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
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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
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