miRNA analysis of prostate cancer by quantitative real time PCR: Comparison between formalin-fixed paraffin embedded and fresh-frozen tissue

doi:10.1016/j.urolonc.2009.05.008

Urologic Oncology: Seminars and Original Investigations

Volume 29, Issue 5, September–October 2011, Pages 533-537

https://doi.org/10.1016/j.urolonc.2009.05.008 Get rights and content

Abstract

Objective

Micro RNA (miRNA) is a class of small noncoding RNA that plays a major role in the regulation of gene expression, which has been related to cancer behavior. The possibility of analyzing miRNA from the archives of pathology laboratories is exciting, as it allows for large retrospective studies. Formalin is the most common fixative used in the surgical pathology routine, and its promotion of nucleic acid degradation is well known. Our aim is to compare miRNA profiles from formalin-fixed paraffin embedded (FFPE) tissues with fresh-frozen prostate cancer tissues.

Methods

The expression of 14 miRNAs was determined by quantitative real time polymerase chain reaction (qRT-PCR) in 5 paired fresh-frozen and FFPE tissues, which were representative of prostate carcinoma.

Results

There was a very good correlation of the miRNA expression of miR-let7c and miR-32 between the fresh-frozen and FFPE tissues, with Pearson's correlation coefficients of 0.927 (P = 0.023) and 0.960 (P = 0.010), respectively. For the remaining miRNAs, the correlation was good with Spearman correlation coefficient of 0.638 (P < 0.001).

Conclusion

Analysis of miRNAs from routinely processed and stored FFPE prostate tissue is feasible for some miRNAs using qRT-PCR. Further studies should be conducted to confirm the reliability of using stock tissues for miRNA expression determination.

Introduction

Identification of the molecular mechanisms related to the development and progression of neoplasms is a vast area of research and has resulted in enormous transformations in clinical practice, permitting the finding of new diagnostic and prognostic markers and, more interestingly, the development of specific target therapy. Ideal clinical specimens for molecular biology study are snap fresh-frozen tissues with preserved DNA, RNA, and proteins. However, this practice of snap freezing requires a special structure and organization that many hospitals and laboratories do not have [1].

The most common fixative for human samples is a formaldehyde solution at a concentration of 3.7%, which is referred to as 10% formalin and leads to extensive crosslinking of all tissue components. As a consequence, nucleic acids suffer different grades of fragmentation depending on the conditions of the tissue fixation, processing, and storing [2].

The archives of pathology laboratories are an enormous source of human samples, allowing for huge retrospective studies, which are important in the study of cancer since many years of follow-up are imperative to prove the role of a new molecular marker.

Micro RNA (miRNA) is a class of small noncoding RNA of 19 to 25 nucleotides, whose major role is the regulation of gene expression. Half of them are located in the so-called “fragile sites” of chromosomal DNA, which are frequently deleted, amplified, or rearranged in many cases of cancer. miRNAs have been shown to interfere in key cellular functions, such as cell proliferation, cell differentiation, and apoptosis, abnormalities that are hallmarks of cancer, which suggests that miRNA might be a new class of genes involved in human tumorigenesis [3].

Our aim is to study the possibility of using formalin fixed, paraffin embedded (FFPE) material from the archives to identify miRNA profiles. To achieve this goal, we compared the expression of 14 miRNAs in paired fresh-frozen and FFPE tissues representative of prostate carcinoma.

Section snippets

Patients

Five patients underwent open radical prostatectomy to treat prostate cancer from December, 2007 to January, 2008. The median patient age was 70 years, and ranged from 54 to 77 years. The median prostate specific antigen (PSA) was 8.7 ng/mL, and ranged from 4.2 to 13.3 ng/mL. The median Gleason score was 8, and varied from 8 to 9, and the median tumor volume was 8 cm³, and ranged from 2.6 to 10.6 cm³. All patients were staged pT3.

The fresh surgical specimen was sent to the laboratory no longer

Quality of recovered RNA

The integrity and quantity of recovered miRNA was analyzed using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA). As shown in Fig. 1, the concentration of miRNA was similar in both the frozen tissues and FFPE specimens. Additionally, the 260/280 absorbance ratio of absorbance was around 2.0 for both samples, indicating good quality miRNA. The 260/230 ratio was 1.26 for fresh tissue and 0.93 for paraffin embedded tissue. The 230 nm is related to solvent contaminants and, as

Discussion

The formalin-fixed, paraffin embedded clinical samples in the archives of pathology laboratories are a rich source of information regarding neoplasm behavior. The possibility of analyzing nucleic acid from these specimens makes research more flexible and less expensive, since it does not requires special specimen handling or infrastructure.

Rupp and Locker [10] were the first to extract RNA from paraffin embedded tissue for northern hybridization, and since their work a large amount of progress

References (25)

T.E. Godfrey et al.
Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5′ nuclease quantitative reverse transcription-polymerase chain reaction
J Mol Diagn
(2000)
F. Meng et al.
MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer
Gastroenterology
(2007)
K.J. Livak et al.
Analysis of relative gene expression data using real-time quantitative PCR and the 2(−ΔΔC_T) method
Methods
(2001)
U. Lehmann et al.
Real-time PCR analysis of DNA and RNA extract from formalin-fixed and paraffin-embedded biopsies
Methods
(2001)
V. Vincek et al.
A tissue fixative that protects macromolecules (DNA, RNA, and protein) and histomorphology in clinical samples
Lab Invest
(2003)
A.K. El-Nagger
Methods in molecular surgical pathology
Semin Diagn Pathol
(2002)
G.A. Calin et al.
Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers
Proc Natl Acad Sci USA
(2004)
G.A. Calin et al.
A MicroRNA signature associated with prognosis and progression in chronic lymphocytic leukemia
N Engl J Med
(2005)
L.X. Yan et al.
MicroRNA miR-21 overexpression in human breast cancer is associated with advanced clinical stage, lymph node metastasis, and patient poor prognosis
RNA
(2008)
S. Ambs et al.
Genomic profiling of microRNA and messenger RNA reveals deregulated microRNA expression in prostate cancer
Cancer Res
(2008)

K.R. Leite et al.

Change in expression of miR-let7c, miR-100, and miR-218 from high grade localized prostate cancer to metastasis

Urol Oncol

(2009 Apr 15)

G.M. Rupp et al.

Purification and analysis of RNA from paraffin-embedded tissues

Biotechniques

(1988)

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Original articlemiRNA analysis of prostate cancer by quantitative real time PCR: Comparison between formalin-fixed paraffin embedded and fresh-frozen tissue

Abstract

Objective

Methods

Results

Conclusion

Introduction

Section snippets

Patients

Quality of recovered RNA

Discussion

J Mol Diagn

Gastroenterology

Methods

Methods

Lab Invest

Methods in molecular surgical pathology

Semin Diagn Pathol

Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers

Proc Natl Acad Sci USA

A MicroRNA signature associated with prognosis and progression in chronic lymphocytic leukemia

N Engl J Med

MicroRNA miR-21 overexpression in human breast cancer is associated with advanced clinical stage, lymph node metastasis, and patient poor prognosis

RNA

Genomic profiling of microRNA and messenger RNA reveals deregulated microRNA expression in prostate cancer

Cancer Res

Change in expression of miR-let7c, miR-100, and miR-218 from high grade localized prostate cancer to metastasis

Urol Oncol

Purification and analysis of RNA from paraffin-embedded tissues

Biotechniques

Original article
miRNA analysis of prostate cancer by quantitative real time PCR: Comparison between formalin-fixed paraffin embedded and fresh-frozen tissue