Use of FTA ^® classic cards for epigenetic analysis of sperm DNA

Abstract

FTA® technologies provide the most reliable method for DNA extraction. Although FTA technologies have been widely used for genetic analysis, there is no literature on their use for epigenetic analysis yet. We present for the first time, a simple method for quantitative methylation assessment based on sperm cells stored on Whatman FTA classic cards. Specifically, elution of seminal DNA from FTA classic cards was successfully tested with an elution buffer and an incubation step in a thermocycler. The eluted DNA was bisulfite converted, amplified by PCR, and a region of interest was pyrosequenced.

Assessment of dried blood spots for DNA methylation profiling

“…These cards contain chemicals that lyse cell membranes and denature proteins, thus protecting DNA from degradation. Samples collected on these cards can be stored at room temperature for several years 8 prior to DNA extraction, thus avoiding the financial and logistical issues associated with freezer storage. Whatman FTA ® cards have been validated for genetic studies 9– 11 ; however, the effect of storing samples in this manner on the epigenome is less well-established.…”

“…Whatman FTA ® cards have been validated for genetic studies 9– 11 ; however, the effect of storing samples in this manner on the epigenome is less well-established. Previous studies provide support for the possibility of profiling DNA methylation in samples stored on Whatman FTA ® cards 8, 12 ; however, these studies were limited by their focus on either a small number of individuals (n = 2) 12 or a single gene of interest 8 and a relatively short time interval between sample collection and DNA extraction (maximum of approximately two years). Moreover, to the best of our knowledge, no previous study has compared genome-wide DNA methylation profiles obtained from dried blood spots (DBSs) stored on Whatman FTA ® cards with those from DNA from matched whole blood samples frozen in EDTA tubes (henceforth referred to as “EDTA samples”).…”

Assessment of Dried Blood Spots for DNA Methylation Profiling

“…These cards contain chemicals that lyse cell membranes and denature proteins, thus protecting DNA from degradation (https://www.sigmaaldrich.com/catalog/product/sigma/whawb120205?lang=en&region=GB). Samples collected on these cards can be stored at room temperature for several years [8] prior to DNA extraction, thus avoiding the financial and logistical issues associated with freezer storage. Whatman FTA ® cards have been validated for genetic studies [9–11] ; however, the effect of storing samples in this manner on the epigenome is less well-established.…”

Simple Workflow for Genome and Methylation Analyses of Ejaculated Bovine Spermatozoa with Low Sperm Input

“…Simplified methods for gDNA extraction of sperm followed by successful bisulfite conversion, PCR amplification and downstream sequencing are needed to improve the workflow in clinical settings and provide more rigorous analyses with minimal processing times. However, extraction of DNA from sperm presents unique challenges that differ from somatic cells, including an acrosomal barrier and protamine compaction of chromatin that often results in low DNA yield [6][7][8]. Some protocols have been developed to address these challenges; however, most involve prolonged incubation times, proprietary reagents and high numbers of total sperm input that may be incompatible with available material [9].…”