Poor RNA quality after Trizol extraction? First, understand these signs.

RNA quality directly affects subsequent experiments.

After extracting RNA from animal tissues, many people proceed directly to quantitative PCR or RNA sequencing. Whether the RNA is intact and whether it is contaminated with proteins or organic solvents will largely affect the results. The Trizol extraction workflow is well established, but different operational details can lead to differences in RNA quality. Learning to check a few basic indicators can help you identify problems more quickly.

Purity ratios: A260/A280 and A260/A230

UV spectrophotometry is a commonly used preliminary detection tool in the laboratory. A260/A280 is generally used to assess protein contamination, and the ratio for pure RNA is mostly around 1.8 to 2.0. If it is significantly lower, it may indicate considerable protein residue; if it is higher, the RNA may be degraded or the nucleic acid species may be complex. A260/A230 is usually used to assess residual salts, phenol, or carbohydrates, and the ideal value is often close to 2.0; when it is lower, it indicates that organic solvents or salts have not been thoroughly removed. These values can be affected by the sample diluent and instrument condition, so a conclusion cannot be drawn based on a single value alone.

What the bands on an electrophoresis gel represent

Agarose gel electrophoresis can provide a more intuitive view of RNA integrity. In RNA from eukaryotic animal cells, the 28S and 18S bands are usually relatively clear, and the 28S brightness is generally higher than that of 18S. If obvious smearing appears, it indicates that the RNA may be degraded; if there are bands near the loading well, it may indicate genomic DNA contamination. For animal tissue samples, slight degradation does not necessarily affect some PCR experiments, but for experiments with higher requirements such as sequencing, integrity is more critical.

Common causes of degradation and contamination

Tissue left for too long after excision: RNases act rapidly, so it is recommended to collect and snap-freeze the tissue as soon as possible.

Insufficient grinding or homogenization: local tissue is not completely lysed, reducing subsequent extraction efficiency.

Use of pipette tips and centrifuge tubes not treated with RNase-free conditions: exogenous RNases may introduce degradation.

Aspirating too much aqueous phase: this easily carries over DNA and protein from the interphase, causing contamination.

Insufficient washing steps: residual ethanol or salt affects purity and downstream reactions.

How to improve and store

If RNA quality is found to be unsatisfactory, you can troubleshoot step by step from tissue collection, grinding, chloroform phase separation, aqueous phase aspiration, and washing. Try to use fresh or properly frozen tissue for each extraction, and keep reagents and consumables clean. After RNA is dissolved, it is recommended to aliquot and store it at low temperature to avoid repeated freeze-thaw cycles. For important samples, after extraction, first perform a small-volume quality check before deciding whether to proceed to downstream experiments. In this way, even if problems are encountered, subsequent waste of time and cost can be reduced.