For qRT-PCR analysis of human LECs, total RNA was isolated by RNeasy Mini kit or RNeasy Micro kit (QIAGEN) and 0.5 μg was reverse transcribed using oligo dT (SuperScript III First-Strand Synthesis System, Invitrogen) or SuperScript® VILO cDNA Synthesis Kit according to the manufacturer’s instructions. For analysis of sorted mouse LECs, total RNA was extracted by RNeasy Micro kit (QIAGEN) and all obtained RNA was reverse transcribed using oligo dT (SuperScript III First-Strand Synthesis System, Invitrogen). cDNA was pre-amplified using the TaqMan PreAmp Master Mix Kit. Gene expression levels were analyzed using TaqMan Gene Expression Assay (Applied iBiosystems) and a 7900 HT Fast Real-Time PCR System thermocycler (Applied Biosystems) following manufacturer’s instructions. Relative gene expression levels were normalized to GAPDH or the endothelial marker TEK. The following probes were used: Hs99999905 GAPDH, Hs00176607 FLT4, Hs00216777 ANTXR1, Hs01114113 HEY1, Hs1062014 NOTCH1, Hs01030099 CCNB1, Hs00170014 CTGF, Hs00932747 TGFBI, Hs00704917 GJA4, Hs00187950 EFNB2, Hs00173317 ANKRD1, Hs00231119 GATA2, Hs00945150 TEK, Hs01554629 ERG, Hs04260396 MIK67, Hs01084593 CCNB2, Hs00942540 MYBL2, Hs01557695_m1 BUB1, Hs00155479 CYR61, Ms99999915 Gapdh, Ms00492300 Gata2, Ms01292604 Flt4, Mm00443243 Tek, Mm01214244 Erg, Mm00438670 Efnb2, Mm00433294 Fgfr3, Mm01278617 Mki67, Mm01171453 Ccnb2.
7900 ht fast real time pcr system thermocycler
Manufactured by Thermo Fisher Scientific
The 7900 HT Fast Real-Time PCR System is a thermocycler designed for fast and accurate real-time PCR analysis. It features a 96-well block format and can perform high-throughput gene expression studies and SNP genotyping.
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2 protocols using 7900 ht fast real time pcr system thermocycler
1
qRT-PCR Analysis of Human and Mouse LECs
2
Skin Biopsy RNA Extraction and qRT-PCR Analysis
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RNA was extracted from skin biopsies (~1 cm²) taken from the upper backs of mice (Qiagen #74106). Each RNA sample was collected in 40 μL of ultrapure water (Thermo Fisher #10977). The RNA concentration and purity were determined using a NanoDrop spectrophotometer (Eppendorf). The RNA integrity was analyzed by 1% agarose gel electrophoresis. cDNA was generated using a high-capacity cDNA synthesis kit (Thermo Fisher #4368814) following the manufacturer's instructions.
We used 384-well optical plates to perform real-time RT-PCR using an Applied Biosystems 7900HT Fast Real-Time PCR System thermocycler. The primer sequences used for real-time RT-PCR are shown in SI Appendix, Table S2. The real-time RT-PCR mix included 6.25 μL of 2× SYBR Green mix, 2 μL of diluted cDNA (1:10), 0.25 μL (5 μM) of each primer and 4.25 μL of ultrapure water (Gibco #10977). The cycling conditions included 50°C for 2 minutes, 95°C for 10 minutes and 40 cycles of 95°C for 15 s and 60°C for 60 s. The delta-delta Ct (2-ΔΔCt) method was used to determine the relative quantities of the target genes compared to the reference genes. We used the average of the two reference genes (Gapdh and β-actin) as the final reference.
We used 384-well optical plates to perform real-time RT-PCR using an Applied Biosystems 7900HT Fast Real-Time PCR System thermocycler. The primer sequences used for real-time RT-PCR are shown in SI Appendix, Table S2. The real-time RT-PCR mix included 6.25 μL of 2× SYBR Green mix, 2 μL of diluted cDNA (1:10), 0.25 μL (5 μM) of each primer and 4.25 μL of ultrapure water (Gibco #10977). The cycling conditions included 50°C for 2 minutes, 95°C for 10 minutes and 40 cycles of 95°C for 15 s and 60°C for 60 s. The delta-delta Ct (2-ΔΔCt) method was used to determine the relative quantities of the target genes compared to the reference genes. We used the average of the two reference genes (Gapdh and β-actin) as the final reference.
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