cDNA Experiment Results

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Materials and Methods: Subjects The cDNA was produced by reverse transcriptase from total RNA by using superscript II (Invitrogen). The cDNA were from the lung which was obtained commercially from Ambion, HASM which was extracted in house and HBEC which was obtained commercially from Lonza. Primer Design Oligonucleotide primers were designed by obtaining the reference sequences for uPA, PAI-1 and PAI-2 from National Centre for Biotechnology Information (NCBI). The sequences were checked using NEBcutter (V2.0) to identify the common restriction enzymes sites that are not included in the sequences to be able to clone into pcDNA3. The Oligonucleotide primers were evaluated by using PCR primer stats program. In addition, a kozak sequence was attached to the 5' primer end of each forward primer to ease recombinant expression and include an appropriate restriction site and 'spacer'. Standard PCR condition PCR was carried out in a total volume of 20 µl containing 1x PCR high fidelity buffer (invitrogen), 2 mM of magnesium, 200 µM of deoxyribonucleotide triphosphate (dNTP), 0.03 U of platinum Taq DNA polymerase (invitrogen), 0.2 µM of each forward and reverse primer (Promega), 11.48 µl of water and 4 µl of the extracted genomic DNA. An uPAR cDNA sample from a previous experiment was used as a positive control for the test. In addition, a negative control sample was included by using DNA-free sample. All samples were subjected to the following condition: a denaturation step for 1 minute at 94ºC, followed by 30 cycles of annealing step for 1 minute at 60ºC and extension step for 2 minutes at 72ºC using PCR Biometra TRIO Thermocycler. PCR products were separated on 1% agarose gel and 1xTAE buffer for 45 minutes at 46V. The PCR p... ... middle of paper ... ...e medium and incubated overnight. After 24 hours, three plates were replaced with fresh serum free medium and two plates with complete growth medium. Then, the MTT proliferation assay was performed in three stages. First, one of the free serum plates was tested in the same day. Second, one serum free medium and complete growth medium plates were examined after 24 hours. Third, the remaining serum free medium and complete growth medium plates were analyzed. This assay was carried out by replacing the medium with 0.5 ml yellow MTT solution (Sigma) and incubated for two hours. Then, the MTT solution was replaced with 0.6 ml of MTT solvent (isopropanol) (BDH) and the plates were placed on a gyratory shaker for 15 minutes. Each well was split in triplicate in a 96 well plate in equal volumes. The absorbance was measured using plate reader at a wavelength of 570 nm.
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