Ambion cells to cdna 2 kit

The Ambion Cells-to-cDNA™ II Kit (AM 1723, Life technologies, UK) was used for the isolation of total RNA and cDNA synthesis from cells of each zone according to the manufacturer's instructions. Quantitative RT-PCR is an accurate way of both detecting and quantifying a given DNA sequence and represents the most sensitive way of detecting differences in mRNA expression irrespective of exact cell number. The standard reference gene used for the studies below was GAPDH. The primers for qRT-PCR are listed in Table 1. Quantitative RT-PCR was performed using the LightCycler™ quantitative PCR machine (Roche, Switzerland). Each reaction was composed with 5 μl master mix, 3.7 μl RNase-free water, 0.4 μl forward and reverse primer mix, 0.8 μl template cDNA and 0.1 μl COX (all reagents from Promega, UK). Quantitative RT-PCR was performed using the LightCycler at 95 °C for 5 min, followed by 40 cycles of 94 °C for 15 s, primer specific annealing temperature for 30 s, and 72 °C for 20s, with a single data acquisition step. The crossing point for each transcript was determined using the LightCycler software (Roche, Switzerland). The LightCycler Relative Quantification software (Roche, Switzerland) was used to analyse the data. Each cDNA sample was measured in triplicate with GAPDH as the reference gene and using RNAse-free water as the negative control.

Trizol reagent (Thermo Fisher Scientific) was used to isolate total RNA from cultured cells according to manufacturer’s instructions. The miRCURY LNA Universal cDNA synthesis kit (Exiqon) and miRNA-specific LNA primers (Exiqon) was used for quantification of mature miRNA transcripts by qRT-PCR. Data were normalized to U6 as the housekeeping gene. For mRNA, Ambion Cells-to-cDNA II Kit (Life Technologies) or SuperScript II RT (Thermo Fisher Scientific) was used with data normalised to expression of 18S rRNA ribosomal RNA. Only samples where the housekeeping genes are expressed within median + /− 1 Ct are included in analyses. Primer sequences are listed in Supplementary Table 1. Both miRNA and mRNA relative quantifications were calculated using the ∆∆CT method. Fluorescence for each cycle was analysed by the real-time PCR 7500 system (Applied Biosystems)^{20 (link)}.