012) 61-72
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S. Ghosh, M. Krishna / Mutation Res
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1568 I. J. Radiation Oncology d Bio
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