Magnesil paramagnetic particles

The “Methylation on Beads” (MOB) method was used for the genomic DNA isolation from FFPE tissue specimens and bisulfite conversion [34 (link)]. MOB is a single-tube method for the extraction and analysis of methylated DNA via the use of silica super magnetic beads and improves the extraction efficiency compared with conventional techniques [35 (link),36 (link)]. After deparaffinization and lysis of the samples with proteinase K (Qiagen, Hilden, Germany) in 60 °C for 14 h, the lysate was mixed with 150 μL of magnesil paramagnetic particles (Promega, Madison, WI, USA). After purification of the beads with sequential washes and drying at 90 °C, the short protocol of the EZ DNA methylation Gold kit (Zymo Research, Irvine, CA, USA) was followed for the bisulfite conversion. Samples were incubated with bisulfite conversion reagent initially at 98 °C for 10 min and then at 64 °C for 2.5 h. After bisulfite conversion, DNA was desulfonated, washed, dried, eluted in 100 μL elution buffer, and stored at −20 °C.

Genomic DNA was isolated from tail tips using Proteinase K (Sigma) with isopropanol purification. Hypothalamic tissue was dissected from mouse brains, and RNA was extracted using the RNeasy lipid tissue Mini-kit™ (Qiagen). Reverse transcription was performed on 200 ng mRNA using Superscript III™ (Invitrogen). Oligonucleotide primers for PCR and direct sequencing were designed using the Primer 3 program and PCR was performed under standard conditions using TaqGold (Roche). PCR products were purified using Magnesil Paramagnetic Particles (Promega), and DNA sequencing was performed on an ABI 377 DNA sequencing machine using Big Dye terminator chemistry.

Genomic DNAs from Neisseria gonorrhoeae (ATCC 700825DQ), Chlamydia trachomatis Serovar D strain UW-3/Cx (ATCC VR-885D), Trichomonas vaginalis (ATCC 30001DQ) and Treponema pallidum (ATCC BAA-2642SD) and heat-inactivated N. gonorrhoeae cells (ATCC 19424-IN) were purchased from LGC standards, UK. WarmStart® colorimetric LAMP 2 × master mix (DNA & RNA) was purchased from New England Biolabs. Primers were supplied from Integrated DNA Technologies (IDT). MagneSil® paramagnetic particles were purchased from Promega. SYBR Safe, mineral oil, Tween 20, DNA decontaminant solution, PCR adhesive film and nuclease-free water were supplied from Thermo Fisher Scientific. Guanidine hydrochloride was purchased from VWR. Sigmatrix synthetic urine diluent was procured from Sigma-Aldrich.