Caliper lc 90

Total cellular RNA was extracted from fully matured OCs, reverse transcribed, and analyzed by high-throughput PCR amplification at the Université de Sherbrooke RNomics Platform as previously described [15 (link)],[16 (link)]. Five (5) ng of total RNA was used for each PCR experiment. For the detection screening we used RNA pre-amplified using a linear isothermal RNA amplification (Transplex Whole Transcriptome Amplification Kit, Sigma, Markham, ON) following the manufacturer’s protocol. AS events were characterized by end-point PCR. Primers were designed to flank the AS events, such that following amplification and analysis by microcapillary electrophoresis on Caliper LC-90 instruments (Caliper Life Sciences, Hopkinton, MA), the relative ratio of the isoforms can be deduced [15 (link)]. AS events which were amenable to characterization by high-thoughput PCR, that is, whose isoform sizes differ by between 10 and 450 nucleotides at a particular event, were selected from the RefSeq database [16 (link)]. Automated querying of this highly curated database has identified a genome-wide selection of 5223 AS events which fit the selection criteria from the full set of over 20 K human gene entries [16 (link)].

Liver Cancer cDNA Arrays form Origene TissueScan plates (Rockville, MD) were assessed for the expression of the various transcripts using the manufacturer’s protocol. The plates contained cDNAs from 8 normal and 39 liver cancer tissues. All forward and reverse primers were individually resuspended to 20–100 μM in Tris-EDTA buffer (IDT) and diluted as a primer pair to 1 μM in RNase DNase-free water (IDT). PCR reactions were performed in 10 μl in 96 well plates on a CFX-96 thermocycler (BioRad). The following cycling conditions were used: 3 min at 95 °C; 50 cycles: 15 s at 95 °C, 30 s at 60 °C, 30 s at 72 °C. For every PCR run, control reactions performed in the absence of template were performed for each primer pair and these were consistently negative. The amplified products were analyzed by automated chip-based microcapillary electrophoresis on Caliper LC-90 instruments (Caliper LifeSciences). Amplicon sizing and relative quantitation were performed by the manufacturer’s software.

Gastroesophageal cancer cDNA Arrays form Origene TissueScan plates (Rockville, MD) were assessed for the expression of the various transcripts using the manufacturer's protocol. The plates contained cDNAs from 6 normal and 42 gastroesophageal cancer tissues. All forward and reverse primers were individually resuspended in 20–100 μM stock solution in Tris-EDTA buffer and diluted as a primer pair to 1 μM in RNase DNase-free water. PCR reactions (10 μl) were performed in 96 well plates on a CFX-96 thermocycler (BioRad). For each PCR run, control reactions were performed in the absence of template and carried out for each primer pair. These reactions were consistently negative. The PCR products were finally analyzed using automated chip-based microcapillary electrophoresis on Caliper LC-90 instruments (Caliper LifeSciences). Both amplicon sizing and quantitation were analyzed using the manufacturer’s software.