CHAPTER II MATERIALS AND METHODS 2.1 Chemicals and ...

More documents

Recommendations

Info

33 seconds and incubated at 15 to 30°C for 2 to 3 min. After centrifugation at 12,000×g for 15 min at 4°C, the mixture was separated into a lower red, phenol-chloroform phase, an inter-phase, and a colorless upper aqueous phase. RNA remained exclusively in the aqueous phase, which is about 60% of the volume of TRIzol Reagent used for homogenization. The aqueous phase was transferred to a fresh tube. The RNA was precipitated from the aqueous phase by mixing with isopropyl alcohol, incubated at 15 to 30°C for 10 min and centrifuged at 12,000×g for 10 min at 4°C. The RNA precipitate, often invisible before centrifugation, formed a gel-like pellet on the side and bottom of the tube. After removing the supernatant, the RNA pellet was washed once with 75% ethanol, mixed by vortexing and centrifuged at 7,500×g for 5 min at 4°C. At the end of the procedure, the RNA pellet was dried by air-dry or for 5- 10 min and then dissolved in DEPC-treated water. The quantity and quality of total RNA were assessed by the ratio of A260 /A280. The concentration of RNA was determined by UV-Visible spectrophotometer. An OD of 1 at the wavelength of 260 nm corresponds to approximately 40 µg/ml for RNA. The obtained RNA was diluted 50 times in DEPC-treated water before measuring. The concentration of RNA was calculated by using the formula below: Concentration of RNA (µg/ml) = OD260 x 40 x dilution factor The quality of the RNA is essential to overall success of the analysis. OD260 is frequently used to measure RNA concentration and OD280 is used to measure protein concentration. For further analysis it is imperative that the RNA extracted should have high purity, displaying ratio of OD260/OD280 values between 1.7 and 1.9. Smaller ratio usually indicates contamination of protein or organic chemicals.
34 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
Page 1 and 2: 2.1 Chemicals and materials CHAPTER
Page 3 and 4: Name 24 Table 2.2: Sequences of oli
Page 5 and 6: 2.6 DNA polymerase stop assay 26 DN
Page 7 and 8: 2.9 Cytotoxicity test 28 Cell survi
Page 9 and 10: 30 with 5% CO2. The first plate, as
Page 11: 32 Table 2.3 Primer sequences for d
Page 15 and 16: 36 2.11.1 Preparation of total cell
Page 17 and 18: 38 through polyacrylamide gel. Smal

CHAPTER II MATERIALS AND METHODS 2.1 Chemicals and ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?