Table 3 Advantages and limitations of molecular and genetic methods and techniques used in mucus research

Quantitative RT-PCR [110,111,112]

- Very specific quantitative information on mucin expression at the mRNA level

- Inexpensive and easily applicable to most samples.

- Inability to detect increase in secretion.

- Post-transcriptional modifications are also not detected.

Northern-blot (RNA-blot) assay [110, 111]

- Alternative method for detection of RNA.

- Allows for separation of RNA molecules by size.

- Provides information on number, length, and relative abundance of mRNAs expressed by a single gene

- More laborious, time-consuming and not as sensitive as qRT-PCR.

- Requires large amount of tissue/sample, and high purity and quality of non-degraded RNA, which can be difficult for the large RNA molecules of mucins.

Luciferase reporter and Chromatin immunoprecipitation (ChIP) assay (promoter-binding) [111, 113]

- Luciferase reporter assay is commonly used to study gene expression at the transcriptional level.

- ChIP allows for the specific study of molecular regulation and induction of mucin expression under various conditions.

- Most applicable in cell cultures.

- Does not give quantitative information on mucin expression or secretion.

Using transgenic or knockout animals [89, 114, 115]

- Unique and valuable information on the overall function and/or effects of overexpression/depletion of each mucin throughout the lifespan of an animal model.

- Can be used for determination and verification of mucin-regulation pathways.

- Most often applied in rodents.

- Often have to be used in tandem with other techniques to verify the effect.

- Depending on the model species, can be expensive, time-consuming.