Mx3005p qpcr machine

The sequences of the oligonucleotides used herein are listed in Supplementary Table 3. The preparation of these sequences is described in the Supplementary Methods. Experiments were performed in a 96-well format using a Mx3005P qPCR machine (Agilent) equipped with FAM filters (λ_ex = 492 nm; λ_em = 516 nm) in 100 μL (final volume) of 10 mM lithium cacodylate buffer (pH 7.2) plus 10 mM KCl/90 mM LiCl (F21T, F-DS-T) or plus 1 mM KCl/99 mM LiCl (F-Myc-T, F-Kit-T, F-TERRA-T) with 0.2 μM of labeled oligonucleotide and 0, 1, 5, or 10 µM of BioTASQ. Competitive experiments were carried out with labeled oligonucleotide (0.2 μM), 10 µM BioTASQ, and increasing amounts (0, 15 and 50 equiv.) of the unlabeled competitor ds26. After a first equilibration step (25 °C, 30 s), a stepwise increase of 1 °C every 30 s for 65 cycles to reach 90 °C was performed, and measurements were made after each cycle. Final data were analyzed with Excel (Microsoft Corp.) and OriginPro®9.1 (OriginLab Corp.). The emission of FAM was normalized (0 to 1), and T_1/2 was defined as the temperature for which the normalized emission is 0.5; ΔT_1/2 values are means of triplicates.

Solutions of EV-F3 and additive at either 100× or 1000× molar excesses (corresponding to 0.1 mM or 1 mM final concentration) were aliquoted into a 96-well PCR plate containing SYTO9 dye (Thermo Fisher Scientific) in triplicate with 50 mM HEPES, pH 8.0, 200 mM NaCl buffer, before centrifugation for 3 min at 4 °C. The assay was performed by heating samples in a Mx3005p qPCR machine (Agilent Technologies, USA) from 25 to 97 °C in 1 °C min⁻¹ increments for 30 s in an expanding saw-tooth profile, a refinement of a previously described protocol^{24 (link)}. Fluorescence changes were monitored at 25 °C with excitation and emission wavelengths of 492 and 517 nm, respectively.

Proteins were mixed with 20× SYPRO Orange (Thermo Fisher Scientific) in PBS containing 1 mmol/l DTT, dispensed in a BioRad qPCR plate, and heated from 25 to 95 °C at a rate of 1 °C/min in a MX3005p qPCR machine (Agilent). Fluorescent emission at 610 nm following excitation at 492 nm was measured, with the resulting curve defined in Excel, and the T_m was calculated in GraphPad Prism (GraphPad Software, Inc).

Purified Notum protein (5 µg) in 25 µl assay buffer (10 mM Hepes, pH 7.4, 150 mM NaCl and 6× SYPRO Orange dye, Thermo Fisher Scientific, UK), was mixed with an equal volume of buffer containing either caffeine, or its metabolites: theophylline, paraxanthine, theobromine at 2-fold dilution from 1 mM to 1 µM. The samples were placed in a semi-skirted 96 well PCR plate (4-Titude, UK), sealed and heated in an Mx3005p qPCR machine (Stratagene, Agilent Technologies, USA) from room temperature at a rate of 1 °C/min for 74 cycles. Fluorescence changes were monitored with excitation and emission wavelengths at 492 and 610 nm, respectively. Reference wells, i.e. solutions without compounds were used to compare the melting temperature (Tm). The experiments were performed in triplicate.

FRET-melting experiments were performed in a 96-well format using a Mx3005P qPCR machine (Agilent) equipped with FAM filters (λ_ex = 492 nm; λ_em = 516 nm) in 100 μL (final volume) of CacoK10 (Table S1, ESI†) for F21T or CacoK1 (Table S1, ESI†) for F-Myc-T, F-Terra-T and F-VEGF-T, with 0.2 μM of labeled oligonucleotide (Table 1) and 1 μM of TASQ. Competitive experiments were performed with labeled oligonucleotide (0.2 μM), 1 μM TASQ and either 15 (3 μM) or 50 mol equiv. (10 μM) of the unlabeled competitor calf thymus DNA (CT-DNA). After an initial equilibration step (25 °C, 30 s), a stepwise increase of 1 °C every 30 s for 65 cycles to reach 90 °C was performed, and measurements were made after each cycle. Final data were analyzed with Excel (Microsoft Corp.) and OriginPro®9.1 (OriginLab Corp.). The emission of FAM was normalized (0 to 1), and T_1/2 was defined as the temperature for which the normalized emission is 0.5; ΔT_1/2 values are means of 3 triplicates.

The pull-down experiments were performed as above (cf. 3a), with the following modifications: (a) the oligonucleotide used was changed for a 97-nt ODN (Table 1) described in Jamroskovic et al.^{37 (link)} and adapted in Mitteaux et al.^{38 (link)} at the center of which the G4-forming sequence d[(GGGCA)₄] is included; (b) the buffer was replaced by the G4RP buffer (Table S1, ESI†); (c) the incubation time was changed for 2 h at 25 °C; (d) the output was changed for qPCR analyses: polymerase reactions were carried out in triplicate in a 96-well format using a Mx3005P qPCR machine (Agilent) equipped with FAM filters (λ_ex = 492 nm; λ_em = 516 nm) in 20 μL (final volume) of G4-1R primer (1 μL, 300 nM, Table 1), TASQ/ODN mixture (3.7 μL) in 10 μL iTaq™ Universal SYBR® Green Supermix (Bio-Rad) + KCl (5.3 μL, 100 mM). After a first denaturation step (95 °C, 5 min), a two-step qPCR comprising a denaturation step (85 °C, 10 s) and a hybridization/elongation step (60 °C, 15 s) for 33 cycles was performed, and measurements were made after each cycle. Final data were analyzed with OriginPro®9.1 (OriginLab Corp.). The starting emission (1st qPCR cycle) of SYBR Green (FI) was set to 2200 and the FI at the 33th cycle was used for calculation. Three biological triplicates (n = 3) were used.

100 ng RNA extracted from BeWo cells treated with 0.5% DMSO or 100 µM forskolin (as a positive control for the induction of syncytialization^[68]) plus neat conditioned basal medium from watermelon EV naive or exposed Caco‐2 cells for 48 h was reversed transcribed using the AffinityScriot QPCR cDNA Synthesis kit (Agilent Technologies, USA). qPCR of syncytialization genes was performed using the Brilliant III Ultra‐Fast SYBR Green QPCR Master Mix (Agilent Technologies, USA) and a Mx3005p qPCR machine (Agilent Technologies, USA)+‐. Gene expression was expressed as ΔΔCt, with 18s used as the housekeeping gene.