Parkinsonism Quality Control in Mitochondria Determined by Real-Time Reverse Transcription Polymerase Chain Reaction

The primary causes of mitochondrial disease are nuclear DNA mutations, mitochondrial DNA mutations, combined nuclear and mitochondrial DNA defects, and random events. A prominent indicator of Parkinsonism is loss of black pigmentation. It falls to less than 80% due to the substantia nigra's loss of dopaminergic neurons. There was a correlation between the amount of black pigmentation loss and the degree of motor impairment.

Since reverse transcriptase was utilized to produce cDNA, reverse transcription is done after RNA extraction. A microliter of total RNA supplemented with nuclease-free, random hexamers, IORT Buffer, and Multi-Scribe Reverse Transcriptase was utilized to produce cDNA. In a thermal cycler, the reaction was run for the following durations: 10 minutes at 25°C, 120 minutes at 37°C, 5 seconds at 85°C, and finally 4°C.

The information was presented as a v-value, indicating the degree of enrichment of the target gene relative to the reference gene. Previous studies with mice injected with functional HtrA2/Omi protease activity showed comparable results, such as increased mtDNA deletions leading to early brain cell aging, reduced mitochondrial function, and induced up-regulation of CHOP.

These results show that the cell viability of the genotypes exhibits a dmg concentration-dependent effect, indicating that cellular content depletion may be caused by high content ratios of drug treatment with 6-OHDA. CHOP is up-regulated following treatment with 6-OHDA neurotoxin. Previous studies conducted in vitro have demonstrated that treatment with a model based on 6-OHDA neurotoxin activates pathways associated with mitochondrial stress, which further promotes neuronal death. In rat brain cells, 6-OHDA reversibly inhibited the activities of complexes I and IV, resulting in dopaminergic neurodegeneration—a characteristic that distinguishes PD pathogens. Consequently, the quantity of living cells will directly correspond with the concentration of dmg, thereby corroborating the findings of this investigation.

When compared to WT cells, real-time PCR can typically show that the deletion of HtrA 2 significantly upregulates the expression of Hsp60. This may be related to a lower threshold for mitochondrial stress in HtrA2 KO cells, which would account for the significant up-regulation of Hsp60 in HtrA2 KO cells after ADEP4 and ACP5 treatment, in comparison to WT cells.


Key words:

RT: real time, PCR: polymerase chain reaction, WT: wild type, dNTP: deoxynucleotide triphosphate