Quantstudio 7 flex real time pcr system machine

For assessment of the level of mRNAs containing CGG repeats, transfection of COS7 with 100×CGG construct and ASOs and total mRNA isolation was performed as described in “miRNA level quantification”. Next, cDNA was synthesized with the use of GoScript^TM Reverse Transcriptase System (Promega) according to the manufacturer’s instructions with the exception of the first-strand synthesis reaction temperature which was 37 °C. We used an anchored oligo(dT) primer containing oligo dT tract which allowed for exclusive reverse transcription of polyA+ RNAs. qPCR was performed with the use of iTaq™ Universal SYBR^® Green Supermix (Bio-Rad) according to manufacturer’s instructions and analyzed with the use of QuantStudio 7 Flex Real-Time PCR System machine (Thermo Fisher Scientific). To ensure amplification of 100×CGG construct mRNA but not DNA following primers were used: forward primer complementary to the 3′ part of GFP sequence and universal reverse (UR) primer. Primers for amplification of endogenous mRNA of FMR1 and NUB1 were set in 3′UTR of transcripts. Ct values were normalized against GAPDH and amplified with gene-specific forward primer set in 3′ part of mRNA and UR primer. All primers are listed in Supplementary Table S2. Fold differences in expression level were calculated according to the 2^−ΔΔCt method.

For quantification of the effect of RNase H1 insufficiency and ASOs on FMR1 mRNA and pre-mRNA level, fibroblasts were seeded on a 12-well plate and transfected at ~80% of confluency with siRNAs (future synthesis) at a final concentration of 15 nM. siRNA sequences are listed in Supplementary Table S2. After 24 h, the 11-nucleotide long (CGG^norm/- (2), CGG^norm/CGG^exp, CGG^exp/CGG^exp) or 9-nucleotide long (CGG^norm/- (1)) ASOs at 200 nM final concentration were delivered. Fibroblasts were harvested 48 h post ASOs delivery. The isolation of RNA was performed with the Total RNA Zol-Out™ D kit (A&A Biotechnology). In total, 300–500 ng of the total RNA was used for reverse transcription with GoScript^TMReverse Transcriptase (Promega) and random primers (Promega). qPCR was performed with the use of iTaq™ Universal SYBR^® Green Supermix (Bio-Rad) according to the manufacturer’s instructions and analyzed with the use of QuantStudio 7 Flex Real-Time PCR System machine (Thermo Fisher Scientific) with primers listed in Supplementary Table S2. Ct values were normalized against GAPDH. Fold differences in expression level were calculated according to the 2^−ΔΔCt method.