Computational protocol: Oestrogen receptor beta isoform expression in sporadic colorectal cancer, familial adenomatous polyposis and progressive stages of colorectal cancer

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[…] Three pair of primers flanking the ESR1 gene were designed. Primer pair 1 amplifies the transcript variants a, b, d, f, k and l (ERβ1, ERβ2 and ERβ4 isoforms); primer pair 2 amplifies transcripts a and g (ERβ1 and ERβ5 isoforms), and primer pair 3 amplifies the transcript variants b, l and k, which are translated into the ERβ2 isoform (Table  and Fig. ). Three endogenous references (PUM1, EIF2B1, and POP4) were used in the RT-qPCR assays (Table ). The primers were designed using Primer-Blast (available at http://www.ncbi.nlm.nih.gov/tools/primer-blast/), and Primer 3 was selected based on the study by Yang et al., 2016 []. Fig. 1 Total RNA samples were digested with 1 U of DNase I (amplification grade, Life Technologies) in 10X DNase I Reaction Buffer. The reactions were performed in a PTC-100 thermal cycler (Peltier-Effect Cycling - MJ Research) for 15 min at room temperature; the enzyme was then inactivated by heating at 70 °C for 10 min. The cDNA synthesis was performed in a final volume of 20 μL containing 5X first-strand buffer (250 mM Tris–HCl pH 8.3, 375 mM KCl and 15 mM MgCl2), 10 mM of each dNTP, 0.5 μg/μL oligo (dT), 0.1 M dithiothreitol and 200 U of reverse transcriptase (SuperScriptTM III, Invitrogen). Reverse transcription was performed at 42 °C for 60 min followed by inactivation at 70 °C for 15 min. The ABI Prism 7900 Sequence Detection System automatic thermocycler was used for RT-qPCR amplification. All reactions were assembled by robotic pipetting into 384-well plates with QIAgility (Qiagen, Courtaboeuf, France) in a total volume of 12.5 μL of Power SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA), 20 ng of cDNA and 200 nM of each primer. All samples were analysed in duplicate and subjected to the following cycling conditions: an initial temperature of 95 °C for 10 min and 45 cycles of 95 °C for 15 s and 60 °C for 1 min. The amplification quality was verified by analysing the dissociation curve (specificity) to discriminate primer-dimers from low levels of transcript expression. A duplicate of no template control (NTC) was included in each PCR amplification for each primer pair, all showing negative signals (Cq = 45 or small dimer amplification detection Cq > 42). The values obtained ​​for all samples were normalised by determining the ratio of the gene of interest to the reference gene. The mean Cq (quantification cycle) was used for normalisation. Samples with a mean Cq greater than the mean + 1 SD of the geometric mean of the Cqs of the three reference genes were excluded (8 fresh frozen and 30 FFPE samples). The model proposed by Pfaffl [] was used for data normalisation. [...] ESR2 expression data in colon carcinomas versus normal tissues (log2 + 1 reads per million) was assessed from The Cancer Genome Atlas (TCGA: level 3 RNA sequencing database) (t test). An isoform specific analysis of ESR2 was implemented using Isoform Expression View (normal-tumor comparison) of the ISOexpresso tool (http://wiki.tgilab.org/ISOexpresso/) []. […]

Pipeline specifications

Software tools Primer-BLAST, ISOexpresso
Databases TCGA Data Portal
Applications RNA-seq analysis, qPCR
Organisms Homo sapiens
Diseases Neoplasms, Adenomatous Polyposis Coli, Colorectal Neoplasms, Adenomatous Polyps