Taqman rna to c 1 step kit

Example 4

Human umbilical vein endothelial cells (HUVEC) were prepared in a 0.5% FBS-supplemented EBM2 culture medium so to reach a cell density of 10×10⁵ cells/ml and cultured in a moist 5% CO₂ atmosphere at 37° C. A cosmetic materials library was used for the candidate drugs. The candidate drugs were added and culturing was performed for 6 hours. After culturing, the culture medium was removed, and RNA was extracted from the cells using RNeasy mini kit (QIAGEN). The concentration of the extracted RNA was measured by NanoDrop, and RNase-free water was used to prepare 100 ng/ml thereof. TaqMan RNA-to-C 1-Step Kit (Applied Biosystems) was used to quantify the prepared RNA by real-time PCR (Roche Lightcycler 480II) using primers for the VE-cadherin gene.

When extracts of Siberian ginseng, Calendula officinalis, and Cistanche salsa were used as the candidate drugs, expression of the VE-cadherin gene significantly increased compared to the control (p<0.05: FIG. 4). These extracts were selected as substances having VE-cadherin expression promoting activity.

Example 5

Human umbilical vein endothelial cells (HUVEC) were prepared in a 0.5% FBS-supplemented EBM2 culture medium so as to reach a density of 10×10⁵ cells/ml and were cultured in a moist 5% CO₂ atmosphere at 37° C. The candidate drugs were added and culturing was performed for 6 hours. After culturing, the culture medium was removed, and RNA was extracted from the cells using RNeasy mini kit (QIAGEN). The concentration of the extracted RNA was measured by NanoDrop, and RNase-free water was used to prepare 100 ng/ml thereof. TaqMan RNA-to-C 1-Step Kit (Applied Biosystems) was used to quantify the prepared RNA by real-time PCR (Roche Lightcycler 480II) using primers for the integrin α5 gene.

When Siberian ginseng, yeast and Cistanche extracts were used as the candidate drugs, the expression of the integrin α5 gene increased significantly compared to the control (p<0.05: FIG. 5). These extracts were selected as substances having firmness improving activity.

Example 3

Vascular endothelial cells from young subjects (aged 0) and old subjects (aged 50) were inoculated onto a 6-well plate at a cell density of 2×10⁵ cells/well and left standing overnight. The following day, RNA was extracted therefrom using RNeasy mini kit (QIAGEN). The concentration of the extracted RNA was measured using NanoDrop, and RNase-free water was used to prepare 100 ng/ml thereof. TaqMan RNA-to-C 1-Step Kit (Applied Biosystems) was used to quantify the prepared RNA by real-time PCR (Roche Lightcycler 480II) using primers for the following genes. Using β-actin (b-actin: Cat #Hs01060665_g1) as an internal standard, a significant difference in expression levels was observed with respect to VE-cadherin (VE-Cadherin: Cat #Hs00170986_m1) and integrin α5 (Integrin alpha5: Cat #Hs01547673_m1) between cells derived from young subjects and cells derived from old subjects (FIGS. 3A to D: *: P<0.05), whereas with respect to integrin α3 (Integrin alpha3: Cat #Hs01076879_m1) and integrin α6 (Integrin alpha6: Cat #Hs01041011_m1) a significant difference in expression levels was not observed.