Super sybr green kit

Total RNA was extracted using TRIzol Reagent (Invitrogen, CA, USA) following the manufacturer’s protocol and was reverse-transcribed into complementary DNA (cDNA) using a Superscript Reverse Transcriptase Kit (Transgene, France). Super SYBR Green Kit (Transgen, France) was used to carry out real-time PCR in ABI7300 real-time PCR system (Applied Biosystems).

RNA was extracted with TRIzol reagent (Invitrogen, CA, USA), and the isolated RNA was reverse-transcribed into complementary DNA (cDNA) using a Superscript Reverse Transcriptase Kit (Transgene, France) according to the standard instructions. qPCR was carried out on an ABI7300 real-time PCR system using a Super SYBR Green kit (Transgene, France). We used the following specific primer pairs to quantify the expression of the genes that encode the proteins indicated: VEGFA (5′-AGGGCAGAATCATCACGAAGT-3′ and 5′-AGGGTCTCGATTGGATGGCA-3′); E-cadherin (5′- ATTTTTCCCTCGACACCCGAT-3′ and 5′-TCCCAGGCGTAGACCAAGA-3′); Vimentin (5′-AGTCCACTGAGTACCGGAGAC-3′ and 5′-CATTTCACGCATCTGGCGTTC-3′); Slug (5′-CGAACTGGACACACATACAGTG-3′ and 5′-CTGAGGATCTCTGGTTGTGGT-3′); GAPDH (5′GAGAGACCCTCACTGCTG-3′ and 5′-GATGGTACATGACAAGGTGC-3′); β-actin (5′-CATGTACGTTGCTATCCAGGC-3′ and 5′-CTCCTTAATGTCACGCACGAT-3′); Tubulin (5′-AAGATCCGAGAAGAATACCCTGA-3′ and 5′-CTACCAACTGATGGACGGAGA-3′). Gene amplification was carried out according to the standard instructions: 95˚°C for 10 min; and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Relative quantification was accomplished by normalizing the expression levels of the other genes to GAPDH, with all tests carried out in duplicate.

After isolation using TRIzol reagent (Thermo Fisher), the RNA was reversely transcribed into cDNA with the help of a Superscript reverse transcriptase kit (Transgene) according to standard instructions. qPCR was conducted on an ABI7300 real-time PCR system (ABI7300, USA) using the Super SYBR Green kit (Transgene). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an endogenous control for ATF3 and AKT1, and U6 as an endogenous control for miR-222. PCR primers were synthesized by Sangon (Shanghai, China). The specific primer sequences are as follows: miR-222: forward, 5′-CCCTCAGTGGCTCAGTAG-3′, reverse, 5′-CCACCAGAGACCCAGTAG-3′; ATF3: forward, 5′-CTCTGCGCTGGAATCAGTCA-3′, reverse, 5′-CCTCGGCTTTTGTGATGGA-3′; AKT1: forward, 5′-TCCTCCTCAAGAATGATGGCA-3′, reverse, 5′-GTGCGTTCGATGACAGTGGT-3′; U6: forward, 5′-CTCGCTTCGGCAGCACA-3′, reverse, 5′-AACGCTTCACGAATTTGCGT-3′; GAPDH: forward, 5′-AGTGGCAAAGTGGAGATT-3′, reverse: 5′-GTGGAGTCATACTGGAACA-3′.

Total RNA was extracted using TRIzol Reagent (Invitrogen, CA, USA) following the manufacturer’s protocol and was reverse-transcribed into complementary DNA (cDNA) using a Superscript Reverse Transcriptase Kit (Transgene, France). Super SYBR Green Kit (Transgen, France) was used to carry out real-time PCR in ABI7300 real time PCR system (Applied Biosystems). Following specific primer was used: GAPDH (5′-GAGAGACCCTCACTGCTG-3′ and 5′-GATGGTACATGACAAGGTGC-3′); vimentin (5′-AGTCCACTGAGTACCGGAGAC-3′ and 5′-CATTTCACGCATCTGGCGTTC-3′); fibroblast activation protein (FAP) (5′-ATGAGCTTCCTCGTCCAATTCA-3′ and 5′-AGACCACCAGAGAGCATATTTTG-3′); ACTA2 (5′-AAAAGACAGCTACGTGGGTGA-3′ and 5′-GCCATGTTCTATCGGGTACTTC-3′). PCR was performed according to instruction: 95 °C for 10 min; and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The relative gene expression levels for vimentin, FAP and ACTA2 were normalized against that of GAPDH in each sample, and each sample was run in triplicate and averaged.