A protocol for good quality genomic DNA isolation from formalin-fixed paraffin-embedded tissues without using commercial kits

DNA isolation from formalin fixed paraffin embedded (FFPE) tissues for molecular analysis has become a frequent procedure in cancer research. However, the yield or quality of the isolated DNA is often compromised, and commercial kits are used to overcome this to some extent. We developed a new protocol (IARCp) to improve better quality and yield of DNA from FFPE tissues without using any commercial kit. To evaluate the IARCp performance, we compared the quality and yield of DNA with two commercial kits, namely NucleoSpin DNA FFPE XS (MN) and QIAamp DNA Micro (QG) isolation kit. Total DNA yield for QG ranged from 120.0 to 282.0 ng (mean 216.5 ng), for MN: 213.6 to 394.2 ng (mean 319.1 ng), and with IARCp the yield was much higher ranging from 775.5 to 1896.9 ng (mean 1517.8 ng). Moreover, IARCp has also performed well in qualitative assessments. Overall, IARCp represents a novel approach to DNA isolation from FFPE which results in good quality and significant amounts of DNA suitable for many downstream genome-wide and targeted molecular analyses. Our proposed protocol does not require the use of any commercial kits for isolating DNA from FFPE tissues, making it suitable to implement in low-resource settings such as low and middle-income countries (LMICs).

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Automation of genomic DNA isolation from formalin-fixed, paraffin-embedded tissues

Pathology - Research and Practice ◽

10.1016/j.prp.2012.08.008 ◽

2012 ◽

Vol 208 (12) ◽

pp. 705-707 ◽

Cited By ~ 22

Author(s):

Soya S. Sam ◽

Kimberly A. Lebel ◽

Cheryl L. Bissaillon ◽

Laura J. Tafe ◽

Gregory J. Tsongalis ◽

...

Keyword(s):

Genomic Dna ◽

Dna Isolation ◽

Genomic Dna Isolation ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

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DNA isolation from achieved formalin-fixed paraffin-embedded tissues in a series of 212 thyroid carcinoma cases: the influence of preanalytical factors on DNA quantity and purity

Journal of Investigative Medicine ◽

10.1136/jim-2019-001134 ◽

2019 ◽

Vol 68 (3) ◽

pp. 792-798

Author(s):

Adela Nechifor-Boilă ◽

Claudia Banescu ◽

Ancuta Elena Zahan ◽

Valeriu Moldovan ◽

Emoke Szasz ◽

...

Keyword(s):

Tumor Volume ◽

Dna Isolation ◽

Storage Period ◽

Routine Practice ◽

Dna Concentration ◽

Maximal Diameter ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Ffpe Tissues ◽

Formalin Fixed

ObjectiveThe present study aimed to investigate the influence of several important preanalytical factors (storage period of the tumor block, maximal diameter of the tumor circled area, tumor volume and tumor fraction) on the isolated DNA from formalin-fixed paraffin-embedded (FFPE) tissues in a series of thyroid carcinomas.DesignOur study included 212 FFPE blocks, archived in the Department of Pathology, Târgu-Mureș Emergency County Hospital for up to 10 years. DNA isolation was performed using a commercially available kit (MasterPure DNA purification kit, Epicentre). The DNA parameters (concentration and purity) were determined using a spectrophotometer and the Qubit 2.0 Fluorometer (Thermo Fisher Scientific) for an accurate and sensitive DNA quantification.ResultsThe mean DNA concentration and purity for the study cases were 489.3±372.6 ng/µl and 1.667±0.1912, respectively. The DNA concentration was correlated with the maximal diameter of the tumor circled area (p<0.0001), the tumor volume (p<0.0001) and tumor fraction (p=0.0462). No statistically significant differences both in terms of DNA concentration (p=0.374) and purity (p=0.125) in relation with the storage period of the tumor blocks were observed. When using a fluorometric quantification method, the DNA concentration was lower (mean DNA concentration: 47.15±32.85 ng/µl), but similar correlations with the morphological factors were observed. Apart for three cases, the real-time PCR amplification of the BRAF gene was successfully assessed in all cases.ConclusionThe maximal diameter of the tumor circled area, tumor volume and tumor fraction are important morphological factors that correlate with the DNA concentration and should be carefully assessed in routine practice prior to performing DNA isolation from FFPE tissues.

