Mirvana s isolation kit

Quantitative real time PCR was performed on total RNA extracted with mirVana’s isolation kit (Ambion) from differentiating iPS cells. Purified RNAs were used to generate the corresponding cDNAs, which served as PCR templates for mRNA quantification. Quantitative RT-PCR assays were performed in duplicate on cDNA samples obtained from the retro-transcription reaction diluted 1:20 in 384-well optical plates (Kisker Biotech) using the ABI Prism 7900 HT Sequence Detection System (Applied Biosystems). The reactions were carried out using 20xTaqMan gene expression assays for genes and 2xTaqMan Universal PCR Master Mix (Applied Biosystems). The reactions were conducted using the following parameters: 50 °C for 2 min, 95 °C for 10 min, 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The fold change was determined using the eq. 2^-ΔΔCT. Primers used in iPS cell differentiation experiments and Tau R3/R4 analysis can be seen in Supplementary Table 3.

Quantitative real time PCR was performed on total RNA extracted with a mirVana’s isolation kit (Ambion, Austin, TX, USA) from the frontal cortex of human samples. Purified RNAs were used to generate the corresponding cDNAs, which served as PCR templates for mRNA quantification. PCR amplification and detection were performed with the ROCHE LightCycler 480 detector, using 2× SYBR GREEN Master Mix (Roche, Basel, Switzerland) as reagent, following the manufacturer’s instructions. The reaction profile was: denaturation–activation cycle (95 °C for 10 min) followed by 40 cycles of denaturation–annealing–extension (95 °C for 10 min, 72 °C for 1 min, 98 °C continuous). mRNA levels were calculated using the Light Cycler 480 software (Roche). Data were analyzed with SDS v. 1.9.1 Software (Applied Biosystems, Madrid, Spain) following the 2^−ΔΔCT method of Applied Biosystems. Primers were as follows: RELN (5’-actctgtcaacagctcaagc-3’) and (3’-tggtcaattgcccagctttg-5). The results were normalized for the expression levels of the housekeeping gene, GAPDH (5’-tccaaaatcaagtggggcga -3’ and 3’-tctccatggtggtgaagacg -5’), which were quantified simultaneously with the target gene in each sample.