Turbo dna free rnase free dnase

For the qPCR analyses, RNA was harvested from cultured neurons using Nucleospin RNA XS (Takara) or RNAiso Plus reagent (Takara), followed by the removal of contaminating DNA using Turbo DNA-free (RNase-free DNase; Ambion). Total RNA (1–2 μg) was reverse transcribed using random hexamers and the PrimeScript first strand cDNA Synthesis Kit (Takara). Reverse transcription-quantitative PCR (RT-qPCR) was performed using a StepOnePlus qPCR system (Applied Biosystems) with Power SYBR Green PCR Master Mix (Applied Biosystems) and the comparative CT method. For relative quantification using qRT-PCR, transcript levels were normalized to that of Gapdh and relative quantification values were calculated. The oligonucleotide primers used for the qPCR are listed in Table S1.
For the abundance ratio of the SF to LF, the percentage of the SF variant was largely estimated from the CT value (CT^SF) and directly compared to that of the LF (CT^LF) at the same threshold set for the CT value. The CT^LF + CT^SF values for the total transcript levels were set to 100%.

RNA was isolated with RNAiso Plus reagent (TaKaRa, Tokyo, Japan), followed by removal of contaminating DNA using Turbo DNA-free (RNase-free DNase, Ambion). Two micrograms of total RNA was reverse transcribed using random hexamers and ImProm-II (Promega, Madison, WI, USA). For semi-quantitative PCR, DNA fragment intensities were quantified using an image analyser (FAS-III, Toyobo, Osaka, Japan) and ImageGauge software (Fujifilm, Valhalla, NY, USA). The value for the intensity of each Nfasc isoform band was normalized to that of total Nfasc (ex24-25). Quantitative PCR (qPCR) was performed on a StepOnePlus qPCR system (Applied Biosystems, Waltham, MS, USA) with Power SYBR Green PCR Master Mix (Applied Biosystems) and the comparative C^T method. For the relative quantification by qRT-PCR, transcript level was normalized to that of Gapdh. On the other hand, transcript levels of each Nfasc isoform was normalized to that of total Nfasc (ex24-25), to avoid confounding by differences in amount of total Nfasc between groups. All the oligonucleotide primer sequences used for semi-quantitative PCR and qRT-PCR are shown in Table 1. Primers for Nrxn1/2/3 have been previously described^{14 (link)}.