Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Systematic comparison of quantity and quality of RNA recovered with commercial FFPE tissue extraction kits.

Author(s): Dube S, Al-Mannai S, Liu L, Tomei S, Hubrack S, Sherif S, Jabeen A, Ahmed EI, Sanchez A, Mifsud W, Bedognetti D, Hendrickx W, Raynaud CM

Publication: J Transl Med, 2025, Vol. 23, Page 11

PubMed ID: 39762904 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the yield and integrity of RNA obtained from formalin-fixed, paraffin-embedded (FFPE) tonsil, appendix and lymph node specimens following extraction with seven different kits. Tonsils from three patients with tonsillitis, appendices from three patients with appendix inflammation, and three lymph nodes from three patients with B-cell lymphoma were surgically removed and cut into 0.5 × 1 × 1 cm blocks. Tissue blocks were fixed in 10% neutral buffered formalin for 18-48 h and embedded in paraffin. FFPE blocks were stored at room temperature (<25°C) for 6-12 months. 81 sections (20 µM thick) were cut from each block and serially placed into 27 tubes until each of the 27 tubes contained three sections. RNA was extracted in triplicate specimens from each block using each of seven kits: RNeasy FFPE, ReliaPrep FFPE Total RNA Miniprep System, Norgen FFPE RNA Purification, GenElute FFPE RNA Purification, PureLink FFPE RNA Isolation, AllPrep DNA/RNA FFPE, and High Pure FFPET RNA Isolation Kits. For kits that did not include a deparaffinization solution, xylene was used; the minimum elution volume was chosen for each kit. RNA was quantified using a NanoDrop Spectrophotometer and Perkin Elmer RNA labchips.

   Summary of Findings:

   RNA recovery from FFPE tissues was highest when extraction was with the ReliaPrep FFPE Total RNA Miniprep System for all three tonsil specimens, all three lymph node specimens, and one of the three appendix specimens. For the remaining two appendix specimens, the RNA yield was highest using the PureLink FFPE RNA Isolation Kit.  When all specimen types were considered together, the ReliaPrep FFPE Total RNA Miniprep System recovered significantly more RNA than each of the other kits: GenElute FFPE RNA Purification Kit (P<0.01), Norgen FFPE RNA Purification Kit (P<0.001), PureLink FFPE RNA Isolation Kit (P<0.0001), RNeasy FFPE Kit (P<0.00001), AllPrep DNA/RNA FFPE Kit (P<0.00001), and High Pure FFPET RNA Isolation Kit (P<0.00001). Extraction with the PureLink FFPE RNA Isolation Kit led to the highest RNA quality score (RQS) for two of three tonsil specimens and comparable RQSs for the ReliaPrep FFPE Total RNA Miniprep System and High Pure FFPET RNA Isolation Kits for the third tonsil specimen. Among appendix specimens, extraction with the High Pure FFPET RNA Isolation Kit resulted in the highest RQS for one specimen, and a RQS that was comparably high relative to the PureLink FFPE RNA Isolation Kit for the third specimen. Isolation with the High Pure FFPET RNA Isolation Kit resulted in the highest RQS for one lymph node specimen, and a comparable RQS to the ReliaPrep FFPE Total RNA Miniprep System for other two lymph node specimens. When specimen types were considered together, RNA isolated with the High Pure FFPET RNA Isolation Kit, PureLink FFPE RNA Isolation Kit and ReliaPrep FFPE Total RNA Miniprep System yielded higher mean RQSs than the other methods examined, but the mean RQS was significantly higher in RNA extracted using the PureLink FFPE RNA Isolation Kit than the ReliaPrep FFPE Total RNA Miniprep System (P<0.01).  While the High Pure FFPET RNA Isolation Kit, PureLink FFPE RNA Isolation Kit and ReliaPrep FFPE Total RNA Miniprep System all yielded RNA with a comparably high percentage of fragments >200 nt (DV₂₀₀) from two of the three tonsil specimens, extraction with the High Pure FFPET RNA Isolation Kit yielded RNA with the highest DV₂₀₀ from the third tonsil specimen. The High Pure FFPET RNA Isolation Kit yielded the highest DV₂₀₀ from two of three appendix specimens, with comparable DV₂₀₀ obtained from the third appendix specimen with the PureLink FFPE RNA Isolation Kit. The High Pure FFPET RNA Isolation Kit and ReliaPrep FFPE Total RNA Miniprep System yielded the highest DV₂₀₀ from two of the three lymph node specimens, with the highest DV₂₀₀ obtained from the third lymph node using the High Pure FFPET RNA Isolation Kit. Overall, the High Pure FFPET RNA Isolation Kit yielded the highest DV_200, but the difference was not significant when compared to the ReliaPrep FFPE Total RNA Miniprep System. When all factors (yield, RQS and DV₂₀₀) were considered together, the High Pure FFPET RNA Isolation Kit was the most consistent, although the High Pure FFPET RNA Isolation Kit, PureLink FFPE RNA Isolation Kit and ReliaPrep FFPE Total RNA Miniprep System all performed well.  However, the ReliaPrep FFPE Total RNA Miniprep System yielded significantly higher quality x quantity score (relative yield x DV₂₀₀) than any other kit evaluated (P<0.002, all).

   Biospecimens

   • Tissue - Tonsil
   • Tissue - Lymph Node
   • Tissue - Appendix

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Lymphoma
   • Other diagnoses

   Platform:

   Analyte	Technology Platform
   RNA	Spectrophotometry
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	RNeasy FFPE ReliaPrep FFPE Total RNA Miniprep System Norgen FFPE RNA Purification GenElute FFPE RNA Purification PureLink FFPE RNA Isolation AllPrep DNA/RNA FFPE High Pure FFPET RNA Isolation
   Biospecimen Acquisition	Biospecimen location	Tonsil Appendix Lymph

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