Alternative Quantitative Digital Analysis of Agarose Gel PCR Products for Detection of Molecular Markers in Livestock

Suyatno Suyatno; Anita Hafid; Ferdy Saputra; Teguh Ari Prabowo

doi:10.14334/jitv.v30i1.3448

Alternative Quantitative Digital Analysis of Agarose Gel PCR Products for Detection of Molecular Markers in Livestock

Suyatno Suyatno, Anita Hafid, Ferdy Saputra, Teguh Ari Prabowo

DOI

https://doi.org/10.14334/jitv.v30i1.3448

Abstract

The conventional polymerase chain reaction (PCR) method has become a prerequisite in molecular biology research. The PCR reaction is easy to prepare and only requires a small portion of the complex target nucleotide sequence, making PCR an easy and accurate method to use in biochemical and molecular analysis. PCR is generally divided into two categories: qualitative PCR and quantitative real-time PCR (RT-qPCR). The RT-qPCR method is more precise but has the disadvantage that it is much more expensive and requires more complicated equipment than conventional PCR. PCR products were visualized using agarose gel electrophoresis, which produced bands. Along with the development of digital technology, the resulting bands can be analyzed using digital software commonly used to analyze photos, such as ImageJ from the NIH. The trial results using ImageJ software to analyze CD44 in comparison with the housekeeping gene ?-Actin demonstrated that gene expression can be quantified. Quantitative measurements of CD44 and ?-Actin expression were obtained by comparing the percentage of their respective peak plots. Analysis showed that CD44 expression was higher than ?-Actin when evaluated with ImageJ software. These findings align with RT-qPCR results, which require more advanced PCR equipment and reagents. The semi-quantitative PCR analysis method using ImageJ offers a practical alternative for livestock and veterinary laboratories with limited budgets and resources.

Keywords

gene expression quantification, molecular biology, veterinary laboratories, RT-PCR, ImageJ Software

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References

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