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Characterization of epididymal and testicular histologic lesions and use of immunohistochemistry and PCR on formalin-fixed tissues to detect Brucella canis in male dogs

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638720986883 ◽

2021 ◽

pp. 104063872098688

Author(s):

Andrea M. Camargo-Castañeda ◽

Lauren W. Stranahan ◽

John F. Edwards ◽

Daniel G. Garcia-Gonzalez ◽

Leonardo Roa ◽

...

Keyword(s):

Accurate Diagnosis ◽

Testicular Atrophy ◽

Detection Techniques ◽

Formalin Fixed Paraffin ◽

Brucella Canis ◽

Formalin Fixed Paraffin Embedded ◽

Variable Sensitivity ◽

Ffpe Tissues ◽

Formalin Fixed

In male dogs, Brucella canis frequently causes epididymitis, ultimately resulting in testicular atrophy and infertility. Although B. canis predominantly affects the epididymis, the misleading term “orchitis” is still commonly used by clinicians. Of additional concern, diagnosis in dogs remains challenging because of variable sensitivity and specificity of serologic assays and fluctuations in bacteremia levels in infected dogs, reducing the sensitivity of blood culture. We describe here the histologic lesions in the scrotal contents of 8 dogs suspected of being infected with B. canis and clinically diagnosed with orchitis. We explored the possibility of using immunohistochemistry (IHC) and real-time PCR (rtPCR) in formalin-fixed, paraffin-embedded (FFPE) tissues to detect the presence of B. canis. Epididymitis of variable chronicity was identified in all 8 dogs, with only 3 also exhibiting orchitis. Using rtPCR, the presence of B. canis was identified in 4 of 8 dogs, with 3 of these 4 dogs also positive by IHC. These results suggest that rtPCR and IHC are promising techniques that can be used in FFPE tissues to detect B. canis when other detection techniques are unavailable. Additionally, accurate recognition of epididymitis rather than orchitis in suspect cases could aid in accurate diagnosis.

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Sarcoma Subgrouping by Detection of Fusion Transcripts Using NanoString nCounter Technology

Pediatric and Developmental Pathology ◽

10.1177/1093526618790747 ◽

2018 ◽

Vol 22 (3) ◽

pp. 205-213 ◽

Cited By ~ 4

Author(s):

Javal Sheth ◽

Anthony Arnoldo ◽

Yunan Zhong ◽

Paula Marrano ◽

Carlos Pereira ◽

...

Keyword(s):

Rt Pcr ◽

Fusion Transcripts ◽

Pediatric Sarcoma ◽

Formalin Fixed Paraffin ◽

Pediatric Sarcomas ◽

Formalin Fixed Paraffin Embedded ◽

Ffpe Tissues ◽

Nanostring Technology ◽

Future Work ◽

Formalin Fixed

Background NanoString technology is an innovative barcode-based system that requires less tissue than traditional techniques and can test for multiple fusion transcripts in a single reaction. The objective of this study was to determine the utility of NanoString technology in the detection of sarcoma-specific fusion transcripts in pediatric sarcomas. Design Probe pairs for the most common pediatric sarcoma fusion transcripts were designed for the assay. The NanoString assay was used to test 22 specific fusion transcripts in 45 sarcoma samples that had exhibited one of these fusion genes previously by reverse transcription polymerase chain reaction (RT-PCR). A mixture of frozen (n = 18), formalin-fixed, paraffin-embedded (FFPE) tissue (n = 23), and rapid extract template (n = 4) were used for testing. Results Each of the 22 transcripts tested was detected in at least one of the 45 tumor samples. The results of the NanoString assay were 100% concordant with the previous RT-PCR results for the tumor samples, and the technique was successful using both FFPE and rapid extract method. Conclusion Multiplexed interrogation for sarcoma-specific fusion transcripts using NanoString technology is a reliable approach for molecular diagnosis of pediatric sarcomas and works well with FFPE tissues. Future work will involve validating additional sarcoma fusion transcripts as well as determining the optimal workflow for diagnostic purposes.

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Sodium Alginate versus Plasma Thrombin Cell Blocks in Diagnostic Cytopathology: A Comparative Analysis

Acta Cytologica ◽

10.1159/000519336 ◽

2021 ◽

pp. 1-7

Author(s):

Shruti Gupta ◽

Upasana Gautam ◽

Shaily Susheilia ◽

Baneet Bansal ◽

Radha Uppal ◽

...

