The Use of Laser Microdissection in the Identification of Suitable Reference Genes for Normalization of Quantitative Real-Time PCR in Human FFPE Epithelial Ovarian Tissue Samples

Cai, Jing; Li, Tao; Huang, Bangxing; Cheng, He Ming; Ding, Hui; Wu, Dong; Xiao, Mi; Liu, Ling; Wang, Zehua

doi:10.1371/journal.pone.0095974

Cited by 20 publications

(21 citation statements)

References 20 publications

(27 reference statements)

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“…In the present study, the DNA yield for samples extracted by WAX was higher than that extracted by QIA at short DNA lengths, and the amount of fragmented DNA tended to be underestimated, although the amplicon was shorter than the template DNA (18) and the DNA extracted by WAX included some PCR inhibitors (7). Developments in the last decade have allowed fragmented DNA to be used (9,(22)(23)(24); it is therefore important to obtain as much DNA as possible from limited FFPE samples (25,26). WAX may therefore have some advantages over QIA with regards to DNA yield.…”

Section: Discussionmentioning

confidence: 99%

Estimation of age-related DNA degradation from formalin-fixed and paraffin-embedded tissue according to the extraction methods

Watanabe

Hashida

Yamamoto

et al. 2017

Experimental and Therapeutic Medicine

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Abstract.Techniques for the extraction and use of nucleic acids from formalin-fixed and paraffin-embedded (FFPE) tissues, preserved over long time periods in libraries, have been developed. However, DNA extracted from FFPE tissues is generally damaged, and long-term storage may affect DNA quality. Therefore, it is important to elucidate the effect of long-term storage on FFPE tissues and evaluate the techniques used to extract DNA from them. In the present study, the yield, purity, and integrity of DNA in FFPE tissue samples was evaluated. Two DNA extraction techniques were used: A silica-binding DNA collection method using QIAamp DNA FFPE Tissue kit (QIA) and a total tissue DNA collection method using a WaxFree DNA extraction kit (WAX). A total of 25 FFPE tissues from lung adenocarcinomas were studied, which had been surgically resected and fixed at Okayama University Hospital prior to examination and subsequent storage at room temperature for 0.5, 3, 6, 9 and 12 years. Extracted DNA was quantified using ultraviolet absorbance, fluorescent dye, and quantitative polymerase chain reaction (qPCR). The quality of the DNA was defined by the absorbance ratio of 260 to 280 nm (A260/280) and Q-score, which is the quantitative value of qPCR product size ratio. The results demonstrated that the yield of total DNA extracted using WAX was significantly greater than when QIA was used (P<0.01); however, DNA extracted using WAX included more contaminants and was significantly more fragmented compared with DNA extracted using QIA (P<0.01). Aging had no significant effect on absolute DNA yield or DNA purity, although it did significantly contribute to increased DNA degradation for both QIA and WAX extraction (QIA P=0.02, WAX P=0.03; 0.5 years vs. 3 years, QIA P<0.01, WAX P=0.03; 9 years vs. 12 years). Both extraction methods are viable depending on whether high yield or high quality of extracted DNA is required. However, due to the increased degradation with age, storage time limits the available DNA in FFPE tissues regardless of the extraction method.

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Section: Discussionmentioning

confidence: 99%

Estimation of age-related DNA degradation from formalin-fixed and paraffin-embedded tissue according to the extraction methods

Watanabe

Hashida

Yamamoto

et al. 2017

Experimental and Therapeutic Medicine

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“…Cox proportional hazard models and log-rank tests were used to calculate HR, 95% confidence interval, and log rank P-value. patient samples, 25 malignant epithelial tumors), and a suitable reference gene for qPCR (43). It should however be noted that only 4/25 cancer samples were characterized as CCC.…”

Section: Discussionmentioning

confidence: 99%

Validation of Novel Prognostic Biomarkers for Early-Stage Clear-Cell, Endometrioid and Mucinous Ovarian Carcinomas Using Immunohistochemistry

