7900ht qpcr platform

The SOD1 mRNA expression qPCR was performed on laterally bisected spinal cords and 15 mg tail tip samples from SOD1 mice. RNA was extracted using the Agencourt RNAdvance Tissue Kit for the Beckman Coulter BioMek FX^P automated liquid handler. RNA quality and quantity were measured using the SpectraMax NanoDrop. cDNA was synthesized using the Applied Biosystems High-Capacity cDNA Reverse Transcription kit. Normalization genes were selected using the Applied Biosystems Mouse Endogenous Control array card. Four control genes per tissue type were chosen; the genes chosen were those with the lowest variation in tissues across a range of SOD1 mouse ages and neurological scores. The spinal cord sample controls were murine Gapdh, Ppia, Tbp, and Ywhaz. The tail tip sample controls were murine Hprt, Polr2a, Ppia, and Ywhaz. A relative quantification qPCR was run using the Applied Biosystems 7900HT qPCR platform using SOD1 (Assay ID Hs00533490_m1, Thermo Fisher Scientific) as the target gene. The resulting Cts were normalized using the control genes and GeNorm software to produce relative SOD1 expression values for each sample.

Using BR Qubit 3 fluorometer, high quality samples were used for the library preparation. Library preparation and barcoding were performed using an Ion Xpress Barcode Adapter 1–16 Kit (Thermo Fischer Scientific). The samples were normalized using Ion Library Equalizer™ (Thermo Fischer Scientific) following manufacturer protocol. The amplified library was quantified using the 7900HT qPCR platform (Thermo Fischer Scientific), and purified using ion chef following manufacturer protocol. Following the standard manufacturer instructions, the samples were sequenced on an Ion Torrent Personal Genome Machine (PGM) sequencer (ThermoFisher Scientific). To identify the germline mutations in our target samples, the Ion 316 chips v2 having 100X coverage was used.