Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Apr 19:1:9.
doi: 10.1186/1755-8794-1-9.

Expression profiling with RNA from formalin-fixed, paraffin-embedded material

Andrea Oberli  1 , Vlad Popovici, Mauro Delorenzi, Anna Baltzer, Janine Antonov, Sybille Matthey, Stefan Aebi, Hans Jörg Altermatt, Rolf Jaggi
Affiliations

Expression profiling with RNA from formalin-fixed, paraffin-embedded material

Andrea Oberli et al. BMC Med Genomics. .

Abstract

Background: Molecular characterization of breast and other cancers by gene expression profiling has corroborated existing classifications and revealed novel subtypes. Most profiling studies are based on fresh frozen (FF) tumor material which is available only for a limited number of samples while thousands of tumor samples exist as formalin-fixed, paraffin-embedded (FFPE) blocks. Unfortunately, RNA derived of FFPE material is fragmented and chemically modified impairing expression measurements by standard procedures. Robust protocols for isolation of RNA from FFPE material suitable for stable and reproducible measurement of gene expression (e.g. by quantitative reverse transcriptase PCR, QPCR) remain a major challenge.

Results: We present a simple procedure for RNA isolation from FFPE material of diagnostic samples. The RNA is suitable for expression measurement by QPCR when used in combination with an optimized cDNA synthesis protocol and TaqMan assays specific for short amplicons. The FFPE derived RNA was compared to intact RNA isolated from the same tumors. Preliminary scores were computed from genes related to the ER response, HER2 signaling and proliferation. Correlation coefficients between intact and partially fragmented RNA from FFPE material were 0.83 to 0.97.

Conclusion: We developed a simple and robust method for isolating RNA from FFPE material. The RNA can be used for gene expression profiling. Expression measurements from several genes can be combined to robust scores representing the hormonal or the proliferation status of the tumor.

