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INNOSC Theranostics and
Pharmacological Sciences BDNF-AS biomarker in multiple sclerosis

2.2. Characteristics of subjects 2.7. Complementary deoxyribonucleic acid (cDNA)

All participants were diagnosed with MS based on the synthesis
guidelines set forth by the International Panel on the cDNA was synthesized from 2.5 µg of total RNA using
Diagnosis of MS, employing the McDonald’s criteria the QIAGEN RT² First Strand Kit (catalog number
(2010). A total of 74 participants were included in the 330404, Hilden, Germany), following the manufacturer’s
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study, comprising 31 cases from the RRMS group, 13 from guidelines. The process involved three stages: (i) genomic
the SPMS group, 10 from the PPMS group, and 20 healthy DNA removal at 42°C for 5 min, (ii) reverse transcription
volunteers as controls. The RRMS and SPMS groups were at 37°C for 60 min to optimize reverse transcriptase
subdivided as follows: 16 with relapsing RRMS, 15 with activity, and (iii) enzyme denaturation at 95°C for 5 min.
remitting RRMS, eight with relapsing SPMS, and five All thermal cycles were performed using the SimpliAmp™
with remitting SPMS. The degree of clinical disability was Thermal Cycler (Applied Biosystems, Singapore).
evaluated using the Kurtzke expanded disability status
scale (EDSS). 34 2.8. Real-time polymerase chain reaction (PCR)
2.3. Exclusion criteria Real-time PCR was used to evaluate the relative gene
expression of the target gene (BDNF-AS) and the
Individuals were deemed ineligible for the research if they housekeeping gene (GAPDH). Pre-designed primers from
had any of the following conditions: cancer, liver disease, QIAGEN (catalog number 330701, Hilden, Germany)
diabetes mellitus, autoimmune disorders, hypertension, were utilized, with the following identifications for
cerebrovascular disease, pregnancy, infectious or each gene: GAPDH (LPH15126A-200) and BDNF-AS
inflammatory diseases, a history of alcohol abuse, or (LPH15814A-200). The amplification was carried out
smoking, based on the specified eligibility criteria.
using QIAGEN’s RT² SYBR Green ROX quantitative PCR
2.4. Demographics and disease characteristics in MS Mastermix (catalog number 330520, Hilden, Germany).
subjects Forty amplification cycles were performed, each
consisting of 15 seconds at 95°C and 1 min at 60°C. After
Clinical data collected from all participants in this study amplification, a melting curve analysis was performed
were as follows: to assess the PCR products. All processes were carried
(i) Frequency of relapses in the previous 3 years out using the Real-Time PCR QuantStudio™ 1 (Applied
(ii) Age and sex
(iii) EDSS score Biosystems, Singapore).
(iv) History of family MS or other autoimmune diseases 2.9. Relative gene expression
(v) Current disease status (relapse/remitting) at the time
of sampling The relative expression of BDNF-AS was calculated for
(vi) History and duration of MS each sample using the double-delta cycle threshold (Ct)
(vii) Patient’s clinical history. method. The Ct values of all samples were normalized
against GAPDH. The fold change for each sample was
2.5. Specimen collection calculated and compared between patients and normal
Five milliliters of blood were drawn from both MS patients controls to investigate the relative expression of BDNF-AS.
and the 20 healthy volunteers. The blood samples were 2.10. Statistical analysis
incubated for 30 min at 37°C and then centrifuged for
10 min at 3000 rpm. The resulting serum was divided Statistical analyses were conducted using Statistical
into two fractions and stored at −80°C until required for Package for the Social Sciences Statistics (IBM, version 26,
analysis. USA). A normality test was performed on all data to
differentiate between non-parametric and parametric
2.6. RNAs extraction data. The non-parametric Kruskal–Wallis test was utilized

RNAs were extracted using the QIAGEN serum and plasma to evaluate variations among groups. Multiple comparison
RNA extraction kit (catalog number 217184, Hilden, tests were applied to identify the source of any significant
Germany). A 200 µL serum aliquot was processed according differences between groups. The non-parametric
to the manufacturer’s instructions. Approximately 14 µL of Spearman’s correlation test was used for correlation
RNA was eluted, and the purity and concentration were analysis. Receiver operating characteristic (ROC) curve
assessed using the One/One Microvolume ultraviolet-Vis analysis was also performed. A P < 0.05 was considered
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C
NanoDrop™ Spectrophotometer (ThermoFisher, Singapore). statistically significant. Microsoft software was used to
The eluted RNA was stored at −80°C until needed. create all graphs and tables.

Volume 8 Issue 1 (2025) 93 doi: 10.36922/itps.4407