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#small RNA sequencing

2 public questions tagged with this topic.

Transcript analysis

Which sequencing approach is best for small RNAs like miRNA?

Micro RNAs are approximately 22 nucleotide regulatory RNAs derived from hairpin precursors and lack poly-adenine tails. Whole transcriptome and messenger RNA sequencing protocols often include size selection steps that discard RNAs below 200 nucleotides or enrich poly-adenylated species, causing loss of micro RNAs. Small RNA sequencing employs specialized library preparation, preserving 18-35 nucleotide species, ligating adapters directly to 3 prime and 5 prime ends of total small RNA, reverse transcribing without fragmentation, and sequencing. This approach allows precise identification, quantification, discovery of novel micro RNAs and analysis of isoforms and modifications.

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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Transcriptome analysis

What type of sequencing is used to target small RNAs like miRNA and siRNA?

Small RNA sequencing is a specialized RNA-seq workflow designed for non-coding RNAs of 18–40 nucleotides such as microRNAs and small interfering RNAs. After total RNA extraction, RNAs are size-selected by gel or beads to enrich the small fraction, followed by ligation of 5′ and 3′ adaptors that exploit the characteristic 5′ phosphate and 3′ hydroxyl ends. Reverse transcription and PCR generate libraries that preserve strand information. Unlike mRNA sequencing which depletes small RNAs through poly(A) selection and size exclusion, this approach accurately quantifies miRNA expression, isoforms, and modifications.

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.

View Details