LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

CHAPTER 2
MATERIALS AND METHODS
expression between the arrays. The minimum correlation between the samples was 0.987, which
was above the acceptable criterion of 0.8.
2.6 Semiquantitative Reverse transcriptional polymerase chain reaction (RT-PCR)
Total RNA from all the groups of cells (1 x 10 6 cells) was extracted after required time periods
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 β-actin, NF-κB
(p65), iNOS, p21, p53, ATM, DNA-PK, ATR, MLH1, Rad52, GADD45α, Toll-like receptor 3
(TLR3), Ferredoxin reductase (FDXR), Chk1, Chk2, Bcl-2 or Bax gene using gene specific
primers (Table 2.6). The PCR condition were 94 o C, 5 min initial denaturation followed by 30
cycles of 94 o C 45 s, 55 – 62 o C 45 s (depending on the annealing temperature of specific primers
given in Table 2.6), 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).
2.7 Immunofluorescence staining
Cells were grown in cover slips which were kept in 35mm petri dishes and irradiated as
mentioned above. Cell layer were washed at different time period after irradiation in PBS and
fixed for 20 min in 4% paraformaldehyde at room temperature. Afterwards, the cells were
washed twice in PBS. For immunofluorescence staining, cells were permeabilized for 3 min in
0.25% Triton X-100 in PBS, washed two times in PBS and blocked for 1 h with 5% BSA in
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