Two hours method for RNA and DNA co-extraction from blood of coronary artery disease patients: Fast, simple and economical technique

Background TRIzolTM is widely used for RNA and DNA extraction. However, this method is laborious and time-consuming. The objective of this study was to validate a time-effective and labor-saving protocol. Methods The TRIzol method was used to separate the aqueous phase, protein, and phenol layer of bone marrow samples from 12 patients with hematological diseases. Subsequently, RNA and DNA were extracted from the aqueous layer containing RNA and phenol layer containing DNA, respectively, using magnetic bead extraction kits. The quantity and purity of extracted RNA and DNA were examined using a NanoDrop spectrophotometer. Quantitative fluorescence PCR amplification of the ABL1 gene was performed to verify the effectiveness of the extracted RNA and DNA for downstream experiments. RNA and DNA from another 16 bone marrow samples were extracted to compare the performance of the two methods. Results Co-extraction of RNA and DNA was completed within 1 h. The data showed that RNA and DNA yield ranged from 13.1 to 204.5 ng/µL and 33.1 to 228.8 ng/µL, respectively. The A260/A280 ratios of RNA and DNA samples ranged from 1.82 to 2.01 and 1.73 to 1.91, respectively. RNA and DNA extracted using this scheme exhibited ideal performance in quantitative fluorescence PCR. The present protocol showed better quality and effectiveness in extracting RNA and DNA compared to the TRIzol method. Conclusions This protocol for RNA and DNA co-extraction is fast, labor-saving, and high throughput. It can be adopted for routine molecular biology analyses, particularly for non-reproducible specimens.

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Optimizing total RNA extraction method for human and mice samples

Zeng,

Tang,

Chen

et al. 2024

PeerJ

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Background Extracting high-quality total RNA is pivotal for advanced RNA molecular studies, such as Next-generation sequencing and expression microarrays where RNA is hybridized. Despite the development of numerous extraction methods in recent decades, like the cetyl-trimethyl ammonium bromide (CTAB) and the traditional TRIzol reagent methods, their complexity and high costs often impede their application in small-scale laboratories. Therefore, a practical and economical method for RNA extraction that maintains high standards of efficiency and quality needs to be provided to optimize RNA extraction from human and mice tissues. Method This study proposes enhancements to the TRIzol method by incorporating guanidine isothiocyanate (GITC-T method) and sodium dodecyl sulfate (SDS-T method). We evaluated the effectiveness of these modified methods compared to the TRIzol method using a micro-volume UV-visible spectrophotometer, electrophoresis, q-PCR, RNA-Seq, and whole transcriptome sequencing. Result The micro-volume UV-visible spectrophotometer, electrophoresis, and RNA-Seq demonstrated that the GITC-T method yielded RNA with higher yields, integrity, and purity, while the consistency in RNA quality between the two methods was confirmed. Taking mouse cerebral cortex tissue as a sample, the yield of total RNA extracted by the GITC-T method was 1,959.06 ± 49.68 ng/mg, while the yield of total RNA extracted by the TRIzol method was 1,673.08 ± 86.39 ng/mg. At the same time, the OD260/280 of the total RNA samples extracted by the GITC-T method was 2.03 ± 0.012, and the OD260/230 was 2.17 ± 0.031, while the OD260/280 of the total RNA samples extracted by the TRIzol method was 2.013 ± 0.041 and the OD260/230 was 2.11 ± 0.062. Furthermore, q-PCR indicated that the GITC-T method achieved higher yields, purity, and greater transcript abundance of total RNA from the same types of animal samples than the TRIzol method. Conclusion The GITC-T method not only yields higher purity and quantity of RNA but also reduces reagent consumption and overall costs, thereby presenting a more feasible option for small-scale laboratory settings.

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Two hours method for RNA and DNA co-extraction from blood of coronary artery disease patients: Fast, simple and economical technique

Cited by 3 publications

References 24 publications

Association of leukocyte telomere attrition in coronary artery disease in Pakistani population: A case-control study with meta-analysis

Association of leukocyte telomere attrition in coronary artery disease in Pakistani population: A case-control study with meta-analysis

Simultaneous Extraction of Bone Marrow RNA and DNA from Patients with Hematologic Diseases Using a Combined Magnetic Bead Method within 1 Hour

Optimizing total RNA extraction method for human and mice samples

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