Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Patel, Palak; Selvarajah, Shamini; Boursalie, Suzanne; How, Nathan; Ejdelman, Joshua; Guérard, Karl-Philippe; Bartlett, John M.S.; Lapointe, Jacques; Park, Paul C.; Okello, John B. A.; Berman, David M.

doi:10.3791/54299

Cited by 31 publications

(29 citation statements)

References 25 publications

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“…44,47 We modified previously described protocols for real-time MSP (qMSP) assays according to MIQE guidelines [42][43][44]48 and quantified changes in 15 DNA methylation alterations (Table S3) in DNA samples collected from three RP cohorts. 45,49 Briefly, individual DNA samples (50 ng) were bisulfite-converted according to the manufacturer's protocol (EpiTect Bisulfite Kit; Qiagen). A mastermix was prepared that contained one of 15 primer pairs (400 nM; Thermo Fisher Scientific) and probe sets (150 nM; Thermo Fisher Scientific) (Table S3) was determined by generating standard curves as described previously (Table S3).…”

Section: Qmsp Analysismentioning

confidence: 99%

A three‐gene DNA methylation biomarker accurately classifies early stage prostate cancer

et al. 2019

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Background We identify and validate accurate diagnostic biomarkers for prostate cancer through a systematic evaluation of DNA methylation alterations. Materials and methods We assembled three early prostate cancer cohorts (total patients = 699) from which we collected and processed over 1300 prostatectomy tissue samples for DNA extraction. Using real‐time methylation‐specific PCR, we measured normalized methylation levels at 15 frequently methylated loci. After partitioning sample sets into independent training and validation cohorts, classifiers were developed using logistic regression, analyzed, and validated. Results In the training dataset, DNA methylation levels at 7 of 15 genomic loci (glutathione S‐transferase Pi 1 [GSTP1], CCDC181, hyaluronan, and proteoglycan link protein 3 [HAPLN3], GSTM2, growth arrest‐specific 6 [GAS6], RASSF1, and APC) showed large differences between cancer and benign samples. The best binary classifier was the GAS6/GSTP1/HAPLN3 logistic regression model, with an area under these curves of 0.97, which showed a sensitivity of 94%, and a specificity of 93% after external validation. Conclusion We created and validated a multigene model for the classification of benign and malignant prostate tissue. With false positive and negative rates below 7%, this three‐gene biomarker represents a promising basis for more accurate prostate cancer diagnosis.

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Section: Qmsp Analysismentioning

confidence: 99%

A three‐gene DNA methylation biomarker accurately classifies early stage prostate cancer

et al. 2019

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“…[91]. It could be postulated that using fresh frozen tissue samples of PLGA tumour would yield more high quality nucleic acids, and this could have altered our results [91].…”

Section: Study Limitationsmentioning

confidence: 93%

Expression of Human Tissue Kallikreins (KLKs) in Polymorphous Low Grade Adenocarcinoma (PLGA)

Cox

2014

Journal of Oral and Maxillofacial Surgery

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Polymorphous low grade adenocarcinoma (PLGA) is the second most common malignant salivary gland tumour of the minor salivary glands. Human tissue kallikreins (KLKs) are a family of highly conserved serine proteases expressed by various tissues throughout the body. KLKs have become powerful tumour markers for the diagnosis of the cancer patient (e.g. PSA (KLK3)). The literature demonstrates a link between KLKs and salivary gland neoplasms. The purpose of this study is to determine levels of KLK mRNA in tissue samples of formalin fixed paraffin embedded polymorphous low grade adenocarcinoma (PLGA). Secondly, we wish to determine if KLK expression is limited to tumour cells alone.Nineteen cases of PLGA were reviewed . A diagnosis of PLGA was confirmed, demographic data was collected, and formalin fixed paraffin-embedded PLGA and normal salivary gland tissue samples were obtained. RNA isolation was achieved, followed by conversion to complementary DNA via reverse transcription. Synthesized DNA primers were added to target kallikrein DNA and through PCR, the quantitative level of expression of KLKs 1-15 was recorded. Samples exhibiting high and low KLK expression were selected for immunohistochemistry staining, using a standard protocol.Results from PCR data reveals statistically significant increase in the mean KLK mRNA expression for KLK1, KLK4, KLK10, KLK12 and KLK15 in PLGA tissue samples, as compared with normal salivary gland tissue (Mann Whitney U test, p<0.05).Immunohistochemistry results demonstrate tumour specific staining. Notably, all samples demonstrating relatively higher KLK mRNA expression showed equivalent or increased staining grade scores relative to the low KLK mRNA expression samples, with respect to a specific KLK.In tissue samples of polymorphous low grade adenocarcinoma there is an increase of KLK1, KLK4, KLK10, KLK12 and KLK15 mRNA relative to normal salivary gland tissue. Furthermore, the tumour cells stain positively and specifically for kallikreins.

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“…DNA isolation from FFPE tissue is a decisive step toward a successful examination. Recent advances in technological developments have made generation of genetic information of DNA and RNA from these kinds of challenging samples possible [110][111][112][113][114].…”

Section: Collection Devices and Paraffin Embedded Tissuesmentioning

confidence: 99%

Most Common Medico-Legal Autopsy-Related Human and Nonhuman Biological Samples for DNA Analysis

Pádár¹,

Zenke²,

Kozma³

2018

Post Mortem Examination and Autopsy - Current Issues From Death to Laboratory Analysis

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The identification and individualization of biological evidences is crucial to actual criminal investigations. In spite of the differences at the national level, all the legal processes attribute particular importance to forensic DNA analysis. However, none of the qualified results from any professional laboratory can produce substantive, valuable evidence with insufficient quality of samples and/or problems with provision of a pristine and controlled environment. The methodology and efficiency of sampling are distinct in case of living persons and in medico-legal autopsy and crime scenes. This chapter is a short overview from the basic introductory information up to ongoing research, and in accordance with constraints on the chapter size, it briefly discusses the important topics of sample collection at medico-legal autopsy for DNA analysis. The content sorts the major types of samples, reviews the common methods of sampling and the potential risk of poor sampling or contamination transfer. The corpses can be more or less degraded, which in special cases (e.g., paraffin embedded tissues, drowned, burning and/or buried cadaver) allow only for analysis of highly degraded samples. The samples can be associated with tissues of a corpse (e.g., blood, soft tissues, bone, tooth, hair) and/or additional extraneous tissues and remains, which are often mixed (e.g., blood, saliva, semen, vaginal fluid, debris of fingernails) on the corpse.

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Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Cited by 31 publications

References 25 publications

A three‐gene DNA methylation biomarker accurately classifies early stage prostate cancer

A three‐gene DNA methylation biomarker accurately classifies early stage prostate cancer

Expression of Human Tissue Kallikreins (KLKs) in Polymorphous Low Grade Adenocarcinoma (PLGA)

Most Common Medico-Legal Autopsy-Related Human and Nonhuman Biological Samples for DNA Analysis

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