et al. 2020

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Early-stage (I and II) ovarian carcinoma patients generally have good prognosis. Yet, some patients die earlier than expected. Thus, it is important to stratify early-stage patients into risk groups to identify those in need of more aggressive treatment regimens. The prognostic value of 29 histotype-specific biomarkers identified using RNA sequencing was evaluated for early-stage clear-cell (CCC), endometrioid (EC) and mucinous (MC) ovarian carcinomas (n = 112) using immunohistochemistry on tissue microarrays. Biomarkers with prognostic significance were further evaluated in an external ovarian carcinoma data set using the web-based Kaplan-Meier plotter tool. Here, we provide evidence of aberrant protein expression patterns and prognostic significance of 17 novel histotype-specific prognostic biomarkers [10 for CCC (ARPC2, CCT5, GNB1, KCTD10, NUP155, RPL13A, RPL37, SETD3, SMYD2, TRIO), three for EC (CECR1, KIF26B, PIK3CA), and four for MC (CHEK1, FOXM1, KIF23, PARPBP)], suggesting biological heterogeneity within the histotypes. Combined predictive models comprising the protein expression status of the validated CCC, EC and MC biomarkers together with established clinical markers (age, stage, CA125, ploidy) improved the predictive power in comparison with models containing established clinical markers alone, further strengthening the importance of the biomarkers in ovarian carcinoma. Further, even improved predictive powers were demonstrated when combining these models with our previously identified prognostic biomarkers PITHD1 (CCC) and GPR158 (MC). Moreover, the proteins demonstrated improved risk prediction of CCC-, EC-, and MC-associated ovarian carcinoma survival. The novel histotype-specific prognostic biomarkers may not only improve prognostication and patient stratification of early-stage ovarian carcinomas, but may also guide future clinical therapy decisions.

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“…In studies investigating reference genes for RT-qPCR experiments using FFPE, MRPL19 has also been shown to be among the top-ranked reference genes for FFPE samples from both breast [20] and lung cancer patients [7] . PPIA has been found to be among the most suitable reference genes for normalization in human FFPE epithelial ovarian tissue samples [21] and from fresh frozen tissue samples in colon cancer [22] . SF3A1 has been found to be among the most stable genes in fresh frozen breast cancer specimens [23] .…”

Section: Discussionmentioning

confidence: 99%

The Importance of Reference Gene Analysis of Formalin-Fixed, Paraffin-Embedded Samples from Sarcoma Patients — An Often Underestimated Problem

Aggerholm‐Pedersen

Safwat

Bærentzen

et al. 2014

Translational Oncology

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Objective: Reverse transcription quantitative real-time polymerase chain reaction is efficient for quantification of gene expression, but the choice of reference genes is of paramount importance as it is essential for correct interpretation of data. This is complicated by the fact that the materials often available are routinely collected formalin-fixed, paraffin-embedded (FFPE) samples in which the mRNA is known to be highly degraded. The purpose of this study was to investigate 22 potential reference genes in sarcoma FFPE samples and to study the variation in expression level within different samples taken from the same tumor and between different histologic types. Methods: Twenty-nine patients treated for sarcoma were enrolled. The samples encompassed 82 (FFPE) specimens. Extraction of total RNA from 7-μm FFPE sections was performed using a fully automated, bead-base RNA isolation procedure, and 22 potential reference genes were analyzed by reverse transcription quantitative real-time polymerase chain reaction. The stability of the genes was analyzed by RealTime Statminer. The intrasamples variation and the interclass correlation coefficients were calculated. The linear regression model was used to calculate the degradation of the mRNA over time. Results: The quality of RNA was sufficient for analysis in 84% of the samples. Recommended reference genes differed with histologic types. However, PPIA, SF3A1, and MRPL19 were stably expressed regardless of the histologic type included. The variation in ∆Cq value for samples from the same patients was similar to the variation between patients. It was possible to compensate for the time-dependent degradation of the mRNA when normalization was made using the selected reference genes. Conclusion: PPIA, SF3A1, and MRPL19 are suitable reference genes for normalization in gene expression studies of FFPE samples from sarcoma regardless of the histology.

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The Use of Laser Microdissection in the Identification of Suitable Reference Genes for Normalization of Quantitative Real-Time PCR in Human FFPE Epithelial Ovarian Tissue Samples

Cited by 20 publications

References 20 publications

Estimation of age-related DNA degradation from formalin-fixed and paraffin-embedded tissue according to the extraction methods

Estimation of age-related DNA degradation from formalin-fixed and paraffin-embedded tissue according to the extraction methods

Validation of Novel Prognostic Biomarkers for Early-Stage Clear-Cell, Endometrioid and Mucinous Ovarian Carcinomas Using Immunohistochemistry

The Importance of Reference Gene Analysis of Formalin-Fixed, Paraffin-Embedded Samples from Sarcoma Patients — An Often Underestimated Problem

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