DEVELOPMENT OF MULTIPLEX RT-QPCR IN URINE SPECIMEN FROM LUPUS NEPHRITIS PATIENTS
Keywords:
Multiplex Reverse Real-Time PCR Technique, Urine, Lupus Nephritis, Multiplex Reverse Real-Time PCR Technique, Urine, Lupus NephritisAbstract
This study developed an in-house Multiplex RT-qPCR method to measure mRNA from four gene sets: Set 1 (18s rRNA, MCP-1), Set 2 (IP-10, VEGF), Set 3 (FoxP3, NGAL), and Set 4 (GATA3, TBX-21). The method was applied to urine
samples from LN patients for clinical analysis.
Method: Comparison between the Multiplex and Single RT-qPCR methods used ΔCt and correlation values, evaluated with linear regression. Urine samples from 15 LN patients (Active and Non-active groups) were tested.
Conclusion: Results indicated ΔCt values for VEGF, NGAL, and TBX-21 were acceptable; 18s rRNA, IP-10, and GATA-3 were slightly outside limits; MCP-1 and FoxP3 showed variability with many values outside limits. Correlation tests revealed no significant differences for genes 1, 2, and 4 between methods, except for low correlation in FoxP3 (Group 3). Comparing mRNA levels with LN patient characteristics, MCP-1, IP-10, VEGF, NGAL, and TBX-21 were significantly higher in active LN and lower in non-active LN. Conversely, GATA-3 levels were higher in non-active LN and lower in active LN. Due to RT-qPCR inefficiency, FoxP3 mRNA levels were not analyzed in LN patients.
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