(M1330-06-38) Comparison of Manual and Automated Nucleic Acid Extraction Methods

Purpose: The traditional RNA isolation techniques using phenol/chloroform extraction are time consuming and prone to sample cross contamination and PCR inhibition from phenol/chloroform carryover. The KingFisher Flex is an automated instrument for high-throughput extraction and purification of DNA, RNA, proteins and cells. The instrument functions without any dispensing or aspiration. There are few studies that compare the performance of manual and automated RNA extraction methods in preclinical animal models. This study compares commercially available manual and automated RNA extraction kits used for extraction of RNA from blood and tissue samples from three different species mouse, rat and monkey.

Methods: RNA was manually extracted from the blood samples with TRIzol LS Reagent through organic extraction and RNA precipitation. TRIzol Plus Kit was used to manually extracted RNA through spin column from the brain, heart, kidney, lung, ovary, spleen and testis. RNA was automated extracted from the same types of samples with KingFisher Flex system using magnetic bead-based separation.

Results: The performance of manual and automated RNA extraction methods was evaluated in terms of both yield (total RNA amount) and purity (A260/A280 ratio). The average amount of the extracted RNA (µg/1mg of tissue or 100µL of blood) was 1.17 (mouse), 1.29 (rat) and 0.84 (monkey) with KingFisher automated extraction, and 1.84 (mouse), 1.49 (rat) and 0.96 (monkey) with TRIzol manual extraction. The average A260/A280 ratios of the extracted RNA were 1.56 (mouse), 1.59 (rat) and 1.63 (monkey) with KingFisher automated extraction, and 1.52 (mouse), 1.49 (rat) and 1.49 (monkey) with TRIzol manual extraction.

Conclusion: Purity and yield are two important criteria for any RNA extraction process. The quantity RNA obtained from KingFisher automated method was comparatively lower than TRIzol manual method. However, the purity of the extracted RNA from KingFisher automated method was better than TRIzol manual methods. When compared with the time requirements for a large sample size in preclinical animal studies, automated RNA extraction is an attractive alternative to labor-intensive manual methods.

References: 1. Sabat J, Subhadra S, Rath S et al. Yielding quality viral RNA by using two different chemistries: a comparative performance study. Biotechniques. 2021;71:510-515.
2. Jeffries MKS, Kiss AJ, Smith AW, Oris JT. A comparison of commercially-available automated and manual extraction kits for the isolation of total RNA from small tissue samples. BMC Biotechnol. 2014:14: 94.