Magna pure compact na isolation kit 1

DNA was extracted from the bone powder received from UoS using a bead-beating approach. Initially, 700 μl bacterial lysis buffer (Roche UK- Cat. No. 4659180001) was added to the powder and vortexed before being transferred to a bead beating tube (Lysis Matrix B, 2ml tube (116911050, MP Biomedicals). The sample was bead-beaten for 2 minutes at full speed on a Tissue Lyser bead beater (Qiagen- Cat. No. 69980) and then pulse centrifuged (Eppendorf UK centrifuge model 5424). An aliquot (420 μl) was then transferred to a fresh Eppendorf tube and 20 μl of proteinase K (>600mAu/ml) (Qiagen, Cat. No. 19133) was added. The sample was then incubated at 65°C on a dry bath system (StarLab UK) for 10 minutes. Finally, DNA was purified on the MagNA Pure Compact machine (Roche UK) using the Roche MagnaPure Compact DNA_bacteria_V3_2 protocol (MagNA pure compact NA isolation kit I, Roche UK- product 03730964001).

This study compared four different commercial extraction kits, where one of them performed in three different protocols to be able to isolate the nucleic acid in different forms as RNA and DNA, and both are based on the manufacturers’ instructions as shown in Figure 1; (1) the MagNA Pure Compact NA Isolation Kit I (Roche, Penzberg, Germany); (2) the Direct-zol RNA Miniprep Plus Kit (Zymo Research, Irvine, CA, USA); (3) the PowerViral^® Environmental RNA-DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA), which is currently sold under the name “Allprep_PowerViral_DNARNA” (Qiagen, Hilden, Germany); and (4) the DNA-RNA Pathogen Miniprep (Zymo Research Irvine, CA, USA). Regardless of the volume of the starting material, the extracted samples were eluted in 50 µL of DNase-/RNase-free water, and each protocol was evaluated using two independent samples.

Human PBMCs were isolated by density centrifugation using lymphocyte separation solution (nacalai tesque, Inc., Kyoto, Japan). Monkey PBMCs were isolated by density centrifugation using Lympholyte mammal cell separation medium (Cedarlane). DNA was extracted from PBMCs using the MagNA Pure compact NA isolation kit I (Roche Diagnostics) or MagDEA Dx SV (Precision System Science Co., Ltd.). Tissue samples were homogenized by using MagNA Lyser green beads with the MagNA Lyser instrument, and then sample lysates were incubated with 2 mg/mL RNase A (Qiagen) for 15 min at 65°C and DNA was extracted as described above.