The concentration of Shg mRNA in was quantified via real-time PCR with the Smartcycler detection system (Cepheid, Sunnyvale, CA) using SYBR green I (Molecular Probe, Eugene, OR) in three independent experiments.
To corroborate the studies using the RNA molecular probe or UV-Vis spectroscopy, we also conducted experiments with human ribosomal RNA and circular plasmid DNA.
Goat anti-mouse and goat anti-rabbit secondary antibodies conjugated to Alex546 (Molecular Probe) and Cy5 (Jackson Immunoresearch) were used for double-labeling experiments.
The sequence of all other probes (probes class II and III) is indicated in the corresponding figures. 1 mm of rat anti-HA (1/50; Molecular probe, Invitrogen, CA, USA) was used for the 'super-shift' experiments.
We demonstrated by a series of experiments that cy-apt 20 was an excellent molecular probe with a certain degree of biostability and high specificity and sensitivity for molecular recognition and targeting therapy of gastric cancer.
Thus, more recent microarray experiments of single probe experiments include confirmation of the proposed hypothesis by means of molecular biology.
For the DQ Ovalbumin experiments (Invitrogen, Molecular Probes, Inc., Carlsbad, CA) SDCs were incubated simultaneously with Alexa Fluor 594-labeled pcDNA3-Hsp65 (4 µg) and DQ Ovalbumin for varying times (1 min, 24 h and 72 h).
LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Molecular Probes) was used in all experiments to exclude dead cells.
Synthetic mRNAs were resuspended in 30% glycerol and, in selected experiments, TRCD (Molecular Probes, Italy) was added at 5%.
Dead cells were excluded using Live Dead Aqua Dead cell stain (Invitrogen; Molecular probes Carlsbad, CA) for this experiment.
