LIFE09200604007 Tabish - Homi Bhabha National Institute

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Materials and Methods 3.9 Effect of γ-radiation on gene expression profiling Materials: 1. Cobalt-60 isotope source (<strong>Bhabha</strong>tron-II) 2. TRIzol 3. Total RNA 4. Superscript II reverse transcriptase 5. dNTP mix (dATP, dCTP, dGTP and dTTP + amino allyl dUPP) 6. Microcon YM30 columns 7. Sodium bicarbonate 8. Cy3 & Cy5 dyes 9. PCR purification kit 10. Human Cot 1 DNA and yeast tRNA 11. Toronto microarray slides (19K+8K, total 27K genes) 12. Heating block 13. Hybridization chambers 14. Centrifuge 15. GenePix 4200A microarray scanner Method: Whole genome expression profiling was done for 5 MPN and 5 Control cell lines where 10x10 6 cells were seeded in 100 mm plates in 5 ml complete DMEM and irradiated with 5 Gy γ-radiation dose. Cells were allowed to grow for 24 h and were collected by centrifuging at 1500 rpm/ 10 min/ RT. Cells were washed twice with PBS, RNA was isolated using TRIzol protocol as described in section 4.3.1 and RNA integrity was checked on 1.2% reducing agarose gel as described in section 4.3.2. After isolating RNA the following steps were followed to perform whole genome expression profiling. 3.9.1 Microcon concentration of the sample: 25 μg of RNA in 10 μl volume (concentration =2.5 μg/μl) was used for first strand cDNA synthesis using Superscript II reverse transcriptase for each slide. If the concentration of sample is less than 2.5 μg/μl, then it is subjected to Microcon column concentration. For sample concentration volume of the sample was made upto100 μl with nuclease free water and it was loaded on Microcon YM30 column. Sample was centrifuged at 10000 rpm/ 3 min/ RT. The sample was centrifuged till the volume of the 79
Materials and Methods sample was reduced to 10 μl or less. Later the filter containing the sample was inverted into a new tube and centrifuged at 10000 rpm/ 3 min/ RT. Volume of the sample collected was measured and made up to 10 μl. 3.9.2 Preparation of cDNA: Total RNA from test as well as control samples was treated with DNase wherever required as described in section 4.3.3 and later subjected to cDNA synthesis as described below. Reaction mixture-1 RNA (2.5 µg/µl) 10 µl Oligo dT (Invitrogen; 1 µg/µl) 6 µl Random Primer (Invitrogen; 0.5 µg/µl) 2 µl A mixture of Oligo dT and random primers was made and 8 μl was dispensed on each tube containing RNA. The tubes were incubated at 65 °C for 10 min and immediately transferred on ice and incubated for 10 min. Reaction mixture-2 5X FS Buffer 6 µl 0.1M DTT 3 µl 50X aadUTP.dNTP mix 0.6 µl RNase IN 0.5 µl Superscript SSII 1.5 µl D/W 0.4 µl 12 μl of this reaction mixture was added to each tube and was incubated at 42 °C for 1 h in water bath. The reaction was terminated by adding 10 μl 0.5M EDTA. 10 μl of freshly prepared 1 N NaOH was added to degrade RNA the reaction mixture was incubated at 65 °C for 10 min and cooled at RT. 3.9.3 Purification of cDNA To the newly synthesized cDNA 25 μl 1 M HEPES buffer pH 7.0 was added. Reaction volume was made 500 μl with nuclease free water and contents were transferred to microcon column. Samples were centrifuged at 10000 rpm/ 10 min/ RT. Flow through was discarded and again 450 μl nuclease free water was added to the column. Samples were again centrifuged at 10000 rpm/ 10 min/ RT and continued to be centrifuged for small intervals so as to reduce the volume
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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
Page 41 and 42: Review of Literature The yet undeci
Page 43 and 44: Review of Literature Fig. 2: Incide
Page 45 and 46: Review of Literature sustained angi
Page 47 and 48: Review of Literature 2.6 Genotype p
Page 49 and 50: Review of Literature 2.7.2 Biologic
Page 51 and 52: Review of Literature Table 1: Steps
Page 53 and 54: Review of Literature future analysi
Page 55 and 56: Review of Literature Inefficient ce
Page 57 and 58: Review of Literature XRCC1 (X-ray r
Page 59 and 60: Review of Literature 2.9.2 Gene inv
Page 61 and 62: Review of Literature MPO gene polym
Page 63 and 64: Review of Literature important role
Page 65 and 66: Materials and Methods Source of che
Page 67 and 68: Materials and Methods Method: EBV-t
Page 69 and 70: Materials and Methods Immunophenoty
Page 71 and 72: Materials and Methods with 500 μl
Page 73 and 74: Materials and Methods cDNA it can b
Page 75 and 76: 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 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 128 and 129: Results 4.2.5.2 Real time PCR valid
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
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Results Fig. 39: Various combinatio
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Results Table 14: Genotype-phenotyp
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Discussion Introduction Squamous ce
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Discussion stimulated lymphocytes w
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Discussion H2AX is currently the mo
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Discussion or G2/M phase arrest. As
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Discussion differential expression
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Discussion DNA repair genes and MPN
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Chapter 6 Summary and Conclusions
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Summary and Conclusions Statistical
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Bibliography 1. Dikshit, R.; Gupta,
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Bibliography 43. Lei, D.; Sturgis,
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Bibliography 76. Fry, R. C.; Svenss
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Bibliography 115. Kote-Jarai, Z.; M
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Bibliography 149. Hashibe, M.; Bren
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Bibliography 183. Bergamaschi, D.;
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Bibliography 216. Bondy, M. L.; Wan
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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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LIFE09200604007 Tabish - Homi Bhabha National Institute

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