Optimal Reference Genes for Normalization of qRT-PCR Data from Archival Formalin-fixed, Paraffin-embedded Breast Tumors Controlling for Tumor Cell Content and Decay of mRNA.
Author(s): Tramm T, Sørensen BS, Overgaard J, Alsner J
Publication: Diagn Mol Pathol, 2013, Vol. 22, Page 181-7
PubMed ID: 23846446 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 storage duration on the expression of 16 housekeeping genes in FFPE breast tumors by real-time qRT-PCR. RNA was isolated from sections of FFPE tissue using VERSANT, but the storage duration of sections and details of FFPE processing were not specified.
Summary of Findings:
The majority of transcripts were detected in all specimens, but beta- glucuronidase (GUSB), hydroxymethylbilane synthase (HMBS), importin 8 (IPO8), and prosome macropain 26S subunit ATPase 4 (PSMC4) were only detected in 95%, 77.5%, 97.5% and 87.5% of specimens, respectively. The real-time qRT-PCR failures occurred when using RNA isolated from 11 FFPE specimens, 8 of which had been stored for 23-29 years, while 2 were stored for 20-21 years, and 1 was stored for 7 years. Expression levels of CALM2, RPL37A, ACTB, and RPLP0 were shown to be the most stable over time, and HMBS and PSMC4 were the least stable; however, the raw CT value of even the most stably expressed transcripts increased by an average of 0.22 per year, but the normalized values were consistent. The variability in expression levels was mostly attributed to differences between specimens.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
- Neoplastic - Benign
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 1-29 years
Real-time qRT-PCR Specific Targeted nucleic acid CALM2
RPL37A
ACTB
RPLP0
SF3A1
TBP
B2M
PUM1
GAPDH
OAZ1
MRPL19
TFRC
GUSB
HMBS
IPO8
PSMC