Protocols to quantify exon skipping levels

cDNA synthesis. ddPCR was performed as described in Verheul et al. [30]. cDNA was generated from 750 ng of RNA with Transcriptor Reverse Tanscriptase (#03531287001, Roche), random hexamer primers (#11034731001, Sigma-Aldrich), RNasin ribonuclease inhibitor (#N2115, Promega) and dNTPs (#11581295001, Sigma-Aldrich) in a total volume of 20 μl according to the manufacturer’s instructions. As a negative control, one reaction without reverse transcriptase was included.

ddPCR. Taqman assays from Thermo Fisher Scientific were ordered as 20-fold concentrations to quantify dystrophin cDNA products with or without exon 51. Primers and probes for Δ48–50 and Δ52 cell lines were identical to those described by Verheul et al. [30] (for sequences see S1 Table). The probes we used bind to the exon-exon junction (EEJ) of the transcripts with and without skipping of exon 51; for the skipped assay and non-skipped assay of Δ48–50 samples the probes bind to EEJ 47/52 and EEJ 51/52 respectively, and for the skipped assay and non-skipped assay of Δ52 samples the probes bind to EEJ 50/53 and EEJ 51/53 respectively. With this design the probe for the skipped assay is specific only for transcripts which lack exon 51 completely.

Separate reactions to detect the skipped and non-skipped products were prepared in a semi skirted 96-well plate (#0030 128.591, Eppendorf), containing 11 μl of 2x ddPCR Supermix for Taqman assays (#1863023, Bio-Rad), 1.1 μl of 20x Taqman assay, 2.2 μl of undiluted cDNA and 7.7 μl of DNase/RNase-free H₂O per reaction. Taqman assays ‘Skip_del48-50’ and ‘Non-skip_del48-50’ were used for 8036 cells and ‘Skip_del52’ and ‘Non-skip_del52’ for 1531 cells to quantify skipped and non-skipped fragments respectively. One reaction without cDNA was included as negative control. The plate was spun down to collect contents at the bottom of the wells before droplet generation.

Droplets were generated with the automated droplet generator QX200 system (#1864100, Bio-Rad) according to the provided supplier instructions, using ddPCR cartridges (#1864108, Bio-Rad) and 70 μl droplet generation oil for probes (#1863005, Bio-Rad) per 20 μl of sample. After finishing droplet generation, the plate was sealed with tin foil (#1814040, Bio-Rad) for 4 seconds at 170°C using the PX1 PCR plate sealer (#1814000, Bio-Rad).

The samples were amplified in a T100 thermal cycler (Bio-Rad) using the following program: 10 min at 95°C, 40 cycles of 30 sec at 95°C and 1 min at 60°C, 10 min at 98°C and held at 8°C. The heated lid was set to 105°C and the reaction volume to 40 μl. The plate containing the amplified droplets was transferred to the droplet QX200 reader (#1864100, Bio-Rad) to count positive (with cDNA) and negative droplets (without cDNA).

Exon skipping quantification. Data were analysed with QuantaSoft software, version 1.7 (#1864011, Bio-Rad), and could be visualized as 1-D or 2-D plots to show the separation between negative and positive droplets. The fluorescent amplitude threshold was set manually to discriminate between positive and negative droplets. The absolute concentration was represented in copies/μl sample mix and the concentrations of the skipped and the non-skipped assay were used to calculate exon skipping percentages according to the formula: Exon skipping % = (skipped copies/μl)/(skipped copies/μl + non-skipped copies/μl) x 100% [30]. This formula can be applied directly when the same volumes of cDNA were added to the skipped and non-skipped ddPCR reactions (as it was the case in our experiment). If the volumes differ, the target concentration present in the cDNA sample can be calculated with the formula: Target concentration cDNA (copies/μl) = absolute concentration (copies/μl) * total volume PCR reaction (μl)/volume cDNA (μl). In this case the target concentration of the cDNA should be used to calculate the exon skipping percentage.

cDNA synthesis. cDNA synthesis was performed with 300 ng of RNA with the High-Capacity cDNA Reverse Transcription Kit (#4368814, Thermo Fisher Scientific) in a 20 μl reaction volume according to the manufacturer’s instructions. RNaseOUT (#10777019, Thermo Fisher Scientific) was used in a final concentration of 2 U/μl. One reaction was included without transcriptase as a negative control.

