Table 3.

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

Method	Advantages	Limitations
Quantitative RT-PCR [110–112]	- 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.