Transcriptor high fidelity cdna synthesis kit

Following the manufacturer’s instructions, 100 ng of total RNA was reverse transcribed with Transcriptor High Fidelity cDNA Synthesis Kit (Roche Diagnostics GmbH, Mannheim, Germany). With total RNA, we used 60 μM random hexamer primers, 1 mM each of dNTPs, 1× reaction buffer, 20 U RNase inhibitor, and 10 U of Transcriptor High Fidelity Reverse Transcriptase in a total volume of 20 μL. The thermal cycling conditions were 25 °C for 10 min, 60 °C for 60 min, and 85 °C for 5 min.

The brain was removed quickly under anaesthesia and made the serial coronal sections, followed by punching out the hippocampal tissue. Total RNA was extracted by RNeasy Mini Kit (Qiagen, Hilden, Netherlands). Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Basel, Switzerland) was used for reverse transcription. qPCR for Ndufs4 was performed using StepOnePlus Real-Time PCR system (Applied Biosystems, California, USA) using following mouse specific TaqMan Probes: Ndufs4:Mm0656176_ml, Actb: Mm02619580_gl. Actb was used as endogenous control. Relative gene expression was calculated using the 2^−∆∆Ct method.

At different time points of differentiation, RNA samples were extracted by using High Pure RNA Isolation Kit (Roche) and converted into complementary cDNA with Transcriptor High Fidelity cDNA Synthesis Kit (Roche). Real-time quantitative PCR (qPCR) was performed using the StepOne^TM or the ViiA^TM 7 RT-PCR Systems (Applied BioSystems). Taqman^® Gene Expression Assays (20X, Applied Biosystems) were selected for NANOG, PAX6, TBR1, DLX2, NKX2.1, LHX6, FOXG1 and GAPDH. DLL1, HES5, PARVALBUMIN (PV), VGLUT1 and GAPDH analysis were performed using the SYBR Green Master Mix (Nzytech). The results were analyzed with the StepOne^TM or the QuantStudio^TM RT-PCR Software. All PCR reactions were done in duplicate or triplicate and then normalized to the housekeeping gene GAPDH. The fold change was calculated using the 2^ΔCt method and in some graphical results it was calculated relatively to the control condition levels obtained. The representative heatmaps were generated using the web tool Clustvis (Metsalu and Vilo, 2015 (link)).

A genomic segment of MFSD8 spanning exon 5 (hg19, chr4:128864292‐128865864) was amplified from patient DNA using primers with NotI and BamHI recognition sequences (Table S5). PCR products were cloned into the pCR2.1 plasmid (Invitrogen‐Life Technologies, Carlsbad, California). Wild type (WT) and mutant inserts were excised by digestion and subcloned into digested pSPL3_2096 (Invitrogen‐Life Technologies, Carlsbad, California). HEK293T cells were transfected with pSPL3‐MFSD8 constructs using Lipofectamine (Invitrogen‐Life Technologies, Carlsbad, California). Total RNA was extracted after 24 hours (peqGOLD Total RNA Kit, VWR, Radnor, Pennsylvania) and reversely transcribed (Transcriptor High Fidelity cDNA Synthesis Kit, Roche Applied Science, Penzberg, Germany). The cDNA was PCR amplified with pSPL3 exon primers and Sanger sequenced (Applied Biosystems, Foster City, California).

Total RNA was extracted from freshly frozen tumor samples using the ReliaPrep RNA Cell Miniprep System (Promega, USA) and reverse-transcribed using random hexamers and the Transcriptor High Fidelity cDNA synthesis kit (Roche, Germany) as per the manufacturer’s instructions. Quantitative RT-PCR was performed using Power SYBR Green PCR Master Mix (Roche, USA) with previously-published primer sets^{32 (link)} on a ViiA^TM 7 Real-Time PCR System (Applied Biosystems, USA).

Total RNA was isolated from freshly isolated adult flukes using Trizol (Sigma Aldrich, Mannheim, Germany) Reverse transcription was performed using “Transcriptor High Fidelity cDNA Synthesis Kit (Roche Diagnostics GmbH, Mannheim, Germany), using either gene specific primers or oligo dT primers. Starting from a previously deposited cDNA sequence 5’ and 3’ ends of the cDNA were identified via RACE (rapid amplification of cDNA ends) technology, using 5’/3’ RACE Kit, 2nd Generation (Roche Diagnostics GmbH, Mannheim). Recognition sites for XhoI and KpnI were added to the full-length cDNA of FhTauT by PCR. The resulting PCR product was cloned via XhoI and KpnI to peYFP-C1 (Takara Bio Europe, France) generating a transporter tagged with a yellow fluorescent protein at its N-terminus. To generate a non-tagged transporter, FhTauT was also cloned to pcDNA3.1 (Invitrogen, Carlsbad, USA). HsTauT was cloned in a similar way to peCFP-C1 via BamHI and HindIII thereby generating a transporter tagged with the cyan fluorescent protein. Generated sequences were validated by Sanger sequencing (LGC Genomics, Berlin, Germany). The cDNA sequence of FhTauT was deposited at GenBank (NCBI, Bethesda, USA) under the accession number: MG674191.