Identifying the stability of housekeeping genes to be used for the quantitative real-time PCR normalization in retinal tissue of streptozotocin-induced diabetic rats
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Nurul Alimah Abdul Nasir. Centre for Neuroscience Research (NeuRon), Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh Campus, Sungai Buloh, Selangor 47000, Malaysia. nurulalimah@uitm.edu.my

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Supported by grant from Fundamental Research Grant Scheme by Ministry of Higher Education (MoHE) 600-IRMI/FRGS 5/3 (101/2019).

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    Abstract:

    AIM: To investigate the stability of the seven housekeeping genes: beta-actin (ActB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18s ribosomal unit 5 (18s), cyclophilin A (CycA), hypoxanthine-guanine phosphoribosyl transferase (HPRT), ribosomal protein large P0 (36B4) and terminal uridylyl transferase 1 (U6) in the diabetic retinal tissue of rat model. METHODS: The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) in two groups; normal control rats and streptozotocin-induced diabetic rats. The stability analysis of gene expression was investigated using geNorm, NormFinder, BestKeeper, and comparative delta-Ct (ΔCt) algorithms. RESULTS: The 36B4 gene was stably expressed in the retinal tissues of normal control animals; however, it was less stable in diabetic retinas. The 18s gene was expressed consistently in both normal control and diabetic rats’ retinal tissue. That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats. Furthermore, there was no ideal gene stably expressed for use in all experimental settings. CONCLUSION: Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.

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Muhammad Zulfiqah Sadikan, Nurul Alimah Abdul Nasir, Mohammad Johari Ibahim, et al. Identifying the stability of housekeeping genes to be used for the quantitative real-time PCR normalization in retinal tissue of streptozotocin-induced diabetic rats. Int J Ophthalmol, 2024,17(5):794-805

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Publication History
  • Received:August 03,2023
  • Revised:February 23,2024
  • Adopted:
  • Online: April 24,2024
  • Published: