Universal probelibrary upl probe

RT-qPCR was performed to validate the expression of DEGs using cDNAs that were generated from the same RNAs used for microarray analysis. First strand cDNA was synthesized from 3000 ng total RNA using random hexamers and the SuperScript™III Reverse Transcriptase Kit (Invitrogen, USA) according to the manufacturer’s protocol. Primers were designed and probes selected using ProbeFinder version 2.34 (except for Stat1 where ProbeFinder version 2.45 was used) at the Universal ProbeLibrary Assay Design Center (Roche Applied Science http://lifescience.roche.com/). RT-qPCR was performed in triplicate using the LC480 Master Probe Mix (Roche Diagnostics, Switzerland) and Universal ProbeLibrary (UPL) probe (Roche Diagnostics, Australia) according to published methods [29 (link), 36 (link)] (see Additional file 1 for a full list of primers and UPL probes used). Conditions for the RT-qPCR, calculation of quantification cycle for each signal, determination of PCR efficiencies, reproducibility (R² values) and relative quantification of target gene expression in Ts1Cje and disomic samples were performed essentially according to methods described previously [36 (link)]. Successful assays were defined by a PCR efficiency of between 90-110% and an R² values > 0.98.

Cells were collected and pelleted after the indicated treatments. RNA was extracted using a RNeasy mini kit (QIAGEN), total RNA was treated with DNase, and then reverse transcribed using the Maxima First Strand cDNA synthesis kit with dsDNase (Thermo Fisher Scientific). Gene expression was determined using assays designed with the Universal Probe Library (UPL probe) from Roche (www.universalprobelibrary.com). For each qPCR assay, a standard curve was generated to ensure that the efficiency of the assay was between 90% and 110%. Oligonucleotides used in this study and related information is presented in S3 Table. The QuantStudio qPCR instrument (Thermo Fisher Scientific) was used to detect the amplification level. Relative expression comparison (RQ = 2^-ΔΔCT) was calculated using the Expression Suite software (Thermo Fisher Scientific), using the housekeeping genes HPRT and ACTB as controls for the normalization.