Page 18 - TD-3-1

Tumor Discovery Mobile phone effects on testis

derived. Over the course of 8 weeks, the rats were exposed were calculated by entering Ct data into Microsoft Excel. For
to RF-EMF at 900 MHz, 26 V/m, and SAR 0.14 W/kg for statistical analyses, the Gene Globe Data Analysis Center
4 h/day. At the end of the 8-week exposure period, all the (Qiagen, Germany) online analysis program was utilized.
rats from both the control and study groups were sacrificed,
and orchiectomy procedures were performed. Testicular 2.3. Vitality determination using flow cytometry
weight and volume were recorded, followed by storage of Fresh tissue samples from both the control and study
testis tissue for subsequent histopathological examination, groups were cut into pieces and separated in sterile
DNA analysis, and apoptotic cell analysis. Petri dishes. They were centrifuged twice for 5 min at
400 relative centrifugal force, resulting in the suspension
2.2. Real-time polymerase chain reaction (PCR) gene of cells at the end of these processes. After the final
expression analysis centrifugation, the cell pellet was resuspended with 100 μL
Gene expression profiling for IL-1, IL-4, IL-10, TNF- α, of a binding solution, resulting in a 10-fold dilution,
and IFN-γ was conducted using primer-based designed and maintained on ice. Next, 5 μL of Annexin V-FITC
specifically for these genes, following the protocols outlined solution (Beckman Coulter, France) was added to the cell
below. suspension and incubated on ice for 10 min in the dark
following gentle mixing. After incubation, 400 μL of the
2.2.1. RNA isolation from tissue binding solution was added, and readings were obtained
Tissue sections with a thickness of 10 μm were collected, and using a flow cytometry device (Cytoflex, Beckman Coulter,
the isolation protocol was performed in accordance with USA). Subsequently, Annexin V-FITC-positive cells were
the manufacturer’s directions (RNeasy FFPE Kit, Qiagen, selected, and apoptotic and necrotic cells were determined.
Germany). Following isolation, RNA concentration was Cells selected from the side scatter (SSC)/forward scatter
measured. (FS) graphics and analyzed in Annexin V/PI graphics.

2.2.2. cDNA synthesis protocol 2.4. Histopathological examination of testicular
tissues
Ten microliters of RNA were transferred to a 0.2 ml PCR
tube and incubated at 65°C for 5 min in a PCR instrument The testis resection samples intended for histopathological
(VeritiPro Thermal Cycler, ThermoFisher Scientific, USA). examination were initially fixed using 10% formaldehyde.
A cDNA mix was prepared with 7.5 μL of RT mix and Subsequently, five-micron-thick sections were obtained
2.5 μL of reverse transcriptase, followed by initiation of from the paraffin-embedded tissues. These tissue
the cycle in the PCR instrument. The High-capacity cDNA sections, prepared on specialized slides, were stained
Reverse Transcription Kit (ThermoFisher Scientific, USA) with hematoxylin and eosin (H&E) staining using
was used for cDNA synthesis. an automated staining device (Leica ST 5020, Leica,
Germany), followed by automated closure using another
2.2.3. Real-time PCR protocol device (Leica ST 5030, Leica, Germany). In addition, tissue
Real-time PCR experiments were performed using the samples were subjected to histochemical analysis using
GoTaq RT-PCR kit (Promega, USA) and SYBR Green Masson’s trichrome method. Immunohistochemical (IHC)
chemistry (Bio-Rad, USA). The primer sequences used in staining was performed on five-micron-thick sections of
this study are provided in the Appendix. Both procedures formalin-fixed, paraffin-embedded tissue using a SALL4
were conducted according to the manufacturer’s antibody (Cellmark 6E3 Mouse Monoclonal Antibody,
instructions. A mixture comprising 20 μL RT-PCR mix Cell Marque, USA). The BenchMark XT visualization
and 5 μL cDNA was dispensed into each of the reaction system with enzymatic digestion ISH protease 2 (Ventana,
tubes, which contained samples, negative control, positive USA) and the iView Blue Detection Kit (Ventana, USA)
control, and standards, respectively. Subsequently, the were employed for visualization. Evaluation of H&E
RT-PCR protocol was started in the PCR instrument. and histochemical specimens was conducted using an
Upon completion of the RT-PCR protocol, the data Olympus BX46 light microscope. Resection samples were
obtained were analyzed using the delta cycle threshold assessed for seminiferous tubule structure, Johnson score,
(Ct) (comparative Ct method [∆Ct], 2-DDCt) method. Ct tunica albuginea thickness, interstitial edema, and Leydig
values for both the control and study groups were obtained cells. Tunica albuginea thickness and seminiferous tubule
from experiments performed using the qRT-PCR Rotor- diameter were measured using the Aperio ImageScope
Gene Q (Qiagen, Germany) device to determine changes program. Seminiferous tubule damage, interstitial edema,
in the expressions of IL-1, IL-4, IL-10, TNF- α, and IFN- γ and Leydig cells were scored semiquantitatively on a scale
genes. The changes in the expressions of the relevant genes ranging from 0 to 3.

Volume 3 Issue 1 (2024) 3 https://doi.org/10.36922/td.1703