Keyword(s):

Sodium Alginate ◽

Molecular Analysis ◽

Low Cost ◽

Nuclear Marker ◽

Fine Needle Aspirates ◽

Dna Yield ◽

Morphological Evaluation ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

Background: Cell blocks (CBs) are an essential adjunct in cytopathology practice. The aim of this study was to compare 2 techniques of CB preparation – plasma thrombin (PT) method with sodium alginate (SA) method for overall cellularity, morphological preservation, obscuring artefacts, immunocytochemistry (ICC), suitability for molecular analysis, and cost of preparation. Design: A total of 80 fine-needle aspirates from various sites and serous effusion samples were included. Of these cases, by random selection, 40 each were prepared by PT method and SA methods, respectively. The haematoxylin-eosin-stained sections from the formalin-fixed, paraffin-embedded CBs from both methods were evaluated in a blinded fashion by 2 cytopathologists and scored for cellularity, artefacts, and morphological preservation and analysed by χ2 test with Yates correction. We evaluated 6 cases from each method by ICC for a range of membrane, cytoplasmic and nuclear marker expression. DNA was extracted from four cases to evaluate their utility for molecular analysis. Results: CB sections from PT and SA techniques showed comparable cellularity and excellent cytomorphological preservation. Blue gel-like artefacts were common in the SA technique but did not interfere with morphological evaluation. ICC staining results were also similar. DNA yield and utility for PCR were also comparable. The SA-CB cost half that of PT-CB (USD 0.4 vs. USD 1). Conclusion: SA technique of CB preparation is an excellent low-cost alternative to PT method for CB preparation.

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Molecular Markers for Prostate Cancer in Formalin-Fixed Paraffin-Embedded Tissues

BioMed Research International ◽

10.1155/2013/283635 ◽

2013 ◽

Vol 2013 ◽

pp. 1-15 ◽

Cited By ~ 9

Author(s):

Tamara Sequeiros ◽

Marta García ◽

Melania Montes ◽

Mireia Oliván ◽

Marina Rigau ◽

...

Keyword(s):

Prostate Cancer ◽

Molecular Markers ◽

Tumor Grade ◽

Developed Countries ◽

Response To Therapy ◽

Tissue Samples ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Ffpe Tissues ◽

Formalin Fixed

Prostate cancer (PCa) is the most frequently diagnosed type of cancer in developed countries. The decisive method of diagnosis is based on the results of biopsies, morphologically evaluated to determine the presence or absence of cancer. Although this approach leads to a confident diagnosis in most cases, it can be improved by using the molecular markers present in the tissue. Both miRNAs and proteins are considered excellent candidates for biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues, due to their stability over long periods of time. In the last few years, a concerted effort has been made to develop the necessary tools for their reliable measurement in these types of samples. Furthermore, the use of these kinds of markers may also help in establishing tumor grade and aggressiveness, as well as predicting the possible outcomes in each particular case for the different treatments available. This would aid clinicians in the decision-making process. In this review, we attempt to summarize and discuss the potential use of microRNA and protein profiles in FFPE tissue samples as markers to better predict PCa diagnosis, progression, and response to therapy.

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Clostridium perfringens–Associated Necrotic Enteritis-Like Disease in Coconut Lorikeets (Trichoglossus haematodus)

Veterinary Pathology ◽

10.1177/0300985820971788 ◽

2020 ◽

pp. 030098582097178

Author(s):

Llorenç Grau-Roma ◽

Mauricio Navarro ◽

Sohvi Blatter ◽

Christian Wenker ◽

Sonja Kittl ◽

...

Keyword(s):

Clostridium Perfringens ◽

Toxin Gene ◽

Necrotic Enteritis ◽

Gene Encoding ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Ffpe Tissues ◽

Polymerase Chain ◽

Beta Toxin ◽

Formalin Fixed

Several outbreaks of necrotic enteritis-like disease in lorikeets, from which Clostridium perfringens was consistently isolated, are described. All lorikeets had acute, segmental, or multifocal fibrinonecrotizing inflammatory lesions in the small and/or the large intestine, with intralesional gram-positive rods. The gene encoding C. perfringens alpha toxin was detected by PCR (polymerase chain reaction) on formalin-fixed, paraffin-embedded (FFPE) tissues in 20 out of 24 affected lorikeets (83%), but it was not amplified from samples of any of 10 control lorikeets ( P < .0001). The second most prevalent C. perfringens toxin gene detected was the beta toxin gene, which was found in FFPE from 7 out of 24 affected lorikeets (29%). The other toxin genes were detected inconsistently and in a relatively low number of samples. These cases seem to be associated with C. perfringens, although the specific type involved could not be determined.

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