Single round PCR. The PCR reactions contained final concentrations of 1 x buffer (#10966–034, Thermo Fisher Scientific), 0.2 mM dNTPs (#4030, Takara), 0.4 μM forward primer (Eurogentec), 0.4 μM reverse primer (Eurogentec) (for sequences see S1 Table), 1.5 mM MgCl₂ (#10966–034, Thermo Fisher Scientific), 0.04 U/μl Platinum Taq DNA polymerase (#10966–034, Thermo Fisher Scientific) and 1 μl of cDNA in a total volume of 25 μl. The PCR was run for 2 min at 94°C, 30 cycles of 45 sec at 94°C, 45 sec at 60°C and 80 sec at 72°C, then 5 min at 72°C and cooled down to room temperature. For 8036 cells (DMDΔ48–50) the expected fragment sizes using primers h45F and h52R (S1 Table) were 539 bp (non-skipped) and 306 bp (skipped) products. For 1531 cells (DMDΔ52) the expected fragment sizes were 800 bp (non-skipped) and 567 bp (skipped) with primer pair h49F and h53R1 (S1 Table).

Exon skipping quantification. PCR products were quantified with the Agilent high sensitivity DNA kit (#5067–4626, Agilent) on an Agilent 2100 Bioanalyzer according to the manufacturer’s instructions. The exon skipping percentages were calculated as the ratio of skipped transcripts and total transcripts according to the formula: Exon skipping % = (molarity skipped transcripts)/(molarity skipped transcripts + molarity non-skipped transcripts) x 100%.

cDNA synthesis. cDNA synthesis was performed with 400 ng of RNA with Transcriptor Reverse Transcriptase (#03531287001, Roche), using random hexamers (#SO142, Thermo Fisher Scientific), RNAsin ribonuclease inhibitor (#N2515, Promega) and dNTPs (#10297018, Thermo Fisher Scientific) in a total volume of 20 μl according to the manufacturer’s instructions, with minor adaptations: i) a lower concentration of random hexamers (final concentration 2 ng/μl) was used and ii) cDNA was prepared at 42°C. For one reaction, no transcriptase was included as a negative control.

Nested PCR. For the amplification 25 μl PCR reactions containing 1 x Supertaq PCR buffer (# TPRB, Sphaero Q), 0.2 mM of each dNTP (#10297018, Thermo Fisher Scientific), 0.4 μM forward primer (h47F1, Eurogentec, see S1 Table), 0.4 μM reverse primer (h54R, Eurogentec, see S1 Table), 0.025 U/μl Taq DNA polymerase (#11146165001, Roche) and 3 μl undiluted cDNA were prepared. Samples were run in a PCR machine for 5 min at 94°C, 20 cycles of 40 sec at 94°C, 40 sec at 60°C and 80 sec at 72°C, then 7 min at 72°C and cooled down to room temperature.

For the nested PCR, 1.5 μl of the first PCR product was mixed with 48.5 μl PCR master mix with final concentrations of 1 x Supertaq PCR buffer (# TPRB, Sphaero Q), 0.2 mM dNTPs (#10297018, Thermo Fisher Scientific), 0.4 μM forward primer (h47F2, Eurogentec, see S1 Table), 0.4 μM reverse primer (h53R2, Eurogentec, see S1 Table) and 0.025 U/μl Taq DNA polymerase (#11146165001, Roche). The 50 μl PCR reactions were run for 5 min at 94°C, 32 cycles of 40 sec at 94°C, 40 sec at 60°C and 60 sec at 72°C, then 7 min at 72°C and cooled down to room temperature. The PCR products were visualised by agarose gel electrophoresis after samples were mixed with 5 μl orange G (10-fold stock solution: 2 g/L Orange G, 30 v/v% glycerol). Five μl were loaded onto a 2% TBE-agarose gel containing 0.3 μg/ml ethidium bromide. The gel was run for 1 h at 100 V in 1xTBE buffer (10.8 g/L of Tris base, 5.5 g/L of Boric acid, 2 mM EDTA). Expected PCR fragment sizes were 475 bp (non-skipped) and 242 bp (skipped) for Δ48–50 cells and 754 bp (non-skipped) and 521 bp (skipped) for Δ52 cells and were checked with a 100 bp ladder (#SM0322, Thermo Fisher Scientific).

Exon skipping quantification. All pictures of the agarose gels obtained at the different laboratories were collected by one laboratory and analysed by one researcher with the ImageJ software. The peak area of the skipped and the non-skipped fragments were determined and corrected for their amplicon sizes. Exon skipping percentages were calculated as exon skipping % = corrected peak area of the skipped fragment/(corrected peak area of the skipped fragment + corrected peak area of the non-skipped fragment) x 100%.

cDNA synthesis and first round PCR. The cDNA synthesis and first round of PCR was prepared with the GeneScript RT PCR system from Quantig (#GS003) to transcribe single-stranded RNA into double-stranded DNA followed by amplification. Four hundred (400) μg of RNA was used for a 25 μl reaction volume containing 200 μM dNTPs, 1X reaction buffer, 300 nM forward primer (h47F2, S1 Table), 300 nM reverse primer (h54R, S1 Table), and 1.25 U Accurase/MMLV reverse transcriptase/RNase inhibitor. The reactions were run for 30 min at 45°C, 5 min at 92°C followed by 20 cycles of 30 sec at 92°C, 30 sec at 60°C and 45 sec at 68°C, finalized for 10 min at 68°C and held at 4°C. One sample without enzyme was used as a negative control.

