Transcriptor high fidelity reverse transcription kit

Complementary DNAs (cDNAs) were synthesized with Transcriptor High Fidelity Reverse Transcription kit (Roche, Basel, Switzerland) using the same number of samples from the isolated RNAs. Reverse transcriptase PCR protocol with Oligo (dt) primers for single chain cDNA synthesis was used as per the manufacturer’s protocol. qPCR analysis was performed with SYBR Green Master Mix (Roche, Basel, Switzerland). All primer sequences are shown in Table 2. The mix is optimized for SYBR Green reactions, and the experiments were performed in Light Cycler 480-II (Roche) qRT-PCR. β-actin was used as an internal control. The relative quantification analysis was performed by delta-delta-Ct method as reported previously (10 (link)).

Sciatic nerves were prepared from P 1, 4 or 9 C57BL/6J mice as described above, dissociated in 700μl Qiazol using a Tissue Ruptor (Qiagen) and total RNA was extracted using the miRNeasy Mini Kit (Qiagen). Reverse transcription of mRNAs was performed with the Transcriptor High Fidelity Reverse Transcription Kit and qPCR was performed with the Taqman Universal Master Mix (all Roche Applied Science). SncRNA715 was reverse transcribed by the TaqMan MicroRNA Reverse Transcription Kit with stem-loop RT primers specific for sncRNA715 or snoRNA135 sequence (Applied Biosystems, order no. sncRNA715 PN4427975, snoRNA135 PN440887) and amplified with the Taqman Universal Master Mix (Roche Applied Sience) with specific primers and probes for the indicated sncRNAs (Applied Biosystems). The crossing points were used for relative quantification based on the _ΔΔCt method using REST software [27 ]. SnoRNA135 was used as a reference gene. PCR products and the 10 bp DNA Step Ladder (Promega) were separated on 4% agarose gels and stained with ethidium bromide.

Total RNA of Oli-neu cells and primary OPCs were extracted using either the RNeasy Mini Kit or miRNeasy Mini Kit (both Qiagen). Reverse transcription of mRNAs was performed with the Transcriptor High Fidelity Reverse Transcription Kit (Roche Applied Science). SncRNA715 was reverse transcribed by the TaqMan MicroRNA Reverse Transcription Kit with stem-loop RT primers specific for sncRNA715 sequence (Applied Biosystems) and amplified with the Taqman Universal Master Mix (Roche Applied Science) with specific primers and probes for sncRNA715 (Applied Biosystems). RT-PCR primers specific for rat MBP: 5′-AACATTGTGACACCTCGAACA-3′ and 5′-TGTCTCTTCCTCCCCAGCTA-3′. PCR of Ago1-4 mRNAs was carried out using the Pfu-DNA Polymerase according to manufacturer’s protocol. Primers were specifically designed to differentiate the four different murine Ago proteins (Ago1: 5′-AGCGGCAGGTGCCTAC-3′ and 5′-GGATCTGGCCACTGACC3-′, Ago2: 5′-ATATGCC TTCAAACCTCCACC-3′ and 5′-CCAGGGCCTGGATCGTC-3′, Ago3: 5′-ACAAGCCTGTCAGCACTAAC-3′ and 5′-CAGAC TTCTAGTGGCAGGTATG-3′, Ago4: 5′-GGAGCTCCTCTACA GCCAGG-3′ and 5′-CAAGCCGGGCATAATATGC-3′. Ago1-4 PCR products and 100 bp DNA Step Ladder (Promega) were separated on 1% agarose gels and stained with ethidium bromide (EtBr). RT-PCR products of Mbp mRNA and sncRNA715 were separated on 4% agarose gels and stained with EtBr.