Superscript 2 rnase h reverse transcriptase enzyme

HMEC1 or BAEC (2 × 10⁵) were seeded on six-well cell culture dishes. Next day, cells were washed with PBS, incubated with endothelial cell basal media and either PBS or sEVs (40 μg/mL) isolated from PC3 cells (4, 8 and 16 h for BAEC or 2, 4, 8, 16 and 24 h for HMEC1). After incubation, cells were washed with PBS, trypsinised and total RNA from HMEC1, BAEC or PC3 PrCa cells (positive control for β6 mRNA expression) was isolated using the Qiagen RNeasy Kits (Qiagen, Valencia, CA, USA) as per manufacturer’s protocol. RNA (1 μg) was reverse transcribed with random hexamer oligos (Invitrogen) and SuperScript II RNase H-reverse transcriptase enzyme (Invitrogen). Subsequently, for real-time PCR analysis, complementary DNA (cDNA) was amplified using the QuantStudio 12 K Flex Real-Time PCR system. The gene expression of β6, BIRC5 and GAPDH were profiled using primers for β6 (forward primer, 5ʹ-GGTCTCATCTGGAAGCTACTGGTGTCA-3ʹ; reverse primer, 5ʹ-GGTCTCCCAGATGCACAGTAGGACAACC-3ʹ), BIRC5 (forward primer, 5ʹ-GACTTGGCTCGATGCTGTGG-3ʹ; reverse primer, 5ʹ-TACGCCAGACTTCAGCCCTG-3ʹ) and GAPDH (forward primer, 5ʹ-GGGAAGGTGAAGGTCGGAGT-3ʹ; reverse primer, 5ʹ-GTTCTCAGCCTTGACGGTGC-3ʹ). The relative mRNA expression was calculated using the 2ΔΔCT method. Each reaction was carried out in triplicate; mean and standard error of mean were calculated using Excel (Microsoft) software.