Hello,

microRNAs can be isolated by Trizol from cancer cells. However, for high-quality miRNA with very good integrety, we had really good experiences with the small RNA isolation kit from machery and nagel.

Hi Samar,

TRIzol is an effective reagent for the extraction of miRNAs due to its ability to preserve and isolate all types of RNAs. However, careful handling is required to minimize phenol contamination, which can interfere with downstream applications. Optimizing the precipitation and washing steps - specifically by using higher concentrations of ethanol or isopropanol - can significantly enhance the recovery and enrichment of small RNA species.

Dear Dr. Samar Sami Alkafaas

If you use TRIzol, you will have to use a phenol-chloroform extraction method which can turn out to be harsh and may lead to degradation of RNA, especially the smaller RNAs like miRNAs which you are interested in. It can also be less efficient at isolating specific miRNA populations. For challenging samples or when high yields of specific low-abundance miRNAs are crucial, use of dedicated small RNA isolation kits might be preferable. The commercial kits are designed with specific protocols and reagents for miRNA isolation, So, they will offer better yield, purity, and reproducibility. These kits often include steps to enrich smaller RNAs and minimize degradation which will be at your advantage.

If you decide to use TRIzol, consider incorporating modifications to improve efficiency, such as including an RNA carrier like glycogen, and using spin columns instead of liquid phase precipitation, as this might help to avoid biases in miRNA recovery.

Regards,

Malcolm Nobre

In response to the answer of Malcolm Nobre:

If the RNA extraction method used in a given laboratory yields unsatisfactory results, the issue is attributable to technical error rather than a limitation of the method itself. When standard precautions and best practices for RNA handling are strictly followed, no RNA extraction method should inherently result in poor RNA quality or degradation.

The primary causes of RNA degradation during isolation from biological samples include:

• Deviation from the established RNA extraction protocol.
• Contamination of reagents, laboratory consumables, or the work environment with RNases.
• Improper collection, handling, or storage of source material prior to extraction.

The inclusion of an RNA carrier, such as glycogen, may be beneficial when extracting RNA from challenging samples characterized by low RNA abundance or/and the majority of RNA content consists of small non-coding RNAs (e.g., from extracellular vesicles such as exosomes). However, this is not necessary for typical cancer cell samples, which are generally not considered difficult in terms of RNA yield. These samples typically contain sufficient quantities of long RNAs (e.g., mRNA, rRNA), which can effectively serve as endogenous carriers for small RNAs, negating the need for additional carriers like glycogen.

It appears there may be some confusion or imprecision regarding the RNA extraction process, particularly in the context of spin column-based methods. Spin columns cannot function independently of a prior liquid-phase precipitation step. Their ability to capture RNA is contingent upon the efficient precipitation of nucleic acids during the earlier phase of extraction. If precipitation is suboptimal, the columns will fail to retain the RNA, especially small RNA species.

Thus, the liquid-phase precipitation step is critical, particularly for the recovery of short RNAs. This step is typically performed using ethanol (final concentration 75–80%) or isopropanol (final concentration 50–60%). The efficiency of RNA precipitation is influenced primarily by two factors:

• Time: Longer incubation times generally enhance RNA precipitation.
• Temperature: Lower temperatures favor RNA precipitation.

However, excessive incubation times or overly low temperatures can promote the co-precipitation of insoluble salts and other contaminants, which significantly compromise RNA purity and downstream applications.

Based on our experience, optimal conditions for this precipitation step are as follows: incubation at –20 °C for up to 1 hour. For samples with high RNA content, shorter incubation (5–15 minutes) is typically sufficient and helps minimize contaminant precipitation.