7900ht instrument

For a subset of Cross-Center samples (Penn, n = 101 and OSU, n = 111), LTL was also measured using the quantitative PCR method developed by Cawthon, modified for compatibility with the Applied Biosystems 7900 HT instrument [30 (link)](S2 Protocol). Assays were carried out in triplicate, and center samples were batch analyzed to minimize inter-assay variation. The T/S ratios of each experimental sample relative to the reference sample were generated using the comparative CT (cycle threshold) method[30 (link)]. T/S ratios and LTL kb were compared for quality control comparisons.

Total RNA was extracted from PBMC and fibroblasts using TRIzol (Invitrogen). RNA was transcribed into cDNA in a 20 μL mixture containing 1 μg of total RNA and PrimeScript™ RT Master Mix (Takara) according to the manufacturer's instructions. Quantitative real-time PCR was performed on Applied Biosystems 7900 HT Instrument (Applied Biosystems, CA, USA) following our published procedures [7 (link)]. The primer sequences were summarized in Table 3. All samples were assayed in triplicate. Relative gene expression was determined by the 2^−ΔΔCt method.

Total RNA of liver samples was extracted using the TRIzol reagent (Invitrogen, USA) and reverse transcribed to cDNA according to the manufacturer's instructions (TaKaRa Biotechnology Co., Ltd., Dalian, China). The mRNA expression in liver tissue was quantified using the SYBR Green detection system in 7900HT instrument (Applied Biosystems, Forster, CA, USA). The primer sequences are listed in Table 2.

Neurospheres were collected before plating on PLL (undifferentiated NSCs) or after differentiation for 1 or 4 days. RNA was extracted using the RNeasy Micro Kit (Qiagen) and quantified with a Nanodrop-1000 Spectrophotometer (NanoDrop Technologies, Inc). qRT-PCR was performed as described in ref. 36 (link). Complementary DNA was analyzed by qRT-PCR (5 ng RNA equivalent/reaction) using 0.2 µM target-specific primers and QuantiFast SYBR Green (Qiagen). Primers were validated by qRT-PCR analysis on serial dilutions of a positive control complementary DNA to assess their specificity and efficiency. Forty qRT-PCR cycles were performed on 7900HT instrument (Applied Biosystems) equipped with the Sequence Detection Systems (SDS) 2.3 software. Data were normalized using the expression of the housekeeping genes Actin and HPRT1 as in ref. 36 (link). C_t values were converted into fold-expression values relative to the control using qBase^PLUS software (Biogazelle).

Gene and miRNAs expression levels were validated by qRT-PCR on both training (array set) and validation set of patients. qRT-PCR have been performed using Sybr Green protocol (Qiagen, Milano, Italy) on an Applied Biosystems 7900HT instrument. Experiments were run in triplicate, using 384-well reaction plates in an automatic liquid handling station (epMotion 5075LH; Eppendorf, Milano, Italy). Raw data was generated with Sodium dodecylsulphate (SDS) Relative Quantification software (version 2.3; Ambion-ABI), data were normalized using the geometric mean of the four independent housekeeping controls (for miRNAs: RNU6B, SNORD61, SNORD72, SNORD68; for genes: ACTB, B2M, PPIA and HPRT1). Two-sided Student's t-test were used to verify among groups mean differences; P-value ≤0.05 was considered statistically significant.

Expression profiling of circulating miRNAs was performed for 20 samples as described above using TaqMan human miRNA arrays and assays in accordance with the manufacturer’s instructions (Taqman Low Density Array Human microRNA, Applied Biosystems, Foster City, CA, USA). In brief, total RNA was reverse transcribed using Megaplex primer pool A (Applied Biosystems) which contained sequence-specific primers for 381 specific miRNAs plus 3 controls (pool A). An additional panel of 384 miRNAs (381 miRNAs and 3 controls, pool B) was performed on a subset of 4 cancers and 4 controls. Real-time quantitative PCR was performed for 667 miRNAs, using A and B microfluidic cards, each containing primers and probes for 381 specific miRNAs plus 3 controls and thermal-cycled on an Applied Biosystems 7900 HT instrument. MiRNA expression data are available from the National Center for Biotechnology Gene Expression Omnibus (GEO) at accession number GSE46355.