LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

SYNOPSIS
2.3 Irradiation
2.3.1] Low LET: For gamma irradiation, cells were exposed to required dose of
60 Co γ radiation in in-house facility Gamma Cell 220 at dose rate of 3.5 Gy/min.
Proton beam irradiation was carried out using the Radiation Biology beam line of
in-house 4 MeV Folded Tandem Ion Accelerator (FOTIA) facility.
2.3.2] High LET: Heavy ion irradiation was carried out using the Radiation
Biology beam line of 16 MV 15UD Pelletron at Inter University Accelerator Centre, New
Delhi.
2.4 Clonogenic Cell Survival Assay
After treatment, cells were seeded out in appropriate dilutions (500 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.
2.5 Semiquantitative Reverse transcriptional polymerase chain reaction (RT-PCR)
Total RNA from 1 x 10 6 cells was extracted after required time periods of irradiation and
RT-PCR was carried out. Briefly, total RNA from A549 cells was extracted after required
time periods of 1, 2, 3 and 4 hours 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 required genes using gene specific primers.
The PCR condition were 94 o C, 5 min initial denaturation followed by 30 cycles of 94 o C
45 s, 55 – 64 o C 45 s (depending on the annealing temperature of specific primers) 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).
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