Identification of methods for use of formalin-fixed, paraffin-embedded tissue samples in RNA expression profiling.

Author(s): Roberts L, Bowers J, Sensinger K, Lisowski A, Getts R, Anderson MG

Publication: Genomics, 2009, Vol. 94, Page 341-8

PubMed ID: 19660539 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 preservation and extraction method on expression analysis on lung, kidney and colon specimens.

   Summary of Findings:

   Deparaffinization solution had no effect on the quantity or quality of RNA isolated from FFPE tissues. RNA yield was highest when RNA was isolated from FFPE sections using Whole Transcript kit, but sufficient RNA was obtained using all kits except the Purelink FFPE kit. RINs and the percentage of positive calls were generally higher and amplification was more efficient and consistent (>40% more yield) when RNA was isolated from frozen instead of FFPE tissue. However, satisfactory microarray results were obtained using FFPE tissue when RNA extraction was done using the RecoverAll, FormaPure and Whole Transcript kits. Satisfactory microarray results could be predicted when the 5' to 3' ACTB ratio was less than 20 and the 5' ACTB CT was within 7 cycles of the standard. The authors report that the RecoverAll kit yielded reproducible results from as few as 3-4 sections as long as twice as much DNAse was used. A correlation coefficient of 0.86 was observed between RNA amplified from frozen specimens using the RampUP protocol and the standard Affymetrix protocol. While the correlation between adjacent sections was 0.975-0.985 for frozen specimens, and 0.887-0.962 for FFPE specimens, the correlation was 0.82-0.89 when frozen specimens were compared to adjacent FFPE specimens extracted with the RecoverAll kit. The correlation with adjacent frozen sections dropped to 0.63-0.89 when the RNA was extracted from FFPE sections using the FormaPure kit. Genes differentially expressed between adjacent sections of normal and tumor tissue had correlation values of 0.99 and 0.98 for frozen and FFPE specimens respectively, while the correlation between frozen and FFPE specimens was 0.89. Real-time PCR confirmed the differential expression of 16 randomly chosen genes between normal adjacent and tumor FFPE tissues, but the magnitude of the change was greater by real-time PCR than microarray. The authors conclude that cancer-related genes can be identified from FFPE tissue.

   Biospecimens

   • Tissue - Lung
   • Tissue - Colorectal
   • Tissue - Kidney

   Preservative Types

   • Formalin
   • None (Fresh)

   Diagnoses:

   • Neoplastic - Carcinoma
   • Neoplastic - Lymphoma
   • Neoplastic - Normal Adjacent

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	RecoverAll for FFPE (Ambion) Paradise Whole Transcript (Arcturus Bioscience) Purelink FFPE RNA isolation (Invitrogen) High Pure RNA paraffin (Roche) FormaPure (Agencourt Bioscience)
   Analyte Extraction and Purification	Deparaffinization	Xylene D-Limonene (Hemo-De)
   Biospecimen Aliquots and Components	Aliquot size/volume	3-4 sections 8 sections
   Real-time qRT-PCR Specific	Targeted nucleic acid	Amino terminal of beta actin Carboxy terminal of beta actin 18S rRNA
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) None (fresh)
   DNA microarray Specific	Signal amplification	RampUP protocol Standard Affymetrix protocol

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