| Authors: |
Adel El-Sayed El-Tarras1,2,3, Hossam Fouad Attia1,4,5,
Mohammed Mohamed Soliman1,4,5,
Mohammed Abdelhamid El Awady1,2,3
and Adnan Abelghani Amin1,6 |
| Journal: |
International Journal of Immunopathology and Pharmacology 29(3) Serum analysis Catalase, GR, GP, and MDA were measured using commercial spectrophotometric analysis kits (Bio- Diagnostic Company, Giza, Egypt). The MAO and acetylcholinesterase (AChE) levels were measured using ELISA commercial kits (San Diego, CA, USA). All of the procedures followed the manufacturers’ instructions. Gene expression RT-PCR analysis of the following genes was performed and the expression levels semi-quantified: GST, GPx, MAO-A, dopamine (DD2R), 5-hydroxytryptamine transporter (5-HTT), and AChE. RNA extraction, complementary deoxyribonucleic acid (cDNA) synthesis and semi-quantitative PCR analysis Total RNA was extracted from the brain tissue samples described previously.24 The RNA concentration and purity were determined spectrophotometrically after measuring the OD at 260 and 280 nm. The RNA integrity was confirmed after running in 1.5% denaturated agarose gel stained with ethidium bromide. The 260/280 optical density ratio of all of the RNA samples was 1.7-1.9. A mixture of 3 μg total RNA and 0.5 ng oligo dT primer (Qiagen Valencia, CA, USA) was used for cDNA synthesis in a total volume of 11 μL using sterilized DEPC water and incubation in the Bio-Rad T100TM Thermal Cycle at 65°C for 10 min for denaturation. Next, 2 μL of 10X RT-buffer, 2 μL of 10 mM dNTPs, and 100 U Moloney Murine Leukemia Virus (M-MuLV) Reverse Transcriptase (SibEnzyme. Ak, Novosibirsk, Russia) were added and the total volume was adjusted to 20 μL with DEPC water. The mixture was then re-incubated in Bio-Rad thermal cycler at 37°C for 1 h, and then at 90 °C for 10 min to inactivate the enzyme. For semi-quantitative RT-PCR analysis, specific primers for the examined genes (Table 1) were designed using the Oligo-4 computer program and were synthesized by Macrogen (Macrogen Company, GAsa-dong, Geumcheon-gu, Republic of Korea). PCR was conducted in a final volume of 25 μL consisting of 1 μL cDNA, 1 μL of 10 pM stock of each primer (forward and reverse), and 12.5 μL PCR master mix (Promega Corporation, Madison, WI, USA), the volume was adjusted to 25 uL using sterilized, deionized water. The PCR was carried out using Bio-Rad T100TM Thermal Cycle with a cycle sequence of 94°C for 5 min for one cycle, followed by 31 cycles (Table 1) consisting of denaturation at 94°C for 1 min, annealing at the specific temperature corresponding to each primer (Table 1) and extension at 72°C for 1 min with an additional final extension at 72°C for 7 min. As a reference, expression of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was examined (Table 1). The PCR products were visualized under UV light after electrophoresis on a 1.5% agarose (Bio Basic, Markham, ON, Canada) gel stained with ethidium bromide in TBE (Tris-Borate-EDTA) buffer. The PCR products were photographed using a gel documentation system. The intensities of the bands were quantified |
