Expression profiling with RNA from formalin-fixed, paraffin-embedded material.

Author(s): Oberli A, Popovici V, Delorenzi M, Baltzer A, Antonov J, Matthey S, Aebi S, Altermatt HJ, Jaggi R

Publication: BMC Med Genomics, 2008, Vol. 1, Page 9

PubMed ID: 18423048 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of RNA extraction method, demodification, and reverse transcription on amplification from FFPE breast tumors.

   Summary of Findings:

   The RNA fragments ranged from 200 to 1000 nucleotides (nts) long when RNA was extracted from FFPE tissue using the author's protocol, but tended to be less than 100 nts when the ncLysis or RNeasy FFPE kits were used. The correlation of the CT values for amplification of 5 housekeeping genes from frozen tissue and FFPE tissue were highest when gene-specific primers were used for reverse transcription instead of random priming. The highest correlation with frozen tissue occurred when RNA was extracted by the author's protocol and reverse transcribed with gene-specific primers. The correlation was lowest when RNA was extracted using the RNEasy kit. The CT difference between frozen and FFPE tissue decreased with amplicon size and was lowest when FFPE RNA was demodified. The CT values were closest to those from frozen tissue when FFPE specimens were demodified by incubation in a tris-based dilution buffer for 20 min at 94 degrees C and pH 8. Similar to frozen tissue, the expression levels of the normalize genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-glucuronidase (GUSB), 60S acidic ribosomal protein P0 (RPLP0), transferrin receptor (TFRC), 60S ribosomal protein L7a (RPL7A), and ubiquitin B (UBB) were found to vary only slightly between the 14 FFPE tumors.

   Biospecimens

   • Tissue - Breast

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Spectrophotometry
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen
   Analyte Extraction and Purification	Analyte isolation method	RNeasy FFPE (Qiagen) ncLysis (Applied Biosystems) Proteinase k digestion, demodification and purification with silica based columns Demodification at various temperatures and pH
   Real-time qRT-PCR Specific	Length of gene fragment	60 bp 109 bp 147 bp
   Real-time qRT-PCR Specific	Targeted nucleic acid	ACTB GAPDH GUSB RPLP0 TFRC RPL7A RPS11 RPS23 UBB

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