Nested PCR. The PCR was prepared with 2 x PCR mastermix from Quantig (#PCRM002RD), 2 μl of PCR product from the first amplification, 200 μM dNTPs, 300 nM forward primer (h47F1, S1 Table) and 300 nM reverse primer (h53R2, S1 Table) in a reaction volume of 25 μl. The cycling conditions for the PCR were 2 min at 92°C followed by 30 cycles of 30 sec at 92°C, 30 sec at 600°C and 1 min at 68°C, followed by 10 min at 68°C and then held at 4°C. Ten (10) μl of the PCR products were loaded on a 2% TBE agarose gel with 1X SYBR Safe Gel Stain (#S33102, Invitrogen) and run against a 100 bp ladder (#BIO-33056, Bioline) at 100V for 45 minutes.

Exon skipping quantification. The peak area was determined with the GeneTools imaging analysis software from Syngene and was corrected for PCR fragment sizes (same fragment sizes as with ImageJ analysis). The following formula was used for the exon skipping quantification: Exon skipping % = corrected peak area of the skipped fragment/(corrected peak area of the skipped fragment + corrected peak area of the non-skipped fragment) x 100%.

cDNA synthesis. This protocol was replicated as previously described by Anthony et al. [31]. cDNA synthesis was performed with SuperScript III First-Strand Synthesis SuperMix (#18080400, Thermo Fisher Scientific). Per reaction, 500 ng of RNA, 1 μl of annealing buffer and 1 μl of random hexamer primers (50 ng/μl) were mixed together and made up to 8 μl with RNase/DNase free water. Reactions were incubated in a thermal cycler at 65°C for 5 min and then immediately placed on ice for at least 1 min. After adding 10 μl of 2X First-Strand Reaction Mix and 2 μl Superscript III/RNase OUT Enzyme Mix, reactions were run for 10 min at 25°C, 50 min at 50°C and 5 min at 85°C. One reaction without transcriptase was used as negative control (-RT reaction). Samples were cooled on ice and diluted 5-fold in RNase/DNase free water for the cDNA pre-amplification.

Pre-amplification PCR. The pre-amplification step was performed using the TaqMan PreAmp Master Mix (#4391128, Thermo Fisher Scientific), according to the manufacturer’s instructions. Taqman assays (see S1 Table) were obtained from Thermo Fisher Scientific (20X concentrations) and were 100-fold diluted by adding 1 μl of the skipped assay (Skip_exon47-52 or Skip_exon50-53) and 1 μl of the non-skipped assay (Non-skip_ exon51-52 or Non-skip_exon51-53) to 98 μl RNase/DNase free water for each cell line. The reaction volume was scaled down to 25 μl containing 12.5 μl PreAmp Master Mix, 6.25 μl mixed assays (skipped and non-skipped with final concentration 0.05X of each), 5 μl of 5-fold diluted cDNA and 1.25 μl RNase/DNase free water. Reactions were run in a thermocycler for 10 min at 95°C followed by 14 cycles of 95°C for 15 sec and 60°C for 4 min. The pre-amplified cDNA was 5-fold diluted to be used as template for performing the qPCR assays.

qPCR. qPCRs were performed on a Roche LightCycler 480 (lab 3), StepOnePlus real-time PCR system from Thermo Fisher Scientific (lab 5) or 7900HT Fast Real-Time PCR system from Applied Biosystems (lab 6). For the amplification of the skipped and non-skipped transcripts, a mastermix for each Taqman probe was prepared, containing 12.5 μl TaqMan Universal PCR master mix (#4369016, Thermo Fisher Scientific), 1.25 μl probe (Non-skip_exon51-52, Skip_exon47-52, Non-skip_exon51-53 or Skip_exon50-53 (see S1 Table)), 6.25 μl 5-fold diluted cDNA from the pre-amplification and 5 μl DNase/RNase free water. As negative controls, one reaction without template and one reaction with the–RT cDNA from the cDNA synthesis were included. Twenty-five μl were pipetted into the wells, and the qPCR plate was sealed and spun down for 2 min at 1000 rpm. The program for the qPCR machines was as follows: 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 sec at 95°C and 1 min at 60°C, and held at 37°C.

Exon skipping quantification. Data were analysed with LinReg [32,33] to calculate the PCR efficiency per individual well. Starting concentrations (N₀) and Ct values were determined and used to calculate exon skipping percentages with the following formula: N₀ skipped transcripts / (N₀ skipped transcripts + N₀ non-skipped transcripts) x 100 [31].