Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Improving gene expression analysis efficacy from formalin-fixed paraffin embedded tissues.

Author(s): Dimaras P, Tasinov O, Ivanova D, Kiselova-Kaneva Y, Stefanova N, Tzaneva M, Ivanova D.

Publication: Folia Med (Plovdiv), 2022, Vol. 64, Page 602-608

PubMed ID: 36045457 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare the yield and purity of RNA extracted using three different extraction methods from sections of a formalin-fixed paraffin-embedded (FFPE) colon biopsy, and to compare real-time RT-PCR cycle threshold (CT) values among two extraction methods, three reverse transcription priming methods, and specimens where preamplification was performed and those where it was not.  One biopsy was collected during a colonoscopy and fixed in 10% neutral buffered formalin for 24 h before being embedded in paraffin and stored at room temperature.  From the FFPE block, 5 µm thick sections were cut and placed three sections to a tube (9 tubes total). The tissue sections were deparaffinized with two changes of xylene and washed in ethanol twice. RNA was extracted in triplicate from the ethanol-deparaffinized sections using three different methods: A) lysis in Quickextract FFPE RNA extraction Lysis buffer at 56°C for 30 min followed by 80°C for 10 min and purification with Zymo research RNA Clean and Concentrator 5 spin columns, B) similar to A with the addition of a phenol-based extraction step using Accuzol after lysis and before column purification, and C) direct extraction with Accuzol followed by column purification. Eluted RNA was stored at -70°C. RNA was quantified by spectrophotometer. RNA was DNase treated, and reverse transcribed using oligo dT, oligo dT and random hexamer primers, or gene-specific primers. To test the effect of preamplification, cDNA was amplified directly or subjected to 5 preamplification cycles or directly amplified by real-time PCR of ribosomal protein L37A.

   Summary of Findings:

   RNA concentration was significantly higher when specimens were lysed in Quickextract FFPE RNA extraction lysis buffer followed by RNA extraction using Accuzol solution and purification with the spin column (Method B) than when lysed in Quickextract and directly purified with spin columns (Method A) (259.87 ± 38.60 versus 163.36 ± 17.96 ng/µL, P<0.05), or when RNA was directly extracted using Accuzol and then purification with the spin column (Method C) (259.87 ± 38.60 versus 15.51 ± 2.06ng/µL, P<0.001).  RNA purity (absorbance at 260/280 nm) was comparable when extraction was with Method A and B (2.03 ± 0.07 and 2.02 ± 0.02, respectively), but slightly lower with Method A (1.83 ± 0.21). CT values were lower when extraction was with Method A rather than B regardless of priming method and inclusion of a preamplification step, but the difference was only significant when reverse transcription was with Oligo dT primers (P<0.01 for both preamplified and not).  CT values were significantly lower when preamplification was performed than when it was not, regardless of extraction method and reverse transcription priming method (P<0.05, all). CT values were significantly lower when gene-specific primers were used than oligo dT, regardless of priming method (P<0.001, all) and lower than oligo dT with random hexamers when no preamplfication step was performed (P<0.001, both extraction methods) or when RNA that was extracted with Method B was preamplified (P<0.05).  CT values were lower when oligo dT and random hexamers were used for priming compared to oligo dT alone, regardless of RNA extraction method and preamplification (P<0.01).

   Biospecimens

   • Tissue - Colorectal

   Preservative Types

   • Formalin

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   RNA	Spectrophotometry
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Lysed in Quickextract and directly purified with spin columns (Method A) Lysed in Quickextract FFPE RNA extraction Lysis followed by RNA extraction using Accuzol and purification with the spin column (Method B) Direct RNA extraction using Accuzol and purification with the spin column (Method C)
   Real-time qRT-PCR Specific	Nucleic acid amplification	Preamplified for 5 cycles Not preamplified
   Real-time qRT-PCR Specific	Priming method	Oligo dT Oligo dT and random hexamers Gene-specific primers

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