Archived formalin-fixed paraffin-embedded (FFPE) blocks: A valuable underexploited resource for extraction of DNA, RNA, and protein.
Author(s): Kokkat TJ, Patel MS, McGarvey D, LiVolsi VA, Baloch ZW
Publication: Biopreserv Biobank, 2013, Vol. 11, Page 101-6
PubMed ID: 24845430 PubMed Review Paper? No
Purpose of Paper
This paper compared quantity and quality of genomic DNA, total RNA and total protein extracted from formalin-fixed paraffin-embedded (FFPE) tumor specimens stored for up to 12 years with those stored for less than one year.
Conclusion of Paper
The purity of DNA and RNA extracted from FFPE blocks was unaffected by storage duration and was comparable to that from an independent cohort of frozen specimens. RNA integrity, assessed by the RNA quality index (RQI), was not significantly affected by FFPE block storage duration and was similar among lung, thyroid and salivary gland specimens. RNA extracted from an independent cohort of frozen tissue specimens displayed higher RQIs than FFPE specimens stored for less than one year (6.7-7.25 versus 1.9-2.63) but statistical significance was not assessed. Protein fragmentation was more prevalent in FFPE blocks that were stored for 5 years or longer than those stored for less than 1 year, but the difference were not significant.
Studies
-
Study Purpose
This study compared quantity and quality of genomic DNA, total RNA and total protein extracted from FFPE tumor specimens stored for up to 12 years with those stored for less than one year. Malignant tumor specimens from lung (4 cases), thyroid (3 cases) and salivary gland (3 cases) were fixed in neutral buffered formalin for 24-48 h, processed for 10 h, and paraffin-embedded for 20 min. FFPE blocks were stored at 17-22°C with 20-60% humidity for 4 months to 12 years. DNA and RNA were extracted using the AllPrep DNA/RNA FFPE kit. DNA and RNA quantity and quality were assessed by spectrophotometry and gel electrophoresis, respectively. RNA Quality Index (RQI), which uses a 0-10 scale, was calculated for all specimens based on bioanalyzer results. Protein was extracted using the Qproteome FFPE tissue kit and quantity and quality were assessed by BCA assay and microchip electrophoresis, respectively.
Summary of Findings:
Data on the quantity of DNA, RNA and protein extracted was pooled among preservation methods for a given storage duration (FFPE, frozen), and while the concentrations were variable no effect of storage duration was observable. FFPE blocks containing tumor specimens that were stored for different durations had comparable DNA and RNA purity based upon 260/280 nm ratios (DNA: 1.86-2.23; RNA: 1.97-2.18). Further, the purity of DNA and RNA extracted from FFPE specimens did not differ significantly from results obtained with an independent cohort of frozen specimens (DNA: 1.91-2.01; RNA: 2.11-2.16). RNA quality, assessed by RQI, was not significantly affected by FFPE block storage duration for a given tissue type, and was similar among the tissue types examined. While 1 out of 3 FFPE blocks were considered unusable for several storage timepoints based on a RQI less than 1.4, the occurrence of low RQI appeared to be random. RNA extracted from an independent cohort of frozen tissue specimens displayed higher RQIs than FFPE specimens stored for less than one year (6.7-7.25 versus 1.9-2.63) although statistical significance was not assessed. While protein fragmentation was more prevalent in FFPE blocks that were stored for 5 years or longer than those stored for < 1 year, differences were not significant.
Biospecimens
Preservative Types
- Frozen
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Spectrophotometry DNA Electrophoresis RNA Spectrophotometry Protein Colorimetric assay RNA Automated electrophoresis/Bioanalyzer Protein 1D/2D gels RNA Electrophoresis Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 4-9 months
1-2 y
5-7 y
11-12 y
Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Frozen