LIFE09200604007 Tabish - Homi Bhabha National Institute

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Results 4.2.5.2 Real time PCR validation of differentially expressed genes The genes that were found to be differentially expressed in microarray data were validated by real time PCR along with GAPDH as an internal control. The genes included AREG, ARHGAP25, MAFB, MAPK10, TMEFF and ZNF429. Total RNA isolated from MPN and control cell lines after 5 Gy radiation exposure and 24 h time point was subjected to cDNA was synthesis which was used for real time validation of genes. The expression of all the genes was calculated with respect to GAPDH of the individual samples and a ΔCt value was calculated. This ΔCt value was plotted as relative fold change by calculating: Fold change = 2 –ΔCt x 1000 Out of these deregulated genes, ARHGAP25, MAFB, MAPK10, and ZNF429 were up regulated in MPN cell lines while TMEFF was down regulated. Real time validation was conducted on 13 MPN and 7 control cell lines including the cell lines taken for microarray experiment. The difference in the expression of genes that was observed in microarray was also observed in the real time validation to some extent, although it was not statistically significant. Cluster plots showing differential expression of these genes is shown in Fig. 35. Fig. 35: Representative cluster plots showing real time validation of differentially expressed genes. ARHGAP25, MAFB, ZNF429 were up regulated in MPN cell lines while TMEFF was down regulated. 118
Results Objective 3 To genotype selected candidate genes involved in DNA repair, carcinogen metabolism, apoptosis and cell cycle regulation. The association of various gene polymorphisms with cancer has been frequently reported. The candidate genes influencing susceptibility to cancer includes genes falling mainly in important carcinogenesis pathways of carcinogen metabolism, DNA repair, cell cycle regulation and cell death control. Variation in any of these mechanisms could result in accumulation of cell with genetic alteration in critical genes leading to tumourigenesis. In the present study 22 single nucleotide polymorphisms (SNPs) in candidate genes involved in DNA repair, apoptosis, cell cycle regulation and carcinogen metabolism were genotyped and a G Score was calculated from the number of variant alleles. 4.3.1 Genomic DNA extraction High molecular weight genomic DNA was isolated from whole blood or the pellet obtained after isolating PBLs from the whole blood for cell line preparation. The quality of DNA was assessed on 0.8% agarose gels stained with ethidium bromide. Approximately 5-20μg of good quality DNA was obtained from each sample. Extreme care was taken to avoid cross contamination of samples. DNA was diluted to a concentration of 50ng/μl for performing PCR amplification while the stock DNA was stored at 0 °C till further use. 4.3.2 Genotyping of genes by PCR-RFLP Genotyping of 15 SNPs was performed by PCR and PCR-RFLP in 30 samples. Each sample was subjected to PCR for amplification of the genes of interest and to confirm the successful amplification PCR products were run on agarose gel. Multiplex PCR was done to assess the null genotype of GSTT1 and GSTM1 genes. In the PCR, to investigate null genotype, IFN was used as internal control to rule out amplification failure. For RFLP, PCR products were digested with respective restriction enzymes and the digested products were analyzed on 2% agarose gel (Fig. 36a, Fig. 36b, Fig. 36c). Depending upon the obtained digested product the genotype was recorded as 119
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Genotype-Molecular Phenotype Correl
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CONTENTS Page No Synopsis 1 List of
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Synopsis
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Synopsis Genotype-Molecular Phenoty
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Synopsis Materials and Methods: 1.