PubMed Disclaimer

Figures

Figure 1
Figure 1
RNA isolation and characterization. Total RNA was isolated from kryo-sections (lanes A) and from paraffin sections according to the RNeasy FFPE protocol of Qiagen (lanes B), the ncLysis protocol of Applied Biosystems (lanes C) or according to our own protocol (lanes D). Aliquots of each RNA were separated by capillary electrophoresis (Agilent Bioanalyzer) on Nano chips along with RNA ladder (L; Ambion). Shown are RNAs from two representative tumors (Tu#10 and #18).
Figure 2
Figure 2
Comparison of RNAs isolated according to different protocols. RNA was reverse transcribed in the presence of random primers (white boxes) or gene-specific primers (hatched boxes). Gene expression was measured from an equivalent of 4 ng of RNA by QPCR for five reference genes (GAPDH, GUSB, RPLP0, TFRC and UBB). Pearson correlations were computed between matched Cts for the five reference genes and each tumor RNA isolated from FF (A) and FFPE material. Shown are correlations between intact RNA and RNA isolated from FFPE material according to the RNeasy FFPE protocol (A versus B), intact RNA and RNA isolated from FFPE material according to the ncLysis system (A versus C) and intact RNA and RNA isolated according to our own protocol (A versus D).
Figure 3
Figure 3
De-modification of RNA results in higher efficiency during subsequent QPCR. RNA was isolated from FFPE material according to our own protocol and compared to intact RNA derived of FF tissue. RNA samples were reverse transcribed without previous de-modification (labeled "no") or after de-modification at room temperature (1), 94°C and pH 8.0 (2) or 94°C and pH 5.0 (3). Each RNA was tested by QPCR using three amplicons for IGBP5. Primers used code for short (60 bp, □), medium-size (109 bp, ◇) or long amplicons (147 bp, △). Shown are raw Ct values from intact RNAs from FF material and from RNAs derived of FFPE material of the same tumors. The benefit of de-modification is visualized as delta Ct values. They are indicated for short and long amplicons (dotted lines).
Figure 4
Figure 4
Comparison of RNA isolation methods. Shown are the means of raw Cts of five reference genes (GAPDH, GUSB, RPLP0, TFRC, UBB) for intact RNA (◇, FF) and for RNA isolated from matched FFPE material according to the protocols of Qiagen (□, Q), Applied Biosystems (△, AB) and our own (○, own). Individual mean Cts of the 14 tumors and summarized box plots of Cts are shown in panel A and panel B, respectively. Tumors are aligned according to increasing Ct in FFPE-derived RNA (Qiagen protocol).
Figure 5
Figure 5
Reproducibility of RNA isolation from FFPE material. The RNAs were isolated from paraffin blocks according to our own protocol. BM33 and BM36 (panel A) are two separate RNAs isolated from tissue block "BM", D33 and D36 are RNAs isolated from block "D" (panel B). For comparison, 45T and 56T originate from two distinct tumors isolated from one patient (panel C). Gene expression was measured by QPCR for 24 genes and raw Ct values are shown for each gene measured from the two matching RNAs.
Figure 6
Figure 6
Stability of reference gene expression in RNA isolated from FF and FFPE material. Raw Cts are shown for 9 putative reference genes (ACTB, GAPDH, GUSB, RPLP0, TFRC, RPL7A, RPS11, RPS23 and UBB). Results based on intact RNA derived of FF material (A) and based on RNA isolated according to our own protocol from FFPE material (B) are depicted for all the 14 tumors. The Ct values for 4 ER-related genes (BCL2, CEPG1, ESR1 and PGR) are shown for comparison (left).
Figure 7
Figure 7
Comparison of Total scores computed from intact and FFPE-derived RNA. Total scores were computed from normalized expression values based on the results of intact RNA (○) and FFPE-derived RNA (△, own protocol) as described in the Methods section. They are shown separately for each of the 14 tumors.
Figure 8
Figure 8
Module scores. ER, HER2 and proliferation scores were computed from expression values of 14 breast cancers and visualized in a scatter plot. The ER score was determined from four genes, the HER2 score from 2 and the proliferation score from 5 genes (see Methods). Tumors are positioned according to their ER score (x-axis) and HER2 score (y-axis). Proliferation scores are color coded. The histological ER status is indicated by a "-" or "+" sign next to the tumor numbers in the plot. The results were computed from intact RNA derived of FF material (A) and RNA isolated from FFPE material according to our own protocol (B). Individual scores for each tumor are given in Table 2.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, Hastie T, Eisen MB, van de Rijn M, Jeffrey SS, Thorsen T, Quist H, Matese JC, Brown PO, Botstein D, Eystein Lonning P, Borresen-Dale AL. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A. 2001;98:10869–10874. doi: 10.1073/pnas.191367098. - DOI - PMC - PubMed
    1. Sorlie T, Tibshirani R, Parker J, Hastie T, Marron JS, Nobel A, Deng S, Johnsen H, Pesich R, Geisler S, Demeter J, Perou CM, Lonning PE, Brown PO, Borresen-Dale AL, Botstein D. Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci U S A. 2003;100:8418–8423. doi: 10.1073/pnas.0932692100. - DOI - PMC - PubMed
    1. Sotiriou C, Neo SY, McShane LM, Korn EL, Long PM, Jazaeri A, Martiat P, Fox SB, Harris AL, Liu ET. Breast cancer classification and prognosis based on gene expression profiles from a population-based study. Proc Natl Acad Sci U S A. 2003;100:10393–10398. doi: 10.1073/pnas.1732912100. - DOI - PMC - PubMed
    1. Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, Pollack JR, Ross DT, Johnsen H, Akslen LA, Fluge O, Pergamenschikov A, Williams C, Zhu SX, Lonning PE, Borresen-Dale AL, Brown PO, Botstein D. Molecular portraits of human breast tumours. Nature. 2000;406:747–752. doi: 10.1038/35021093. - DOI - PubMed
    1. Rampaul RS, Pinder SE, Elston CW, Ellis IO. Prognostic and predictive factors in primary breast cancer and their role in patient management: The Nottingham Breast Team. Eur J Surg Oncol. 2001;27:229–238. doi: 10.1053/ejso.2001.1114. - DOI - PubMed