CHAPTER II MATERIALS AND METHODS 2.1 Chemicals and ...

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2.10.3 cDNA synthesis by reverse transcription
Total RNA prepared in step 2.10.2 was primed with oligo-(dT)18 primer to
synthesize first-stranded complementary DNA using AMV reverse transcriptase
(Fermentus), according to the manufacturer’s instructions. Briefly, The 20 µl reaction
mixture contained 1 µg of extracted RNA, 0.5 µg of oligo-(dT)18, 1 mM dNTPs, 20
unit of ribonuclease inhibitor, 200 units of reverse transcriptase, and adjusted volume
to 20 µl with DEPC-treated water. The reaction mixture were incubated at94 o C for 5
min, 42 o C for 1 h and then at 70 o
C for 10 min. The extracted RNA was converted to
cDNA in these processes.
2.10.4 PCR optimization
The PCR condition of interested genes (VEGF and GAPDH) was optimized to
yield the amplified products without any nonspecific byproduct. The annealing
temperature was varied within the range from 58using a fixed number of
PCR cycle at 30. Next, the optimum PCR cycle was analyzed by various of numbers
PCR cycle from 15 to 33 cycles that could give the proportional PCR product relative
to its original cDNA. The chosen PCR condition, the annealing temperature and the
number of PCR cycle, was confirmed by 2-fold dilution series of cDNA template: 1x,
0.5x, 0.25x, and 0.125x.
The procedures and optimized protocols are as follows: The 10 µl reaction
mixture contained 1.0 µg of cDNA, 1X buffer (10 mM Tris-HCl pH 9.0, 50 mM KCl
and, 2.0 mM MgCl2),
0.2 mM dNTPs, 2 units of Taq polymerase, and 0.2 µM of
primers. Each reaction mixture was then placed on a Mastercycler gradient thermal
cycler. The temperature profile was as follows: initial denaturation at 95 C for 5