Comparison of Modified Manual Acid-Phenol Chloroform Method and Commercial RNA Extraction Kits for Resource Limited Laboratories

Abstract

Background and Aim. RNA extraction is a commonly used technique in molecular biology. In recent years, commercially available RNA extraction kits have largely replaced conventional approaches. However, these commercial kits are expensive and are not readily available in many resource-constrained institutions and laboratories. This study therefore compared the performance of the conventional acid guanidinium thiocyanate-phenol-chloroform (AGPC) extraction method to QIAamp® Viral RNA Mini Kit (QIAGEN, Cat. No. 52906) and OxGEn RNA Kit (OxGEn Molecular Solutions, GE-009) to build an in-house RNA extraction technique from blood and oral swab samples. Method. In a comparative experimental cross-sectional study, RNA was extracted from oral swabs and blood samples from 25 healthy individuals at the Department of Molecular Medicine, KNUST. RNA was extracted by the manual AGPC extraction method and commercial RNA extraction kits. The quantity (ng/μl) and purities (260/280 nm) of the extracted RNA were measured spectrophotometrically using the IMPLEN NanoPhotometer® N60. The presence of RNA in the extracts was confirmed using 2% agarose gel electrophoresis. Statistical analyses were conducted using R language. Results. The yield of RNA extracted from blood and oral swab samples using modified AGPC was significantly higher compared to the commercial methods ( p < 0.0001). However, the purity of RNA extracted by the manual AGPC method from blood was significantly lower than the commercial methods ( p < 0.0001). Moreover, the purity from oral swabs using the manual AGPC method was significantly lower compared to QIAamp ( p < 0.0001) and the OxGEn kits method ( p < 0.001). Conclusion. The modified manual AGPC method has a very high yield of RNA extracts using blood samples, which could serve as an alternate cost-effective method for RNA extraction in resource-limited laboratories; however, its purity may not be suitable for downstream processes. Moreover, the manual AGPC method may not be suitable for extracting RNA from oral swab samples. Future investigation is needed to improve the purity of the manual AGPC RNA extraction method and also confirmation of the obtained results by PCR amplification and RNA purity verification by sequencing.

Spectrophotometric method for determining the quantity and quality of DNA in animal breeding

“…The phenol-chloroform method for nucleic acid extraction was used in the research because it is reported by researchers such as S.A. Sakyi et al (2023) to be much cheaper than other commercial methods. And in addition, it allows maximizing the yield of DNA from the sample.…”