Nanodrop one microvolume uv

RNA was extracted from eukaryotic cell lines using the Qiagen RNeasy Miniprep following manufacture's specifications (QIAGEN, Germantown, MD). Total RNA from L. johnsonii N6.2 bacterial cells or EV were extracted using Invitrogen RiboPure bacterial RNA extraction kit (Thermo Scientific, Waltham, MA). For the EV suspension in 1x PBS, a 1:10 ratio (V/V) of EV to Lysis/Binding Buffer (100 μL of vesicle suspension to 1 mL Lysis/Binding Buffer). The RNA extracted were assessed for concentration and purity using a NanoDrop One Microvolume UV–vis spectrophotometer (Thermo Scientific, Waltham, MA) and visually analyzed for degradation using a 1% agarose gel. qRT‐PCR was performed using a QuantStudio 6 Flex (Thermo Scientific, Waltham, MA) as described (Teixeira et al., 2022 (link)). Primer sequences used to determine relative transcript abundance are listed in Table 1.

The plasmid containing the target fragment was digested into linear fragments by HindIII. The concentration of digested DNA was measured using a NanoDrop One microvolume UV–Vis spectrophotometre (Thermo Fisher Scientific, USA) for three replicates. Subsequently, 10-fold serial dilutions of the standard linear fragment were prepared as templates to evaluate the linearity relationship (repeated three times for each template) and detection limit range for both ddPCR and qPCR assays. The standard plasmid content was calculated using the Avogadro constant formula as follows: $\text{copy}\text{number}(\text{copies}/\mathrm{\mu }\mathrm{L})=(\mathrm{A}260(\text{ng}/\mathrm{\mu }\mathrm{L})\times {10}^{‐9}\times 6.02\times {10}^{23})/\left(\text{DNA}\text{length}\left(\text{bp}\right)\times 660\right)$