Isolation of high-quality total RNA from small animal articular cartilage for RNA-seq analysis

Researchers from the University of Debrecen highlight the importance of obtaining high-quality RNA for RNA-seq analysis, as poor RNA quality can result in biased and inaccurate sequencing results. They noted that RNA quality can be affected by factors such as tissue handling, RNA extraction methods, and storage conditions.

To evaluate the impact of RNA quality on RNA-seq results, the researchers performed RNA-seq analysis on high-quality and low-quality RNA samples extracted from small animal articular cartilage. They found that low-quality RNA samples resulted in lower mapping rates, reduced transcript coverage, and increased noise in the sequencing data. Additionally, they observed that low-quality RNA samples led to decreased sensitivity in detecting differentially expressed genes.

The researchers emphasize that obtaining high-quality RNA is critical for accurate and reliable RNA-seq analysis. They recommend using RNA extraction methods that minimize RNA degradation, handling tissues carefully to avoid RNA damage, and storing RNA samples at appropriate temperatures to maintain RNA integrity.

Procedure for pulverizing chicken articular cartilage shavings using a cryogenic mill
(SPEX SamplePrep 6775 Freezer/Mill)

(A) Fill the tank of the mill to the fill line with liquid nitrogen. (B) Load cartilage shavings (snap-frozen directly after dissection) in the pre-chilled vial of the instrument. (C) Drop in the chilled impactor. (D) Insert the vial into the chamber. Check the liquid nitrogen level and top it off, if necessary. (E) Run the program as set on the control panel touchscreen display. (F) Remove the vial, open the end plug using the Extractor/Vial Opener, and extract the impactor using the Magnetic Extractor as shown. Immediately proceed to a measuring scale and weigh 100-mg portions of pulverized cartilage into microcentrifuge tubes.

In summary, these researchers highlight the impact of RNA quality on RNA-seq results and emphasizes the importance of obtaining high-quality RNA for accurate and reliable sequencing analysis. The findings underscore the need for careful sample handling and RNA extraction methods to ensure the integrity of RNA samples for successful RNA-seq analysis.

Takács R, Póliska S, Juhász T, Barna KB, Matta C. (2023) Isolation of High-Quality Total RNA from Small Animal Articular Cartilage for Next-Generation Sequencing. Curr Protoc 3(3):e692. [article].

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