Banca de DEFESA: GABRIEL GINANI FERREIRA

Uma banca de DEFESA de DOUTORADO foi cadastrada pelo programa.
STUDENT : GABRIEL GINANI FERREIRA
DATE: 27/02/2025
TIME: 14:00
LOCAL: A definir, em função da banca
TITLE:

“Analysis of serum microRNAs in patients with REM sleep behavior disorder: evaluation of RNA purification and quantification methods via RT-qPCR, and assessment of miR-7 and miR-19b relative expression”

KEY WORDS:

REM Sleep behavior disorder, Parkinson’s disease, microRNA, biomarker, RT-qPCR, miR-7, miR-19b.

PAGES: 100
BIG AREA: Ciências da Saúde
AREA: Medicina
SUMMARY:

The discovery of molecules with altered expression in the peripheral blood of patients diagnosed with neurodegenerative disorders is a significant topic in neurology, both for diagnostic and clinical prognostic purposes. This concept is applicable to REM sleep behavior disorder (RBD), a condition characterized by dream enactment, which can progress to other neuropathologies. Characterizing molecules with aberrant expression will aid in better characterizing this disease and identify individuals at risk of phenoconversion from RBD to synucleinopathies, such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Among the potential blood biomarkers of RBD are microRNAs, which are non-coding RNAs that regulate the content of messenger RNAs at a post-transcriptional level. However, the precise detection of miRNAs, especially those that are less abundant, requires meticulously standardized scientific assays to purify RNAs from other constituents in the serum and to quantify the targets of interest through RT-qPCR. This study comparatively examined two RNA purification methodologies and two RT-qPCR methodologies aimed at quantifying microRNAs relevant to neurobiology and the diagnosis of RBD: miR-7 and miR-19b. Two experimental groups were used: individuals diagnosed with probable RBD and healthy control individuals. Initially, it was observed that RNA purification using the guanidine thiocyanate method (miRNeasy Serum/Plasma Advanced Kit, Qiagen), method 1, yielded significantly superior results compared to the conventional methodology involving aqueous and organic phase separation by phenol-chloroform (miRNeasy Serum/Plasma, Qiagen), method 2. The purification methods 1 and 2 yielded, respectively, 1.46 ng/µL versus 0.75 ng/µL (P<0.05) in the control group, and 2.22 ng/µL versus 0.639 ng/µL (P<0.05) for the group diagnosed with RBD. Regarding the RT-qPCR methodologies, both used the TaqMan system and were executed with commercial kits (Applied Biosystems), with reactions calibrated by the endogenous control miR21 and the exogenous spike-in control miR-39 from C. elegans. Two RT-qPCR systems were compared: the traditional method, which uses target-specific reverse transcription, and the Advanced system, which performs pre-amplification of the RNA transcripts. The Advanced system provided superior results for miR-7, with CT (Threshold cycle) values below 30, while in the traditional system, CT values were close to 35, a value considered limiting for qPCR precision. The expressions of miR-7 and miR-19b were significantly higher in the RBD group compared to the control group, with accuracy values (area under the curve, AUC) of 0.93 (P=0.0176) and 0.89 (P=0.025), respectively, as determined by the ROC (Receiver Operating Characteristics) curve methodology. The combined analysis of miR-7 and miR-19b as a microRNA signature also showed satisfactory accuracy with an AUC of 0.86 (P=0.0374). In conclusion, our study demonstrated that it is both crucial and feasible to refine the purification and qPCR steps to improve the quality of microRNA quantification in the serum of individuals with RBD. The guanidine thiocyanate purification methodology provided superior RNA yields from the serum, and the Advanced amplification system allowed for adequate CT values, particularly for less abundant microRNAs such as miR-7. The two studied targets, miR-7 and miR-19b, individually or combined, calibrated by miR-21 and cel-miR-39, showed the capability to identify RBD patients with accuracy above 85%. Subsequent tests in a new cohort with a larger number of individuals should be conducted to validate this promising miRNA signature, aiming to identify individuals with RBD. Additionally, these new tests will seek to determine if miR-7 and miR-19b can serve as accurate biomarkers for the phenoconversion of RBD into neurodegenerative disorders such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy.

COMMITTEE MEMBERS:
Externo ao Programa - 1307139 - FABIANO JOSE FERREIRA DE SANT ANA - nullExterno ao Programa - 1156673 - FELIPE VON GLEHN SILVA - nullExterno à Instituição - FERNANDO FRANCISCO BORGES RESENDE - UNICEPLAC
Externo à Instituição - RAIMUNDO NONATO DELGADO RODRIGUES
Presidente - 1127261 - RICARDO TITZE DE ALMEIDA