High-throughput quantification of splicing isoforms

Abstract

Most human messenger RNAs (mRNAs) are alternatively spliced and many exhibit disease-specific splicing patterns. However, the contribution of most splicing events to the development and maintenance of human diseases remains unclear. As the contribution of alternative splicing events to diagnosis and prognosis is becoming increasingly recognized, it becomes important to develop precise methods to quantify the abundance of these isoforms in clinical samples. Here we present a pipeline for realtime PCR annotation of splicing events (RASE) that allows accurate identification of a large number of splicing isoforms in human tissues. The RASE automatically designed specific primer pairs for 81% of all alternative splicing events in the NCBI build 36 database. Experimentally, the majority of the RASE designed primers resulted in isoform-specific amplification suitable for quantification in human cell lines or in formalin-fixed, paraffin-embedded (FFPE) RNA extract. Using this pipeline it is now possible to rapidly identify splicing isoform signatures in different types of human tissues or to validate complete sets of data generated by microarray expression profiling and deep sequencing techniques.

Tumor microenvironment–associated modifications of alternative splicing

“…Cell culture, transfection, RNA extraction, and quantitative RT-PCR Cell culture, transfection, RNA extraction, and quantitative RT-PCR were performed as previously described (Brosseau et al 2010;Prinos et al 2011) except for FFPE and LCM samples. LCM samples isolated using several successive 8-µm cuts from FFPE blocs of each LCM sample were deposited on membrane slides (Molecular Machines & Industries).…”

“…The resulting DNA libraries were purified using the QIAquick PCR purification kit (Qiagen) and diluted to a final volume of 12.5 mL. Global (Prinos et al 2011) and isoform-specific (Brosseau et al 2010) quantitative RT-PCR primers were designed and validated as previously described (Brosseau et al 2010). The reliability of the gene expression assay using RNA samples from archived material was evaluated by comparing global gene expression (Supplemental Fig.…”

Dok‐1 overexpression promotes development of γδ natural killer T cells

“…Reactions were performed in triplicate using the following cycling conditions: 10 min at 95 • C; 50 cycles: 15 s at 95 • C, 30 s at 60 • C, 30 s at 72 • C. Relative expression levels were calculated using the qBASE framework [64] and the geometric average of three reference genes, UBC, HPRT1, and GAPDH. Primer design and validation was evaluated as described elsewhere [63]. Primer sequences are available upon request.…”

A single nucleotide polymorphism in exon 3 of the kallikrein 1 gene is associated with large but not small abdominal aortic aneurysm

“…The QuantiTect SYBR ® Green one-step RT-PCR Kit (Qiagen) was used according to the manufacturer's instructions with 100ng of total RNA as template. SYBR ® Green PCR primers were designed using the RASE (Real-time PCR Annotation of Splicing Events) tool [23] for both KLK1 short variant (5'-ATGCTGTGAAGGTCGTGGAGTT-3' and 5'-CACTGGAGATCATCTGGAAATGAGAAA-3', reference sequence NM_002257) Hypertension, coronary heart disease, dyslipidemia, smoking and gender (Queensland group only), in addition to rs5516 genotype, were examined for inclusion in the model. Forward stepwise variable selection procedures were performed where only significant covariates were retained in the models.…”