Transplex whole transcriptome amplification kit

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)].

Total RNA extraction from tissue samples with TRIzol (Invitrogen by Life Technologies) was previously described [30 (link)]. For cDNA synthesis, 200 ng of RNA and the TransPlex Whole Transcriptome Amplification Kit (Sigma-Aldrich) were used, following the manufacturer's instructions. For cell lines, total RNA was extracted using the Illustra TriplePrep Kit (GE Healthcare Bio-science Corporation, NJ, USA), and cDNA was obtained from 1μg of RNA using oligo-dT primers and the H-minus RevertAid cDNA synthesis kit (Fermentas, Ontario, Canada), according to the manufacturer's instructions. Real-time RT-PCR was performed using pre-developed TaqMan^® Gene Expression assays (Applied Biosystems, Foster City, CA, USA). Amplification reactions were carried out in triplicates on a 7500 Sequence Detection System (Applied Biosystems), with GUSB used as a reference gene. Relative expression was obtained using the comparative Ct method [31 ].

Samples were thawed and processed for DNA and RNA extraction using Norgen Biotek Corp. RNA/DNA Purification Plus kits according to Manufacturer’s specifications.
cDNA amplification was performed starting from 100 ng of total RNA using TransPlex Whole Transcriptome Amplification Kit (Sigma-Aldrich Co.). cDNA was then purified using the QIAquick PCR purification kit (Qiagen) and relative quantity was assessed using a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific Inc.) as described previously [14 (link)].
RNA was labeled according to Manufacturer’s instructions and hybridized to Agilent SurePrint G3 Human Gene Expression v2 8x60K microarrays, which include 8,127 lncRNA-dedicated probes as well as probes for 20,560 known coding transcripts. Hybridized chips were then scanned on an Agilent Technologies Inc. G2565C device.

D283-MED cells were cultured in 10 cm dishes. Reverse transcriptome amplification of extracted RNA was conducted using Transplex® Whole Transcriptome Amplification kit (Sigma) from 3 different replicate per time point. NimbleGen 12x135k format array slide was utilised for the microarray experiment, whereby each transcript was represented by 3 probes. Statistical analysis of data was conducted using Matlab (script written by Damon Daniels, University of Manchester), with clustering based on k-means. All microarray raw and normalised data are available on NCBI: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE106959).

Following gene selection, mRNA levels were confirmed in the same group of tissue samples previously indicated. A total of 300 ng was reverse transcribed and amplified using TransPlex® Whole Transcriptome Amplification Kit (Sigma-Aldrich®, Schnelldorf, Germany) with subsequent purification using QIAquick® PCR Purification Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. Expression levels were evaluated using TaqMan® Gene Expression Assays (Applied Biosystems, Foster City, CA, USA), and GUSB was used as a reference gene for normalization, according to the formula: Relative expression = (Target gene mean quantity/Reference gene mean quantity). Ratios were then multiplied by 1,000 for easier tabulation. Each plate included multiple non-template controls and serial dilutions (10×) of a cDNA obtained from human prostate RNA (Ambion) were used to construct a standard curve for each plate. All experiments were run in triplicates.