Realplex2 mastercycler ep gradient s thermal cycler

Total RNA was extracted using the TRIzol reagent (Invitrogen). One microgram of total RNA was used for complementary DNA reverse transcription using the Superscript-III First Strand Synthesis kit (Invitrogen) following the manufacturer's protocol. All real-time PCR reactions were performed in duplicates in a 20 μl volume using SYBR Green Master Mix (Invitrogen) in a Realplex2 Mastercycler ep gradient S thermal cycler (Eppendorf). The PCR program followed for all the primers was an activation at 95 °C for 15 min, followed by 40 cycles of 95 °C for 30 s, 54 °C for 1 min, and 72 °C for 45 s. The reaction was finally put to hold at 4 °C. Subsequently, 18S was used as the reference gene for the mRNA levels. Relative change in gene expression was calculated according to the ΔΔCt method (53 (link), 54 (link)).

Total RNA was isolated from the cell lines using TRIzol (Life Technologies) and quantified. Equal amount of RNA was used for the one-step or two-step qPCR performed using the Superscript III SYBR Green qRT-PCR kits, according to manufacturer’s instructions (Life Technologies). For miRNA, PCR was performed using NCode VILO miRNA cDNA Synthesis and EXPRESS SYBR GreenER miRNA qRT-PCR Kits (Life Technologies), according to the manufacturer’s protocol. The primers (sequences provided in the Supplementary materials and methods; Additional file 7) were designed using Primer 3 [48 (link)] and synthesized by Integrated DNA Technologies (Coralville, IA). PCR was performed using Realplex² Mastercycler ep gradient S thermal cycler (Eppendorf).