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Synopsis loading control. PCR produ
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Synopsis 7.1 Percent cell death aft
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Synopsis slides were then dried and
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Synopsis range 41.77% - 90.95% at 5
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Synopsis 5. Genotyping of genes: Ge
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Synopsis G2/M arrest and delaying e
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Synopsis may have an important bear
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Synopsis References: 1. Bobba, R.;
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Abbreviations MOPS : 3-(N-morpholin
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List of Figures Fig. 28: Percent re
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Chapter 1 Introduction
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Introduction In previous studies fr
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Introduction important carcinogenes
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Review of Literature The yet undeci
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Review of Literature Fig. 2: Incide
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Review of Literature sustained angi
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Review of Literature 2.6 Genotype p
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Review of Literature 2.7.2 Biologic
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Review of Literature Table 1: Steps
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Review of Literature future analysi
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Review of Literature Inefficient ce
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Review of Literature XRCC1 (X-ray r
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Review of Literature 2.9.2 Gene inv
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Review of Literature MPO gene polym
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Review of Literature important role
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Materials and Methods Source of che
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Materials and Methods Method: EBV-t
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Materials and Methods Immunophenoty
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Materials and Methods with 500 μl
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Materials and Methods cDNA it can b
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Materials and Methods Cobalt-60 iso
Page 77 and 78: Materials and Methods specific bind
Page 79 and 80: Materials and Methods h half of the
Page 81 and 82: Materials and Methods phenol and th
Page 83 and 84: Materials and Methods 7. Filter ste
Page 85 and 86: Materials and Methods viz. EXO-SAP
Page 88 and 89: Materials and Methods Table 2: List
Page 90 and 91: Materials and Methods SMAD6 AREG HM
Page 92 and 93: Materials and Methods 3.9 Effect of
Page 94 and 95: Materials and Methods fresh eppendo
Page 96 and 97: Materials and Methods Power SYBR Gr
Page 98 and 99: Results Objective 1 To generate EBV
Page 100 and 101: Results Fig. 8: Frequency of second
Page 102 and 103: Results Table 6: Demographics of he
Page 104 and 105: Results Fig. 10: Cell population do
Page 106 and 107: Results Fig. 12: Cell surface marke
Page 108 and 109: Results 4.1.5 Expression of ATM gen
Page 110 and 111: Results Objective 2 To compare the
Page 112 and 113: Results Fig. 17: Standardization of
Page 114 and 115: Results Fig. 19: Standardisation of
Page 116 and 117: Results The cells were incubated fu
Page 118 and 119: Results 4.2.3.1 Percent G2 delay af
Page 120 and 121: Results 4.2.3.2 Effect of BPDE expo
Page 122 and 123: Results Fig. 29: Comparison of perc
Page 124 and 125: Results Fig. 31: Percent cell death
Page 126 and 127: Results normalization with baseline
Page 130 and 131: Results homozygous wild-type, heter
Page 132 and 133: Results Fig. 36b: Electrophoresis o
Page 134 and 135: Results Fig. 37a: Representative el
Page 136 and 137: Results Table 13: Consolidated geno
Page 138 and 139: Results LCL Genes NAT2(1) NAT2(2) N
Page 140 and 141: Results Fig. 38: Distribution of Ge
Page 142 and 143: Results Fig. 39: Various combinatio
Page 144 and 145: Results Table 14: Genotype-phenotyp
Page 146 and 147: Discussion Introduction Squamous ce
Page 148 and 149: Discussion stimulated lymphocytes w
Page 150 and 151: Discussion H2AX is currently the mo
Page 152 and 153: Discussion or G2/M phase arrest. As
Page 154 and 155: Discussion differential expression
Page 156 and 157: Discussion DNA repair genes and MPN
Page 158 and 159: Chapter 6 Summary and Conclusions
Page 160 and 161: Summary and Conclusions Statistical
Page 162 and 163: Bibliography 1. Dikshit, R.; Gupta,
Page 164 and 165: Bibliography 43. Lei, D.; Sturgis,
Page 166 and 167: Bibliography 76. Fry, R. C.; Svenss
Page 168 and 169: Bibliography 115. Kote-Jarai, Z.; M
Page 170 and 171: Bibliography 149. Hashibe, M.; Bren
Page 172 and 173: Bibliography 183. Bergamaschi, D.;
Page 174 and 175: Bibliography 216. Bondy, M. L.; Wan
Page 176 and 177: Indian J Med Res 135, June 2012, pp
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822 INDIAN J MED RES, JUNE 2012 mar
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824 INDIAN J MED RES, JUNE 2012 (h)
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826 INDIAN J MED RES, JUNE 2012 Tab
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828 INDIAN J MED RES, JUNE 2012 lik
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CASE REPORT Eben L. Rosenthal, MD,
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FIGURE 2. Chromosomal breakage anal
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FA along with ataxia telangiectasia
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