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

Abstract

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).

LGALS3 cfDNA methylation in seminal fluid as a novel prostate cancer biomarker outperforming PSA

“…CfDNA was isolated from blood and seminal plasma, respectively, using Macherey‐Nagel's NucleoSnap or NucleoSpin (for plasma volumes <500 µL) cfDNA kit and the QIAvac 24 Plus vacuum station (Qiagen). The concentration of cfDNA was determined by qPCR targeting LINE‐1 element, as described by Krasic et al 27 Isolation of gDNA from FFPE samples tissue was performed using the protocol by Talukdar et al 28 The gDNA concentration was measured by NanoDrop 2000c spectrophotometer. Regarding FFPE radical prostatectomy samples, gDNA was isolated from both the tumor tissue ( N = 27) and NTT tissue where possible ( N = 21).…”

“…The concentration of cfDNA was determined by qPCR targeting LINE-1 element, as described by Krasic et al 27 Isolation of gDNA from FFPE samples tissue was performed using the protocol by Talukdar et al 28 The gDNA concentration was measured by NanoDrop 2000c spectrophotometer. Regarding FFPE radical prostatectomy samples, gDNA was isolated from both the tumor tissue (N = 27) and NTT tissue where possible (N = 21).…”