改良手动酸-酚-氯仿法与商用 RNA 提取试剂盒在资源有限实验室中的比较。

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

机构信息

Department of Molecular Medicine, School of Medicine and Dentistry, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.

Department of Medical Diagnostics, Faculty of Allied Health Sciences, College of Health Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.

出版信息

Int J Clin Pract. 2023 Jun 9;2023:9593796. doi: 10.1155/2023/9593796. eCollection 2023.

DOI:10.1155/2023/9593796

PMID:37333947

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10275685/

Abstract

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/l) 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 ( < 0.0001). However, the purity of RNA extracted by the manual AGPC method from blood was significantly lower than the commercial methods ( < 0.0001). Moreover, the purity from oral swabs using the manual AGPC method was significantly lower compared to QIAamp ( < 0.0001) and the OxGEn kits method ( < 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.

摘要

方法

在一项比较性实验性横断面研究中，从 KNUST 分子医学系的 25 名健康个体的口腔拭子和血液样本中提取 RNA。通过手动 AGPC 提取方法和商用 RNA 提取试剂盒提取 RNA。使用 IMPLEN NanoPhotometer® N60 分光光度计测量提取的 RNA 的量（ng/l）和纯度（260/280nm）。使用 2%琼脂糖凝胶电泳确认提取物中 RNA 的存在。使用 R 语言进行统计分析。

结果

与商用方法相比，使用改良 AGPC 从血液和口腔拭子样本中提取的 RNA 的产量明显更高（<0.0001）。然而，手动 AGPC 方法从血液中提取的 RNA 的纯度明显低于商用方法（<0.0001）。此外，与 QIAamp（<0.0001）和 OxGEn 试剂盒方法（<0.001）相比，手动 AGPC 方法从口腔拭子中提取的 RNA 纯度明显更低。

结论

改良的手动 AGPC 方法在使用血液样本提取 RNA 方面具有非常高的产量，它可能是资源有限的实验室中提取 RNA 的一种替代经济有效的方法；然而，其纯度可能不适合下游过程。此外，手动 AGPC 方法可能不适合从口腔拭子样本中提取 RNA。需要进一步研究以提高手动 AGPC RNA 提取方法的纯度，并通过 PCR 扩增和 RNA 纯度验证测序来确认获得的结果。