Total RNA from fresh normal and tumour human and mouse tissue samples was extracted, followed by DNase treatment using the AllPrep DNA/RNA/Protein Mini Kit and the RNase-Free DNase set (Qiagen, #80004/#79254), respectively. Total RNA from glioblastoma cell lines was extracted with TRIzol® Reagent (ThermoFisher Scientific, #15596026). In all cases, total RNA concentration and purity were assessed by Nanodrop One Microvolume UV-Vis Spectrophotometer (ThermoFisher Scientific). For qPCR analyses, total RNA was retrotranscribed by using random hexamer primers and the RevertAid RT Reverse Transcription Kit (ThermoFisher Scientific, #K1691). Thermal profile and qPCR analysis to obtain absolute mRNA copy number/50 ng of sample of selected genes are reported elsewhere (Luque et al., 2013 (link), 2015 (link)).
As recently reported (Jimenez-Vacas et al., 2019b (link), 2020 (link)), a qPCR dynamic array based on microfluidic technology (Fluidigm, #BMK-M-48.48) was implemented to determine the simultaneous expression of 48 transcripts in HGA/glioblastoma samples compared to control samples using the Biomark System and the Fluidigm® Real-Time PCR Analysis Software v.3.0.2 and Data Collection Software v.3.1.2 (Fluidigm). Specific primers for human and mouse transcripts including components of the major spliceosome (n = 13), minor spliceosome (n = 4), associated splicing factors (n = 28), PDGFRB pathway-related genes and three housekeeping genes were specifically designed with the Primer3 software (Supplementary Tables 2–4). To control for variations in the efficiency of the retrotranscription reaction, mRNA copy numbers of the different transcripts analysed were adjusted by a normalization factor, calculated with the expression levels of three housekeeping genes [β-actin (ACTB), hypoxanthine guanine phosphoribosyl-transferase (HPRT), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; only for human samples) and peptidylprolyl isomerase-A (Cyclophilin A; only for mouse samples)] (Supplementary Tables 2 and 3 ) and the GeNorm v.3.3 software as previously reported (Luque et al., 2015 (link); Hormaechea-Agulla et al., 2017a (link); Jimenez-Vacas et al., 2019a (link)).
As recently reported (Jimenez-Vacas et al., 2019b (link), 2020 (link)), a qPCR dynamic array based on microfluidic technology (Fluidigm, #BMK-M-48.48) was implemented to determine the simultaneous expression of 48 transcripts in HGA/glioblastoma samples compared to control samples using the Biomark System and the Fluidigm® Real-Time PCR Analysis Software v.3.0.2 and Data Collection Software v.3.1.2 (Fluidigm). Specific primers for human and mouse transcripts including components of the major spliceosome (n = 13), minor spliceosome (n = 4), associated splicing factors (n = 28), PDGFRB pathway-related genes and three housekeeping genes were specifically designed with the Primer3 software (Supplementary Tables 2–4). To control for variations in the efficiency of the retrotranscription reaction, mRNA copy numbers of the different transcripts analysed were adjusted by a normalization factor, calculated with the expression levels of three housekeeping genes [β-actin (ACTB), hypoxanthine guanine phosphoribosyl-transferase (HPRT), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; only for human samples) and peptidylprolyl isomerase-A (Cyclophilin A; only for mouse samples)] (
