Technical Optimization of Total RNA Extraction from Complex Animal Tissues: Evaluation of a Mini-Extraction Protocol Based on the Magnetic Bead Method

1.Abstract

Animal visceral tissues (such as liver, kidney, hati, and intestinal tissues) are rich in endogenous nucleases, polisakarida, lipid, and connective tissue components. As a result, the extraction of total RNA from these tissues has long faced technical challenges such as a high risk of degradation, low product purity, and unstable yields. The traditional phenol-chloroform extraction method is not only cumbersome and time-consuming but also involves toxic organic reagents, making it difficult to meet the demands of high-throughput and automated experiments.

Based on application data from the Shanghai Lingjun Biotech Kit Pengekstrakan RNA Jumlah Sejagat (Kaedah Manik Magnet) in various animal visceral samples, this paper systematically evaluates its performance in small-volume extractions. The results indicate that the kit effectively removes protein and polysaccharide contamination. The A260/A280 and A260/A230 ratios of the extracted RNA both meet the quality requirements for downstream molecular biology experiments, and the yield is sufficient for various applications, such as RT-qPCR and transcriptomic sequencing, providing a safe and efficient solution for RNA extraction from complex tissue samples.

2.pengenalan

The integrity and purity of ribonucleic acid (RNA) are critical prerequisites for molecular biology experiments such as gene expression analysis, transcriptomic sequencing, and non-coding RNA research. Animal visceral tissues—particularly the liver, kidneys, hati, and intestines—are typically considered “difficult samples” for RNA extraction. The liver is rich in glycogen and endogenous RNases; kidney and intestinal tissues contain large amounts of polysaccharides and proteolytic enzymes; and the heart, due to the dense structure of its muscle fibers, does not lysis sufficiently. These factors can all lead to RNA degradation, low yield, or the co-purification of impurities, which in turn affect reverse transcription efficiency and the reliability of quantification results.

Dalam beberapa tahun kebelakangan ini, the magnetic bead method for nucleic acid extraction has gradually emerged as an alternative to traditional centrifugation column and organic solvent methods, thanks to its advantages of operational safety, ease of automation, and consistent product quality. This study evaluated the performance of a universal total RNA extraction kit based on the magnetic bead method, developed by Shanghai Lingjun Biotechnology, in visceral samples from pigs and mice, providing a systematic technical reference for RNA extraction from such samples.

3.Product Technical Principles

This kit is based on surface-modified magnetic bead technology and utilizes the selective adsorption of nucleic acids by magnetic beads in a specific buffer system to purify RNA. The extraction process includes: releasing total RNA from the sample using a lysis buffer; specific binding of nucleic acids to the magnetic beads; removal of protein and salt ion impurities using wash buffers; and elution with a nuclease-free wash to obtain high-purity RNA. This system does not require the use of organic solvents such as phenol and chloroform, thereby avoiding the health risks and waste disposal burdens associated with traditional methods. Selain itu, the magnetic bead method prevents column clogging caused by sample particulates during centrifugation-based filtration, and offers flexible elution volumes (30–100 μL), making it suitable for the concentration and recovery of low-concentration RNA samples.

4.Experimental Data and Analysis

Approximately 5–10 mg each of fish liver, tisu usus, gills, and heart were collected and extracted using the Lingjun Biotech Universal Total RNA Small-Volume Extraction Kit. The results are shown in the table below.

Tidak.	Sampel	260/280	260/230	Conc. (ng/μL)
1	5 mg Hati ikan	1.92	1.54	272.95
2	10mg Fish guts	1.84	2.04	119.15
3	10mg Fish gill	1.92	2.13	276.15
4	10mg Jantung ikan	1.89	1.89	113.70

Electrophoresis Results：

Seperti yang dapat dilihat, the A260/280 ratios are above 1.9, the concentrations are all above 100 ng/μL, dan jalur utama jelas kelihatan.

5.Discussion and Recommendations for Application

5.1 Technical Advantages of the Magnetic Bead Method

Compared to the traditional Trizol method, the magnetic bead method significantly reduces processing time (the entire process can be completed in under 40 minit) and minimizes the risk of RNA degradation due to exposure during the procedure. Selain itu, the magnetic bead method is compatible with automated nucleic acid extraction platforms, menjadikannya sesuai untuk pemprosesan sampel berkemampuan tinggi.

5.2Extraction Strategy

Small-volume extraction is suitable for routine analyses such as RT-qPCR, cDNA library construction, and Northern blot; it requires only a small amount of sample and is ideal for the initial screening of precious samples. For tissues with high fat content or high RNase levels (such as the pancreas), the amount of lysis buffer can be appropriately increased or an RNase inhibitor can be added in advance to optimize extraction efficiency.

6.Kesimpulan

The Shanghai Lingjun Biotech Universal Total RNA Extraction Kit (Kaedah Manik Magnet) demonstrates excellent purity, hasil, and ease of use in the extraction of RNA from animal visceral tissues. This system provides a technical solution that combines efficiency and quality for RNA research on complex biological samples, and the resulting RNA is suitable for a wide range of downstream applications, from RT-qPCR to transcriptomic sequencing.

Pembekal

Shanghai Lingjun Biotechnology Co., Ltd.ditubuhkan pada 2016 yang merupakan pengilang profesional bahan biomagnet dan reagen pengekstrakan asid nukleik.

Kami mempunyai pengalaman yang kaya dalam pengekstrakan dan penulenan asid nukleik, penulenan protein, pemisahan sel, chemiluminescence, dan bidang teknikal yang lain.

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