Profiling of 24-nt phasiRNAs in Rice Anther by Massively Parallel Sequencing and Stem-Loop Reverse Transcription PCR

Methods Mol Biol. 2025:2869:135-146. doi: 10.1007/978-1-0716-4204-7_14.

Abstract

Small RNAs are highly abundant and play important roles in plant reproduction. Profiling of small RNAs in reproductive tissues is a critical step in understanding their biology. Here, we describe a protocol for small RNA profiling in rice anthers, with a focus on an abundantly expressed but little-understood reproductive small RNA class named 24-nucleotide phased secondary small interfering RNAs (24-nt phasiRNAs). The protocol details small RNA library preparation steps for high-throughput sequencing, with a subsequent bioinformatic analysis framework. A low-throughput stem-loop reverse transcription PCR protocol for quick semi-quantification of small RNAs is also included as a complementary approach. The collective protocol, although focused on 24-nt phasiRNAs, should be amenable to other small RNA classes in various plant reproductive tissues.

Keywords: 24-nt phasiRNAs; Reproduction; Small RNAs; Stem-loop RT-PCR.

MeSH terms

  • Computational Biology / methods
  • Flowers / genetics
  • Gene Expression Profiling / methods
  • Gene Expression Regulation, Plant
  • High-Throughput Nucleotide Sequencing* / methods
  • Oryza* / genetics
  • RNA, Plant* / genetics
  • RNA, Small Interfering* / genetics
  • Reverse Transcriptase Polymerase Chain Reaction / methods

Substances

  • RNA, Plant
  • RNA, Small Interfering