LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

CHAPTER 2
MATERIALS AND METHODS
2.4 Clonogenic Cell Survival Assay
After treatment, cells were seeded out in appropriate dilutions (500 cells/60 mm petriplate, 1000
cells/60 mm petriplate or 2000 cells/60 mm petriplate) to form colonies in 14 days. Colonies
were fixed with glutaraldehyde (6.0% v/v), stained with crystal violet (0.5% w/v) and counted
using a stereomicroscope. Colonies containing more than 50 cells were scored as survivors.
Absolute plating efficiency of A549 cells at 0 Gy (sham irradiated control) was 32.3±2.4 %.
Absolute plating efficiency of MCF-7 cells at 0 Gy (sham irradiated control) was 45.2±2.4 %.
2.5 Microarray
Two independent series of experiments were performed with Human Genome U-133 Plus 2.0
microarrays containing 54675 probe sets (Affymetrix, Santa Clara, CA). Microarray analysis was
carried out at Oscimum Biosolution, Hyderabad on paid basis. Total RNA was isolated from
~3 × 10 6 A549 cells with RNeasy Mini Kits (Qiagen) including digestion with RNase-free
DNase I, and its quantity and integrity were checked spectrophotometrically and by
electrophoresis in 1% agarose gels. Materials and methods for microarrays were from
Affymetrix (Santa Clara); double-stranded cDNA was prepared with the GeneChip Expression
3’-Amplification One-Cycle cDNA Synthesis Kit, cleaned using the Sample Cleanup Module,
and biotinylated cRNA was synthesized with GeneChip Expression 3’-Amplification Reagents
for IVT Labeling, cleaned on RNA Sample Cleanup columns, and fragmented at 94 °C for
35 min in Fragmentation Buffer. The biotinylated cRNA was hybridized first to a control Test3
microarray to evaluate its quality and then to a Human Genome U-133 Plus 2.0 microarrays
containing 54675 probe sets, using GeneChip Expression 3’-Amplification Reagents.
Microarrays were stained with streptavidin–phycoerythrin conjugate and scanned in a Gene
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