Page 53 - scheppingen
P. 53

CODING AND SMALL NON-CODING TRANSCRIPTIONAL LANDSCAPE OF TSC
lectual disability (ID; Mild = IQ 55-70; Moderate = IQ 40-55; Severe IQ 25-40) and autism. The majority of TSC patients harbored TSC2 gene mutations (66.6%; Table 1). The tissue specimens were age-matched with the controls (p-value>0.86, two-sided t-test). Using a principal variance component analysis (PVCA) brain region and gender were shown to contribute minimally to the overall variation of the RNA-Seq results.
Perituberal frozen material was available for only 3 out of 10 TSC cases exam- ined and two samples did not pass the minimal quality requirements; thus analysis of per- ituberal tissue was not performed. Histologically normal cortex was obtained at autopsy from 10 controls without a history of seizures or other neurological diseases (cause of death was acute cardiorespiratory failure; Table1). We acknowledge that the choice of the control material is extremely difficult in human studies, particularly in case of pathologies affecting young patients, which limits the number of cases suitable for gene expression studies. We were fortunate to obtain the human postmortem from young controls, as well as autopsy TSC samples.
Tissue was obtained and used in accordance with the Declaration of Helsinki and the AMC Research Code provided by the Medical Ethics Committee and approved by the science committee of the UMC Utrecht Biobank. The local ethical committees of all participating centers gave permission to undertake the study.
Tissue preparation
Cortical specimens from control and TSC patients were snap frozen in liquid nitrogen and stored at -80°C until use for RNA isolation. Additional tissue was fixed in 10% neutral buffered formalin and embedded in paraffin. Paraffin-embedded tissue was sectioned at 5 μm, mounted on pre-coated glass slides (Star Frost, Waldemar Knittel GmbH, Brunschweig, Germany) and used for in situ hybridizations and immunocytochemistry. One representative paraffin block per case was sectioned and processed for hematoxy- lin and eosin (HE) as well as for immunocytochemical staining for a number of neuronal and glial markers to confirm the diagnosis.
RNA isolation
Frozen tissue material was homogenized in Qiazol Lysis Reagent (Qiagen Benelux, Venlo, The Netherlands). The total RNA including the miRNA fraction was isolated using the miRNeasy Mini kit (Qiagen Benelux, Venlo, the Netherlands) according to manufactur- er’s instructions. The concentration and purity of RNA were determined at 260/280 nm using a Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). Samples required an RNA integrity number (RIN) greater than 6.0 for use in down-stream sequencing.
RNA-seq library preparation and sequencing
Library preparation and sequencing were completed at ServiceXS, Plesmanlaan 1D, 2333 BZ, Leiden, Netherlands. The Illumina (San Diego, California, USA) mRNA-Seq and TruSeq Small RNA-Seq sample preparation kits were used to prepare sequencing libraries of mRNA and small RNA, respectively. Briefly, mRNA was selected by oligo-dT magnetic beads, fragmented and subjected to cDNA synthesis. Sequencing adapters were ligated to the cDNA fragments, followed by PCR amplification. Small RNA samples were pro-
 51
three























































































   51   52   53   54   55