Determinants of RNA quality from FFPE samples.
Author(s): von Ahlfen S, Missel A, Bendrat K, Schlumpberger M
Publication: PLoS One, 2007, Vol. 2, Page e1261
PubMed ID: 18060057 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of formalin-fixed paraffin-embedded (FFPE) block storage duration on the integrity and amplificability of RNA from lung carcinoma specimens.
Conclusion of Paper
The duration of ambient temperature FFPE block storage did not affect the mean RNA size observed in electropherograms. However, cycle threshold (CT) values of between 29 and 35 were only reliably observed for each of the three TATA binding protein (TBP) amplicons when specimens were stored for 1 or 2 years and reverse transcribed using a combination of random and oligo dT priming.
Studies
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Study Purpose
This study investigated the effects of FFPE block storage duration on the integrity and amplificability of RNA from lung carcinoma specimens. FFPE blocks had been stored at ambient temperature for 1, 2, 4, 7 or 10 years before sectioning and RNA extraction. RNA was extracted from a single 10 µm section using the RNeasy FFPE kit. RNA was reverse transcribed using Oligo dT priming alone or with random primers in conjunction with the QuantiTect RT kit. RNA integrity was investigated by bioanalyzer and real-time RT-PCR amplification of fragments 128, 227, and 428 nt bp from the 3’ end of the TBP.
Summary of Findings:
Mean RNA size as determined by electropherogram was between 100 and 220 nt with no apparent effects of FFPE storage duration. The authors report no correlation between FFPE block age and RNA yield. A combination of random and oligo dT priming allowed for amplification of all three TBP products with CT values of 29-35 in all FFPE specimens stored for 1 or 2 years and one of the two specimens stored for 4 y, but specimens stored for longer durations did not consistently produce CT values below 35. Additionally, for specimens stored for more than 4 years amplification of the fragment located 428 bp from the 3’ end was specifically hindered. In contrast, when specimens were reverse transcribed with Oligo dT alone the CTs were higher than 35, for both FFPE specimens that were stored for 1 year.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR RNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 1 year
2 years
4 years
7 years
10 years
Real-time qRT-PCR Specific Targeted nucleic acid 128 nt from 3’ end of TBP
227 nt from 3’ end of TBP
428 nt from 3’ end of TBP
Real-time qRT-PCR Specific Priming method Oligo dT primer
Oligo dT and random primers