Linregpcr

Quantitative PCR was performed as previously described (Øverby et al., 2015 (link)). Briefly, fresh plant material was snap-freezed in liquid nitrogen and subsequently grounded from which total RNA was extracted using Spectrum Plant Total RNA Kit. RNase-Free DNase Set was used to prevent DNA contamination. RNA concentration was measured using NanoDrop 1000 (Thermo Scientific). cDNA was synthesized using QuantiTect Reverse Transcription Kit, and qPCR was performed with SYBRgreen in 96-well plate in a Lightcycler 480 (Roche Applied Science) with the following program: preincubation step (95°C, 5 min), 45 amplification cycles (95°C, 10 s; 55°C, 10 s; 72°C, 10 s), and a final melting curve analysis. Cycle threshold values, PCR efficiencies and relative expression values were calculated using Lightcycler 480 Software (Roche), LinRegPCR and REST 2009 (QIAGEN), respectively. At4g24550 (Clathrin) and At4g34270 (TIP41-like) were used as housekeeping genes. Primer sequences are given in Supplementary Table S1.

Fold changes in gene expression were quantified by LinRegPCR (version 2) and Relative Expression Software Tool-RG©-version 3 (QIAGEN, Korea) by means of the amplification efficiencies and cycle thresholds from comparative quantification analysis. By the means of LinRegPCR, the baseline fluorescence was determined and subtracted and PCR efficiencies were then computed for each sample. Afterwards, Cq value and the starting concentration per sample (reported in an arbitrary unit) were measured. The calculated Cq and efficiency values were used for quantification analysis. The quantities of mRNAs in the tissues were standardized to the B2M mRNA and compared between tumor and non-cancerous tissues. The pairwise fixed reallocation randomization test with 2000 iterations in the REST 2009 software was used to express the significances. The level of statistical significance was set at P<0.05. The associations of demographic and clinical data with gene expression levels were assessed using SPSS v.18.0.1 (SPSS Inc., Chicago, IL, USA). The data were presented as the mean±SD. The McNemar’s test was used to compare paired tumor and adjacent non-cancerous tissues (ANCTs). Chi-square and independent t-tests were applied to assess the significance of CCAT2 expression as correlated with clinicopathologic features in breast cancer. Significance was delineated as P<0.05.