Plasma serum circulating and exosomal rna purification kit

Adult brain samples (sample 1–5, Supplementary Materials) were obtained from the Harvard Brain Tissue Resource. Total RNA was extracted from 5 adult frontal lobe samples. To obtain total RNA, tissue pieces were first fixed in OCT and two sections were used for total RNA purification. Total RNA purification for these samples and 6 human blood samples was performed using RiboPureTM RNA purification kit (Ambion) according to the manufacturer instructions. Total RNA from fetal and adult liver, fetal and adult heart, and placenta were purchased from Clonetech. Total RNA from the FFPE tissues was extracted using High Pure FFPET RNA isolation kit (Roche) according to the manufacturer instructions. RNA from the serum was purified using Plasma/Serum Circulating and Exosomal RNA purification Kit (Norgen). RNase R treatment of total RNA was performed after ribosomal RNA depletion. Briefly, 2 μg RNA was added to a mixture of 1x RNase R buffer (Epicentre®), 1 mM MgCl_2, and 1.0 μl (20 U/μl) RNase R enzyme (Epicentre®). The mix was then incubated at 37 C° for 10 minutes. RNase R was then inactivated by adding 1:1 volume of H₂O. All samples are listed in Supplementary Materials.

For each HD and time point, cfDNA and cfRNA was extracted separately (Fig. 1). CfDNA was extracted from 1 ml plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen #55114) according to manufacturer instructions and eluted into 20 μl buffer EB (10 mM Tris–Cl, pH 8.5). CfRNA was extracted from 1 ml plasma using the Plasma/Serum Circulating and Exosomal RNA Purification Kit (Norgen #42800) and eluted into 100 μl nuclease-free deionized water. To remove genomic DNA contamination, the cfRNA samples were treated with 2 MBU of Baseline-ZERO DNase in 1X Baseline-ZERO DNAse buffer (Lucigen #DB0715K) for 20 min at 37 °C, purified using the RNA Clean & Concentrator-5 kit (Zymo #R1013), and eluted in 14 μl nuclease-free water. For no plasma controls, 1 ml nuclease-free deionized water was used in place of plasma. These purified cfDNA and cfRNA samples were used for all subsequent nucleic acid analyses.

Plasma samples received on dry ice from our collaborators were stored at –80 °C until further processing. Total circulating nucleic acid was extracted from plasma ranging in volume from ~215 µl to 1 ml, using a column-based commercially available extraction kit, following the manufacturer’s instructions (Plasma/Serum Circulating and Exosomal RNA purification kit, Norgen, 42800).
Following extraction, cfDNA was digested using Baseline-ZERO DNase (Epicentre) and the remaining cfRNA was purified using an RNA Clean and Concentrator-5 kit (Zymo, R1016) or an RNeasy MinElute Cleanup kit (Qiagen, 74204).

Consistent and reliable blood collection protocols are critical to maintain the integrity of circulating nucleic acid assays. We collected blood in Streck cfRNA tubes containing a stabilising reagent to prevent cell lysis and reduce degradation of RNA (Fernando et al., 2012 (link)).
cfRNA was extracted from 1 mL of plasma using Plasma/Serum Circulating and Exosomal RNA Purification Kit (Norgen, Cat no. 42800). The residual DNA in the cfRNA was digested using RNase-Free DNase I Kit (Norgen, Cat no. 25720). Extracted cfRNA was purified using RNA Clean and Concentrator^TM-5 (Zymo, Cat no. ZYR.R1016), yielding 24 μL of cfRNA per sample.

RNA was extracted from 4 mL of fractionated plasma using a plasma/serum circulating and exosomal RNA purification kit (Norgen Biotek, 42800) according to the manufacturer’s protocol with the following modifications. After fractionated plasma samples were lysed at 60 °C for 10 min and mixed with ethanol in step 2 of the procedure, 10 μL of 10⁶ times diluted ERCC RNA spike-in control mix (Thermofisher, 4456740) was added into each denatured plasma fraction sample (i.e. after combining the plasma fraction samples with denaturing solution in step 1) on ice as an external RNA control for normalization. These ERCC RNA-spiked in plasma fraction samples were followed by centrifugation at 1000 RPM for 2 min. After that point, we followed the manufacturer’s protocol and eluted RNA in 100 μL. To digest trace amounts of contaminating DNA, we treated the RNA with 10X Baseline-ZERO DNase (Lucigen, DB0715K). DNase I treated RNA samples were purified and further concentrated using RNA clean and concentrator-5 (Zymo Research, R1014) according to the manufacture’s protocols. Final eluted RNA was aliquoted and stored at −80 °C immediately.

Blood was collected into EDTA-coated Vacutainers. Plasma was collected from the blood samples after centrifugation at 1 600× g for 10 min at 4 °C and 16 000× g at 10 min at 4 °C. cfDNA was extracted using the Circulating Nucleic Acid Kit (Qiagen). Whole blood genomic DNA was extracted using the DNA Mini Kit (Qiagen) and fragmented using dsDNA Fragmentase (NEB) into average 300 bp. DNA was quantified by Qubit Fluorometer (Life Technologies). Cell-free RNA was extracted using the Plasma/Serum Circulating and Exosomal RNA Purification Kit (Norgen). The extracted cell-free RNA was further digested using Baseline-ZERO DNases (Epicentre) and depleted using Ribo-Zero rRNA Removal Kit (Epicentre) according to a protocol from Clontech.