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#mRNA enrichment

2 public questions tagged with this topic.

frog development

Chordin mRNA enrichment occurs primarily in embryos treated with:

Chordin gene transcription is strictly dependent on organizer induction requiring nuclear beta-catenin, Siamois/Twin and Nodal-related Smad2 signaling. Lithium chloride inhibits GSK3, stabilizing beta-catenin throughout embryo and converting entire marginal zone into organizer tissue expressing high levels of chordin, noggin and goosecoid. UV irradiation abolishes beta-catenin transport and eliminates chordin. Therefore lithium treated embryos exhibit dramatic enrichment of chordin mRNA radially, serving as molecular readout of ubiquitous dorsalization, opposite to ventralized UV embryos where chordin is absent. This illustrates conserved developmental logic of morphogen gradients patterning embryonic axes through Wnt and BMP antagonism.

Ref: Gilbert, Developmental Biology 12th ed., Chapter 10: Organizer genes and lithium dorsalization experiment.

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Sequencing

Which method uses poly-A tail for mRNA enrichment?

Mammalian messenger RNAs are modified with a 3' polyadenosine tail of 100 to 250 nucleotides added post-transcriptionally. Exploiting this feature, poly-T selection uses paramagnetic beads conjugated with oligo-dT sequences that base-pair with poly-A tails under high salt conditions. Non-polyadenylated rRNA and tRNA fail to bind and are washed away, enriching mRNA fraction for library preparation. This approach is essentially an affinity chromatography strategy based on A-T hybridization. Hydrolysis degrades RNA randomly, size exclusion separates by length without specificity, whereas poly-T selection provides mRNA specificity.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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