LIFE09200604007 Tabish - Homi Bhabha National Institute

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Synopsis 8.3 Restriction fragment length polymorphism (RFLP): Restriction digestion of the PCR product with appropriate restriction enzyme was done to analyse the genotype. PCR product from gene specific PCR reactions was incubated with specific restriction enzyme with appropriate buffer. Restriction digestion products were analysed by agarose gel electrophoresis along with DNA ladder. 8.4 SNaPshot reaction: SNaPshot genotyping is based on the dideoxy single-base extension of an unlabeled oligonucleotide primer which binds to a complementary template in the presence of fluorescently labelled ddNTPs and Taq DNA polymerase. The polymerase extends the primer by just one nucleotide, adding a single ddNTP to its 3‟ end. First the desired gene was amplified by PCR using gene specific primers. SNaPshot assay was performed according to manufacturer‟s protocol which consists of three steps: (1) EXO-SAP purification (remove unused dNTPs, primer and primerdimers), (2) SNaPshot reaction (single base extension of the primer) and (3) Post SNaPshot purification (remove unused ddNTPs from SNaPshot reaction. The processed samples were then sequenced in Genetic analyzer for interpretation of SNaPshot results. 8.5 Genotype Score: Genotyping of 22 candidate single nucleotide polymorphisms (SNPs) in 17 candidate genes involved in DNA repair, apoptosis, cell cycle regulation and carcinogen metabolism was done and a Genotype Score (G Score) was created from the number of variant alleles. A value of 2, 1 and 0 was allotted to homozygous variant, heterozygous and homozygous wild type alleles respectively, and a consolidated G Score was calculated taking values of all genes together. 9. Microarray: Whole genome expression profiling was done for 5 MPN and 5 Control cell lines after 5 Gy γ-radiation exposure and 24 h time point using Toronto 27 K slides (University Health Network Microarray Centre, Toronto). RNA was isolated using TRIzol protocol and was used for first strand cDNA synthesis using Superscript II reverse transcriptase for each slide. Indirect labelling of test and reference cDNA samples was done using Cy5 and Cy3 dyes (Amersham), respectively. Labelled samples were added to microarray slides and were kept in hybridization chambers at 42 o C in water bath overnight. After hybridization slides were sequentially washed using reducing concentrations of saline sodium-citrate (SSC) buffer containing SDS. The 9
Synopsis slides were then dried and the fluorescent images of hybridized microarrays were obtained using a GenePix 4200A microarray scanner. Primary data analysis for determining differential expression of genes between was carried out using the Genepix Pro software version 5.0. 10. Quantitative RT-PCR: Differential expression of genes was validated by Quantitative RT-PCR. Invitrogen RT-PCR kit was used to reverse transcribe total RNA using both oligo dT and random primers. The synthesized cDNA was diluted to a final concentration of 10 ng/µl with nuclease free H 2 O to be used as a template for real time reactions. All PCR reactions were performed in duplicate using Power SYBR Green and analysed on ABI Prism 7700 Sequence Detection System. GAPDH was used as endogenous RNA control and each sample was normalized on the basis of its endogenous gene expression. 11. Statistical methods: Phenotypic assays were performed thrice wherever a statistical analysis was done. The differences between MPN patient and control groups, for DNA damage repair assay, cell cycle profile and percent cell death response, were examined by standard unpaired Student‟s t-test or Mann-Whitney U test depending on whether the data followed normal distribution or not, respectively. For statistical analysis of genotyping data G Score was compared between the two groups by unpaired Student‟s t-test. All analyses were performed using the GraphPad Prism V 5.0 and a p-value of ≤ 0.05 was considered significant. For genotype phenotype correlation, statistical dependence between two variables (G Score and phenotypic response) was calculated by applying standard Pearson or Spearman rank correlation. The negative or positive value of correlation coefficient indicated the negative or positive correlation between the variables. A p-value of ≤ 0.05 was taken to be significant. Results: 1. Preparation and characterization of EBV cell line: A total of 34 LCLs from UADT MPN patients (n=24) and healthy controls (n=10) were prepared from PBLs isolated from blood samples. Sixteen UADT MPN patient (n=8) and healthy control cell lines (n=8) were prepared as a part of earlier study in the lab; eighteen MPN patient (n=16) and control cell lines (n=2) have been prepared in the present study. The average population doubling (PD) time of representative LCLs was found to be 24 h. 10
Page 1: Genotype-Molecular Phenotype Correl
Page 7 and 8: CONTENTS Page No Synopsis 1 List of
Page 9 and 10: Synopsis
Page 11 and 12: Synopsis Genotype-Molecular Phenoty
Page 13 and 14: Synopsis Materials and Methods: 1.
Page 15 and 16: Synopsis loading control. PCR produ
Page 17: Synopsis 7.1 Percent cell death aft
Page 21 and 22: Synopsis range 41.77% - 90.95% at 5
Page 23 and 24: Synopsis 5. Genotyping of genes: Ge
Page 25 and 26: Synopsis G2/M arrest and delaying e
Page 27 and 28: Synopsis may have an important bear
Page 29 and 30: Synopsis References: 1. Bobba, R.;
Page 31 and 32: Abbreviations MOPS : 3-(N-morpholin
Page 33 and 34: List of Figures Fig. 28: Percent re
Page 35 and 36: Chapter 1 Introduction
Page 37 and 38: Introduction In previous studies fr
Page 39 and 40: 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
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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
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Materials and Methods specific bind
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Materials and Methods h half of the
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Materials and Methods phenol and th
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Materials and Methods 7. Filter ste
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Materials and Methods viz. EXO-SAP
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Materials and Methods Table 2: List
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Materials and Methods SMAD6 AREG HM
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Materials and Methods 3.9 Effect of
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Materials and Methods fresh eppendo
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Materials and Methods Power SYBR Gr
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Results Objective 1 To generate EBV
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Results Fig. 8: Frequency of second
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Results Table 6: Demographics of he
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Results Fig. 10: Cell population do
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Results Fig. 12: Cell surface marke
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Results 4.1.5 Expression of ATM gen
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Results Objective 2 To compare the
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Results Fig. 17: Standardization of
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Results Fig. 19: Standardisation of
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Results The cells were incubated fu
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Results 4.2.3.1 Percent G2 delay af
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Results 4.2.3.2 Effect of BPDE expo
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Results Fig. 29: Comparison of perc
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Results Fig. 31: Percent cell death
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Results normalization with baseline
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Results 4.2.5.2 Real time PCR valid
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Results homozygous wild-type, heter
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Results Fig. 36b: Electrophoresis o
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Results Fig. 37a: Representative el
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Results Table 13: Consolidated geno
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Results LCL Genes NAT2(1) NAT2(2) N
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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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