Bioline lo rox sybr

Endogenous levels of miRNAs were quantified by real time quantitative PCR (qRT-PCR). Total RNA was extracted from cells using trizol and cDNA was synthesized using the MystiCq microRNA cDNA Synthesis Mix (Sigma Aldrich, St. Louis, MO, USA). Next, Bioline Lo-Rox Sybr (Bioline, Australia) was used to perform qRT-PCR in the ViiA7 RT-PCR machine (Thermo Fisher Scientific) under the following conditions: 95 °C: 10 m followed by 40 cycles of 95 °C: 5 s, 60 °C:10 s and 70 °C: 10 s. Forward primer used for miRNA122a was 5′-TGGAGTGTGACAATGGTGTTTGT-3′ whereas that for miRNA199a was 5′-CCCAGTGTTCAGACTACCTG-3′. Both the miRNAs were expressed as number of copies per 1000 copies of the control microRNA RNU6-1 (MIRCP00001) using the formula (2^ (Ct (RNU6-1)-Ct (miRNA122a/199a)) *1000. MystiCq Universal PCR (MIRUP, Sigma) was used as a reverse primer for the qRT-PCR reactions.

Endogenous expression levels of miRNA122a was quantified by quantitative real time PCR (qRT-PCR) with Bioline Lo-Rox Sybr (Bioline, Alexandria, Australia) using the ViiA7 RT-PCR machine (Thermo Fisher Scientific) at following conditions: 95° C- 2mins followed by 40 cycles of 95° C- 5 s, 60° C -15 s and 70° C -15 s on the cDNA which was synthesized from total RNA isolated with trizol using the MystiCq microRNA cDNA Synthesis Mix (Sigma Aldrich) as per the manufacturer's recommendations. MystiCq Universal PCR (MIRUP, Sigma) and 5′- TGGAGTGTGACAATGGTGTTTGT- 3′ primers were used and the levels if miRNA122a was calculated as number of copies per 1000 copies of human positive control using formula (2^(Ct control-Ct sample))*1000. Similarly, HCC stemness markers CD44, EpCAM, and Oct4 were quantified as number of copies per 1000 copies of GAPDH housekeeping gene using primers listed in Table 1.