Transcriptor high fidelity cdna synthesis kit

Total cellular RNA was prepared from mouse LV isolated cardiomyocytes using RNeasy Mini kit (Qiagen) following manufacturer’s instructions. cDNA was obtained from total RNA using Transcriptor High Fidelity cDNA Synthesis kit (Sigma-Aldrich). Real-time RT-PCR was performed with primers listed in Supplemental Table S1. The QuantStudio Real-Time PCR system (Applied Biosystems) was employed for quantitative RT-PCR. In each case, cDNA was combined with Fast SYBR Green Master Mix (Applied Biosystems) in a 25-μL reaction mix. Cycling conditions were as follows: 95°C for 10 min followed by 40 cycles of amplification (95°C denaturation for 10–15 s, 60°C annealing and extension for 1 min). The melting curve was then obtained. C_t values were normalized with respect to hypoxanthine guanine phosphoribosyl transferase (Hprt).

A human cDNA pool was constructed by reverse transcription-PCR (RT-PCR) with the Transcriptor High Fidelity cDNA Synthesis Kit (Cat. 5081955001, Sigma Aldrich, St. Louis, MO) from RNA extracted from immortalized non-transformed gastric epithelial cell (GES-1). A 650-bp cDNA, encoding the human ALKBH6 open reading frame (nt 280–786 of XM_024451747), was amplified using PCR (Platinum Taq, Invitrogen, Carlsbad, CA). The primers are: 5′-GCGAAGCTTTCACTTGCCCAGCAGG-3′, and 5′-GCTCTAGAGCGGAAATGGCTGGGAG-3′. The amplified product was cloned into pBAD24 (Addgene) as an XbaI-NotI fragment. The recombinant plasmid was transformed into BS87 with electroporation to construct BS87-pBAD24-ALKBH6, and the expression of ALKBH6 was induced by the addition of 0.1% arabinose (Cat. A3256, Sigma Aldrich).

HEK293E sh^TDP-43 cells were plated in 6-well plates and after 24 h were double-transfected with the corresponding TDP-43 construct and a CFTR minigene construct^{11 (link)} at a ratio of 2:1. After 72 h RNA was isolated using the RNeasy Mini Kit (Qiagen) following the indications from the provider. From the isolated RNA cDNA was obtained using the Transcriptor High Fidelity cDNA Synthesis Kit (Sigma) following the protocol of the provider. The cDNA of interest was then amplified via a standard PCR and the product was run in a 2% agarose gel. DNA was visualized using Midori Green from Biozym and the ChemiDoc XRS + Imaging System.