Preparation and Classifying Animals
Male albino Wistar rats weighing 220 ± 20 g were purchased from an Animal laboratory of Tehran University of Medical Sciences. The animals were kept at 22 ± 2 ºC and 55 ± 5% humidity. Standard feeding, and 12-hour light/ 12-hour dark cycle were used. These animals had access to enough water and food, except the time they were taking drugs. The Ethics Committee approved the protocols used in this study of Tehran University of Medical Sciences. The animals were anesthetized and then sacrificed by cervical dislocation. Animals were randomly classified into six groups of six; in the form of experimental groups, non-radiation control groups, and radiation control groups As follows:
- The control group (no radiation and probiotic consumption).
- without radiation, with the use of Lactobacillus Casei probiotics.
- Radiation and use of Lactobacillus Casei.
- No radiation, with probiotic consumption of Lactobacillus acidophilus.
- Pollution and use of Lactobacillus acidophilus.
- With radiation-without probiotics, test124 groups and control groups consumed probiotics as gavage for 28 working days and standard food, respectively.
As each gram of every bacterium CFU/g (. So, for each animal, the calculated equivalent dose of the drug was dissolved in 1 ml of PBS buffer with a pH of about 7.2, Followed by garaging for three times a day for Lactobacillus Casei and once a day for Lactobacillus acidophilus. It was also gavaged at the same level as the PBS buffer for the control group without medication.
At the end of 28thworking day, all groups were exposed immediately after anesthesia, at the Department of Radiation therapy, Cancer Institute, Imam Khomeini Hospital Complex in Tehran, Radiation was performed using a machine with the dose and dose rate of 2 Gy and 100cGY/min respectively. At the same time, FOV was set to 36x36 and SSD was 80cm.
Immediately after exposure, the animals were transferred to the Animal lab of the Tehran University of Medical Sciences and were Dissected By cervical dislocation method, under ethical considerations. After extracting the testicle, it was placed in a 2.5 microtube and transferred immediately to the nitrogen tank. Samples were stored at -80 ℃ until the total RNA was extracted.
Total RNA Extraction from Testicular Tissue
Firstly, the tissue was removed from liquid nitrogen .1ml of Trysol was added to the microtubule containing chopped tissue. Vortexing of cells was then performed to make the sample wholly lysed. In the subsequent stage, incubation was done either at the temperature of the room or by placing on the ice. The next step was phase separation, which included: 1) adding 200 landa cold chloroform. 2) shaking for 15 seconds and 3) incubation on 4C ice.
The microtube was centrifuged in a refrigerated centrifuge machine for 15 minutes at a temperature of 4° C and 12000 rpm. The supernatant was carefully is separated RNAs-Free 1.5 ml tube at 12,000 rpm for 15min centrifuges at a temperature of 4 ° C. After the supernatant was poured into a new microtube, the same amount of isopropanol was added to be slowly mixed in the next stage. The centrifuge was then run at 12,000 rpm for 15 minutes at 4 ° C. after that, we removed the supernatant and added 1 ml of cold ethanol 75 ml. In next step, the centrifuge was run at 7500 rpm for 8 minutes at 4 ° C. Again the superficial fluid was discarded to dissolve the RNA, Which then was dried at room temperature for 5 to 10 minutes, and was suspended by adding 20 to 50 μl of RNase-free water and pipetting, before stirring the microtube for 10-15 minutes in a water bath with the incubation temperature of 55-60 ° C. finally we removed four lambda, two landa from the gel and two lambda for OD reading using a nanodrop machine.
Control of the concentration and quality of RNA extraction steps
The biotech Nanodrop was applied and. The ratio of 280 / 260OD was considered as the sign of the purity of the RNA extracted. The more it became closer to 2, the more the purity of the sample is deemed to be acceptable. Moreover, the 230 / 260OD ratio is regarded as the extraction of RNA contamination with the kit. Likewise, being closer to 2 is also more desirable. Ultimately, to assess the quality of RNA, it was extracted using 1% agarose gel electrophoresis and stained with ethidium bromide.
Hprt was used for the quality control of RNA extraction processes and cDNA synthesis. The samples which were contained a copy of hprt were regarded as positive samples for the next steps. Master mix is then poured into every Micro USB, and following that one microgram of RNA extracted tube is used. RNA sample can be calculated based on the concentration of the sample to be poured into microtubes in the later step. All steps were performed on the ice. The tubes were placed in a thermocycler, and the cDNA synthesis was performed according to the Kit Takara instruction and according to the following table. Then cDNA was synthesized in a freezer -20 ° C.
The primer for Bax, Bcl2, caspase3 genes as well as hprt used in this study was based on the above table principles, The primers were designed using the NCBI and the Gene Runner software, and were developed by Sinacolone with OD 2. According to the Sinacolone protocol, Primers were diluted and elaborated as follows:
First, the appropriate amount of sterile distilled water was added to the primer, to reach a focus of 100 μm. Then, for 5 minutes, they were incubated at room temperature. To obtain a concentration of 10 μM, again, 90 μl sterilized distilled water was added to a ten μL primer. The optimal focus of the introduction for these reactions is one μm.
Steps to Real-Time PCR
In the beginning, the ingredients were removed from the freezer to thaw slightly at room temperature. Subsequently, according to the instructions, the ingredients were carefully mixed, and after adding every one of them, we spin slightly to ensure that it is well mixed. The reaction volume is 20 μL. Streeps (0.1 microtubes) were placed carefully and by using pansies in the Cool Box, which were removed out of the freezer. The 6000 Corbett Rails Time Machine obtain the results.
The mean of each of the variables was statistically analyzed using one way ANOVA and Tukey post hoc test. P <0.001 was considered as meaningful level.