Sybr green protocol

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.

Gene and miRNAs expression levels were validated by qRT-PCR on both training (array set) and validation sets of patients. qRT-PCR have been performed using Sybr Green protocol (Qiagen, Milano, Italy) on an Applied Biosystems 7900HT instrument (Waltham, MA, USA). 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 between groups’ mean differences, then the p-value underwent Benjamini–Hochberg FDR-controlling procedure, and only comparison with adjustedp-value ≤ 0.1 was considered statistically significant and reported in Table 2.