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

From: Strategies for measuring airway mucus and mucins

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.