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Comparison of Fecal MicroRNA Isolation Using Various Total RNA Isolation Kits.

Author(s): Lederer T, Hipler NM, Thon C, Kupcinskas J, Link A

Publication: Genes (Basel), 2024, Vol. 15, Page 498

PubMed ID: 38674432 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the RNA yield, RNA purity, levels of three endogenous miRNA (RNU6b, miR-16 and miR-21), and recovery of spiked-in cel-miR-39 when RNA was extracted from the feces of healthy volunteers using five different extraction kits. Fecal specimens were collected from ten volunteers, aliquoted, and stored at -80°C. RNA was extracted from fecal specimens from 5 volunteers using Trizol, the miRNeasy Mini Kit, the RNeasy Kit without the small RNA enrichment step, and the RNeasy Universal Plus Kit and from the other 5 volunteers using the miRNeasy and miRVana Kits. RNA was quantified by RiboGreen and by quantification of spiked-in cel-miR-39 as well as miR-16, miR-21, and RNU6b by real-time PCR.

   Summary of Findings:

   RNA yield was significantly lower when extraction was with the RNeasy Kit compared to the miRNeasy, RNeasy Universal Plus, or Trizol kits (P<0.001) and when the miRVana Kit was used for RNA extraction compared to the miRNeasy Kit (P=0.0038). RNA yields were very strongly correlated between the RNeasy and the RNeasy Universal Plus Kits (r=0.958, P=0.01). A260/A280 ratios were comparable among the RNA extraction methods evaluated and were strongly correlated between the RNeasy Universal Plus Kit and the miRNeasy Kit (r=0.981, P=0.003).  CT values for RNU6b, miR-16, and miR-21 as well as the spiked-in cel-miR-39 were all higher when eRNA xtraction was with the RNeasy Kit compared to the other kits evaluated (P<0.01, P<0.001, P<0.001 and P<0.001, respectively). miR-16 levels were slightly higher when RNA was extracted with the miRNeasy Kit rather than the miRVana Kit (P=0.01).  CT values were very strongly correlated between any two of the following methods: miRNeasy Kit, the RNeasy Universal Plus kit, and the Trizol Kit (r> 0.95, P<0.0001, all). After normalization to either cel-miR-39 or a combination of cel-miR-39 and miR-16, miR-21 levels did not differ among specimens that underwent RNA extraction with the miRNeasy Kit, the RNeasy Universal Plus Kit, or the Trizol Kit. The real-time RT-PCR assay was highly reproducible, with very strongly correlations between two independent PCR runs, regardless of the extraction kit (r≥0.99, P<0.0001, all). 

   Biospecimens

   • Bodily Fluid - Feces

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Fluorometry
   RNA	Tissue microarray
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Trizol miRNeasy Mini Kit RNeasy Kit without the small RNA enrichment step RNeasy Universal Plus Kit miRVana kit
   Real-time qRT-PCR Specific	Quality metrics	Comparison of two independent runs
   Real-time qRT-PCR Specific	Data handling	Normalized to cel-miR-39 Normalized to cel-miR-39 and miR-16
   Real-time qRT-PCR Specific	Targeted nucleic acid	RNU6b miR-16 miR-21 cel-miR-39

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