LIFE09200604007 Tabish - Homi Bhabha National Institute

Materials and Methods
care that there were no air bubbles. 40 µl 3X SSC buffer was put in the grooves of the
chamber. Chamber was screwed tight and placed in the water bath at 42 o C overnight.
3.9.8 Washing:
Washing Solution
20x SSC SDS Water
2X SSC 50 ml 2.5 ml 500 ml
1X SSC 25 ml 2.5 ml 500 ml
0.2X SSC 5 ml 2.5 ml 500 ml
0.2X SSC 5 ml - 500 ml
The hybridisation chambers were removed from 42 °C water bath and
whipped carefully with tissue paper. The slides were directly opened in 2x SSC with
SDS and coverslips were removed carefully. The slides were kept in slide rack and
washed in 2x, 1x and 0.2x SSC with SDS and 0.2x SSC without SDS for 3, 5, 5, and 3
minutes respectively. After washing the slides were dried by centrifugation at 1500 rpm
/5 min/ RT and the fluorescent images of hybridized microarrays were obtained using a
GenePix 4200A microarray scanner (Axon Instruments) within a couple of hours.
Primary data analysis for determining differential expression of genes between was
carried out using the Genepix Pro software version 5.0 (Axon Instruments).
3.10 Quantitative RT-PCR
Materials:
1. Power SYBR Green
2. cDNA
3. gene specific primers
4. Distilled sterile water
Method:
Differential expression of genes was validated by Quantitative RT-PCR.
Invitrogen RT-PCR kit was used to reverse transcribe 3 µg of total RNA using both
oligo dT and random primers in 30 µl reaction volume as described in section 4.3.4.
The synthesized cDNA was diluted to a final concentration of 10 ng/µl with nuclease
free H 2 O and 4 µl was used as a template for real time reactions. A reaction mixture
containing 40 ng cDNA, 2.5 μM of each forward and reverse primers and 5 µl of 2x
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