Retrieving high-quality genomic DNA from formalin-fixed paraffin-embedded tissues for multiple molecular analyses

Nguyen, Ha Thi Thu; Tatipamula, Vinay Bharadwaj; Ngoc, Duy; Huynh, Thien Chi; Dang, Mai Kim

doi:10.1080/10826068.2021.1923030

Cited by 3 publications

(2 citation statements)

References 24 publications

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“…The chronic usage of CP leads to oxidative stress that culminates in the generation of various classes of ROS, such as superoxide anion, nitric oxide and hydroxyl free radical (18). The platinum substances form complexes with the nitrogen bases of DNA and induce apoptosis (19). ROS plays a crucial role in cellular injury and necrosis via several mechanisms, namely increased lipid peroxidation, decreased endogenous antioxidants, DNA damage and protein denaturation by reacting with thiols (20,21).…”

Section: Research Articlementioning

confidence: 99%

Protective effect of Vitex altissima L.f. bark extract on cisplatin-induced renal injury in Wistar rats

2022

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Cisplatin (CP) is a commonly used chemotherapeutic drug. The major limiting factor in the use of CP is the side effects in normal tissues, including the kidney. Since ancient times, medicinal plants are rich sources of various bioactive constituents used to treat multiple ailments, including drug toxicities. The present work is a preliminary study to explore the renoprotective actions of methanolic extract of Vitex altissima L.f. bark (Va) against CP-induced renal damage in Wistar rats. Va was found to have potent radical scavenging activity than metal ion reducing power properties, compared with ascorbic acid. Further, Va was evaluated for nephroprotective activity in rats induced by CP (8 mg/kg; intraperitoneal) on the 7th day. The animals were grouped (n = 6) and treated with Va (100 and 200 mg/kg) orally for 14 days. The outcomes of the study found that CP significantly (P < 0.001) altered the oxidative stress markers (MDA, SOD and CAT), serum urea and creatinine levels. The administration of Va significantly halted the toxic condition and maintained it towards normal levels. The higher dose of Va significantly (P < 0.001) raised the SOD and CAT levels and halted the MDA levels than the low dose. Also, a higher dose of Va maintained the normal integrity of the histopathological studies of kidneys than a low dose. The present study demonstrates that V. altissima can attenuate the oxidative stress induced by CP by enhancing the endogenous antioxidant levels and depleting the lipid peroxidation levels.

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Section: Research Articlementioning

confidence: 99%

Protective effect of Vitex altissima L.f. bark extract on cisplatin-induced renal injury in Wistar rats

2022

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“…One common technique, called heat-induced antigen retrieval (HIAR), utilizes high heat to weaken and break the covalent bonds created by formalin, thereby allowing the antibodies employed by IHC to bind with their antigen epitope ( Rourke and Padula, 2016 ). Recently, genomic and proteomic studies have sought ways to break the covalent bonds of formalin fixation and free the genetic material ( Al-Attas et al, 2016 ; Berrino et al, 2020 ; Frazer et al, 2020 ; Panchal et al, 2020 ; Nguyen et al, 2022 ) or cellular proteins ( Kawashima et al, 2014 ; Lai et al, 2016 ; Mason, 2016 ; Rourke and Padula, 2016 ; Coscia et al, 2020 ; Griesser et al, 2020 ; García-Vence et al, 2021 ; Pöschel et al, 2021 ; Thacker et al, 2021 ) for analysis, involving prolonged incubation at high heat or enzymatic digestion. To unlock the diagnostic potential of vast repositories of FFPE tissues, it is paramount to harness the power of kinetic assays.…”

Section: Introductionmentioning

confidence: 99%

New protocol for kinetic assay seeding ability recovery “KASAR” from formalin-fixed paraffin-embedded tissues

Hepker

Clabaugh²,

Jin³

et al. 2023

Front. Mol. Biosci.

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The real-time quaking-induced conversion (RT-QuIC) alpha-synuclein (aSyn) protein kinetic seeding assay has been very useful for detecting pathological aggregates in various synucleinopathies including Parkinson’s disease (PD). This biomarker assay relies on fresh frozen tissue to effectively seed and amplify aSyn aggregating protein. With vast repositories of formalin-fixed paraffin-embedded (FFPE) tissues, it is paramount to harness the power of kinetic assays to unlock the diagnostic potential of archived FFPE biospecimens. However, the major challenge posed by significantly reduced amplification of formalin-fixed tissues in the assay suggests that formalin fixation deterred monomer interaction with the sample seed and depressed subsequent protein aggregation. To overcome this challenge, we developed a kinetic assay seeding ability recovery (KASAR) protocol to maintain the integrity of the tissue and seeding protein. For this, we implemented a series of heating steps with the brain tissue suspended in a buffer composed of 500 mM tris-HCl (pH 7.5) and 0.02% SDS after the standard deparaffinization of the tissue sections. Initially, samples from seven human brain samples, including four samples from patients diagnosed with dementia with Lewy bodies (DLB) and three samples from healthy controls without DLB, were compared to fresh frozen samples under three different, but clinically common sample storage conditions: formalin-fixed, FFPE, and FFPE slices cut 5 µm thick. The KASAR protocol was able to recover seeding activity for all positive samples in all storage conditions. Next, 28 FFPE samples from the submandibular gland (SMG) of patients diagnosed with PD, incidental Lewy body disease (ILBD), or healthy controls were tested with 93% of results replicating when blinded. With samples of only a few milligrams, this protocol recovered the same quality of seeding in formalin-fixed tissue as fresh frozen tissue. Moving forward, protein aggregate kinetic assays, in conjunction with the KASAR protocol, can be used to understand and diagnose neurodegenerative diseases more comprehensively. Overall, our KASAR protocol unlocks and restores the seeding ability of formalin-fixed paraffin-embedded tissues for the amplification of biomarker protein aggregates in kinetic assays.

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

et al. 2022

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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's 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 -282.0 ng (mean 216.5 ng), for MN: 213.6 -394.2 ng (mean 319.1 ng), and with IARCp the yield was much higher ranging from 775.5 -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 genomewide 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 lowresource settings such as low and middle-income countries (LMICs).

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Retrieving high-quality genomic DNA from formalin-fixed paraffin-embedded tissues for multiple molecular analyses

Cited by 3 publications

References 24 publications

Protective effect of Vitex altissima L.f. bark extract on cisplatin-induced renal injury in Wistar rats

Protective effect of Vitex altissima L.f. bark extract on cisplatin-induced renal injury in Wistar rats

New protocol for kinetic assay seeding ability recovery “KASAR” from formalin-fixed paraffin-embedded tissues

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

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