We obtained these genes by first performing reverse transcriptase-PCR (RT-PCR) using total RNA from HEK 293, HCT 116 or WI-38 cells.
Negative control samples (no first-strand synthesis) were prepared by performing reverse transcription reactions in the absence of reverse transcriptase.
We validated differential expression of genes involved in processes relevant to metastasis, including BUB-1, a regulator of genomic integrity and mitosis, VIM, a marker of epithelial-to-mesenchymal transition, and the adhesion molecule, CCAM1, by performing quantitative reverse transcriptase PCR analysis (Figure 2).
Since there are currently no specific antibodies available that can distinguish between the different splice variants, we quantified their expression levels by performing a reverse transcriptase and polymerase chain reaction (RT PCR).
In addition, the presence of genomic DNA contamination was excluded by performing reactions without reverse transcriptase.
The absence of genomic DNA was confirmed by performing a no reverse transcriptase (NoRT) control for randomly selected RNA samples, and the absence of contaminations was assessed by including a no template control (NTC) in every run.
Thereafter, by performing macroarray and reverse transcriptase quantitative-polymerase chain reaction (RT qPCR) experiments, gabarapl1 expression was quantified in several histological breast tumour types and in a retrospective cohort of 265 breast cancers.
These additional clones were obtained from the Deutsches Krebsforschungszentrum (Deutsches Ressourcenzentrum für Genomforschung, Berlin, Germany) or created by performing an RT PCR (reverse transcriptase) reaction on RNA from mammary cell lines.
Reverse transcription was performed with reverse transcriptase primed by oligo (dT) primer (Promega, Madison, WI, USA).
To investigate mRNA expression of ECT2 identified as a cancer-related gene by our microarray analysis [6], we performed quantitative reverse transcriptase PCR (qRT-PCR) analysis using six OSCC-derived cell lines (HSC-2, HSC-3, HSC-4, H1, Ca9-22, and Sa3) and human normal oral keratinocytes (HNOKs).
