Detection of un-methylated DNA within different abundances of methylated DNA background by conventional or <i>fast</i>-COLD-MS-PCR.

<p><b>Panel A.</b> Post-PCR melting profile of the 255 bp bisulfite-converted-specific amplicon after conventional PCR or COLD-PCR. Serial dilutions of un-methylated (U) to methylated (M) genomic DNA are depicted (top half). Higher abundances of un-methylated DNA can be discriminated from methylated DNA by the melt peak, whereas lower abundances are only detectable if <i>fast</i>-COLD-MS-PCR replaces conventional PCR. <b>Panel B.</b> Sanger sequencing results of the 0.05% un-methylated (U): methylated (M) DNA sample as amplified by conventional and <i>fast</i>-COLD-MS-PCR are shown (bottom half). Chromatograms are aligned and compared using SeqDoc, and the CpG methylation positions are revealed in the middle panel.</p

Melting profiles of bisulfite-converted DNA from clinical samples following conventional PCR.

<p>Post-PCR melting profiles of the 255 bp bisulfite-converted <i>MGMT</i> gene amplicon after conventional PCR. Examples of fully un-methylated DNA samples isolated from infant blood (<b>Panel A</b>) and glioma samples (<b>Panel B</b>) are depicted. 100% methylated (M) and 100% un-methylated (U) DNA controls are used as reference standards, demonstrating a ∼5°C melting temperature difference among the two. <b>Panel C.</b> A glioma sample with mixed methylation/unmethylation pattern is shown.</p

Melting profiles of bisulfite-converted DNA from clinical samples following <i>fast</i>-COLD-MS-PCR.

<p>The effect of lowering the denaturation temperature in PCR is depicted. <b>Panel A.</b> Glioma sample no. 3 was subjected to different critical denaturation temperature-T_c during <i>fast</i>-COLD-MS-PCR. The modulation of the preferential amplification of the un-methylated DNA fraction is shown. <b>Panel B. </b><i>fast</i>-COLD-MS-PCR performed at a T_c of 84°C demonstrates that the amplification of the methylated DNA fraction is completely inhibited.</p

Comparison of EV quantification by NTA and TEM.

<p>CSF EVs were fractionated into microvesicles (10,000×g) and exosomes (120,000×g) by differential ultracentrifugation and then analyzed by NTA and TEM. <b>(A)</b> Representative TEM images, scale bar = 200nm. <b>(B)</b> Total EV count as determined by NTA and TEM. Fold difference in particle detected between NTA and TEM is denoted.</p

Total EV yield as determined by NTA and TRPS.

<p>Total EV yield as determined by NTA and TRPS.</p

EV quantitative analysis.

<p><b>(A)</b> Schematic representation of protocol used for the isolation of CSF microvesicles and exosomes. <b>(B)</b> In nanoparticles tracking analysis, light scattered by EVs is captured by digital camera over a series of frames. The rate of the particle movement is then used to calculate particle size using the Stokes—Einstein equation. <b>(C)</b> In tunable resistive pulse sensing, EVs change the electrical resistance as they pass through a pore-based sensor resulting in a resistive pulse signal. Signals obtained from the measurements can then be used to calculate the size, concentration and charge of each particle by correlating the signal back to a set of known standards. <b>(D)</b> In Vesicle flow cytometry, EVs were stained with an optimized concentration of a fluorogenic lipophilic probe, di-8-ANEPPS, and detected on a custom high sensitivity flow cytometer. Vesicle diameter was estimated by comparison to di-8-stained liposomes.</p

Comparison of EV quantification by NTA and TRPS.

<p>EVs were isolated from CSF collected from glioblastoma patients by differential centrifugation into microvesicle (10,000×g) and exosome (120,000×g) fractions, and resuspended in PBS. Isolated EVs were analyzed by NTA or TRPS. <b>(A)</b> Size profile of CSF exosomes determined by NTA and TRPS. <b>(B)</b> Size profile of CSF microvesicles determined by NTA and TRPS. <b>(C)</b> Comparison of EV yield by size ranges.</p

Total EV as determined by NTA and VFC.

<p>Total EV as determined by NTA and VFC.</p

EV yield by size range as determined by NTA and TRPS.

<p>EV yield by size range as determined by NTA and TRPS.</p

Comparison of EV quantification by NTA and VFC.

<p>CSF EVs isolated by differential centrifugation into microvesicle (10,000×g) and exosome (120,000×g) fractions were analyzed by NTA or VFC. <b>(A)</b> Size profile of CSF exosomes determined by NTA and VFC. <b>(B)</b> Size profile of CSF microvesicles determined by NTA and VFC. <b>(C)</b> Comparison of EV yield by size ranges.</p