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<p>The abundance of rRNA in cells makes it difficult to enrich mRNAs, especially in prokaryotes, as it lacks a poly(A) tail. Numerous methods and kits have been developed to enrich mRNAs in prokaryotes, which are either organism-specific or have less enrichment efficiency apart from being costly. The current protocol aims to remove bacterial ribosomal RNA via the enzymatic digestion method. 23S rRNA, 16S rRNA and 5S rRNA were amplified from total RNA via reverse transcription, followed by digestion of RNA via RNase H and digestion of DNA via DNase I, which tends to enrich the total mRNA. The off-target digestion was evaluated and confirmed by reverse transcription followed by PCR, and the results were compared with the cDNA amplified products. The presence of rRNA and four genes was confirmed via RT‒qPCR, which revealed 99.99% removal of 23S rRNA and 16S rRNA and 92.84% removal of 5S rRNA. The enriched sample shows an increase in the copy number of mRNA genes of 92.88, 88.66, 85.67 and 84.50% for <i>nor</i>A, <i>mec</i>A, <i>fib</i> and <i>sar</i>A, respectively. This method is not only cost-effective but also efficiently removes rRNA from the total RNA of any prokaryote and can remove any gene from the RNA pool via using the respective gene primers.</p> Graphical Abstract <p></p>

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A Universal, Cost-Effective, and Efficient rRNA Depletion Method for Bacterial mRNA Enrichment

  • 期刊论文

  • 发表日期：

    2026年4月3日

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  • Ravi Chauhan,
  • Hardi Patel,
  • Seema Rawat

摘要

The abundance of rRNA in cells makes it difficult to enrich mRNAs, especially in prokaryotes, as it lacks a poly(A) tail. Numerous methods and kits have been developed to enrich mRNAs in prokaryotes, which are either organism-specific or have less enrichment efficiency apart from being costly. The current protocol aims to remove bacterial ribosomal RNA via the enzymatic digestion method. 23S rRNA, 16S rRNA and 5S rRNA were amplified from total RNA via reverse transcription, followed by digestion of RNA via RNase H and digestion of DNA via DNase I, which tends to enrich the total mRNA. The off-target digestion was evaluated and confirmed by reverse transcription followed by PCR, and the results were compared with the cDNA amplified products. The presence of rRNA and four genes was confirmed via RT‒qPCR, which revealed 99.99% removal of 23S rRNA and 16S rRNA and 92.84% removal of 5S rRNA. The enriched sample shows an increase in the copy number of mRNA genes of 92.88, 88.66, 85.67 and 84.50% for norA, mecA, fib and sarA, respectively. This method is not only cost-effective but also efficiently removes rRNA from the total RNA of any prokaryote and can remove any gene from the RNA pool via using the respective gene primers.

Graphical Abstract