Materials and methods
Human samples
Skin biopsies from 52 consenting CMT1A patients enrolled in the ascorbic acid italian trial were
obtained from the medial aspect of proximal phalanx of the index (Milan and Genoa centres) or
from the tip of the little finger (Naples) and studied. The procedure was performed using a 3 mm
biopsy punch under sterile conditions and local anaesthesia with 1% lidocaine. Seven pairs of skin
biopsies, which were known to have encountered problems of cryopreservation, did not reach the
minimum standard quality of total RNA and were then discarded. Thus, the 45 remaining patients
were analysed in this study.
To evaluate splicing variants tissue distribution, we used Human I MTC Panels (Clontech, USA)
containing cDNAs derived from heart, brain, placenta, lung, liver, skeletal muscle, kidney and
pancreas human tissues. Primary human fibroblast cultures and sorted human lymphocytes were
also used.
Absolute quantification of PMP22 mRNA
In order to verify whether skin biopsy is a suitable tissue to measure PMP22 mRNA levels, we
cloned full length cDNA of the human PMP22 splicing variants in pDrive Cloning Vector through
Qiagen PCR cloning Plus Kit (Qiagen, Italy). The sequences of each plasmid were verified through
sequencing (TibMolBiol, Italy) and are available upon request.
Plasmids containing cDNA of each PMP22 splicing variants were used to perform a titration curve
assay. Pure plasmid preparations (Qiaprep spin miniprep kit, Qiagen, Italy) were gradually diluted
and utilised as standard positive controls in Real-Time quantitative PCR (RT-qPCR) reactions to
calculate the absolute copy number of PMP22 mRNA in skin biopsies and to test PCR reaction
efficiency of our primers (see next paragraph).
Real-time quantitative reverse transcription PCR (RT-qPCR)
Human skin and sural nerve biopsies were homogenised in Qiazol reagent through a high speed
shaking Tissue Lyser II system using the 5 mm stainless steel beads (Qiagen s.r.l. - Italy). Paired
samples (T0 and T24 skin biopsies from the same patient and the age and gender- matched normal
subject), were processed at the same time to minimise experimental errors and handling. RNA
extraction from tissues was performed with miRNeasy mini kit (Qiagen-Italy) according to
manufacturer’s protocols. Total RNA was resuspended in RNase free water and checked for quality
and quantity by Nanodrop technology (NanoDrop products, Wilmington, DE, USA). The quality of
primers used in this study was checked for intron spanning and matching common PCR standard
parameters by BLASTN algorithm. Specificity and efficiency of the amplification product for each
primer pair was verified by the presence of a single peak in RT-qPCR melting curve and by the
achievement to the cut-off value through a titration curve. We have utilised multiple reference
genes to normalise PMP22 mRNA according to MIQE standard guidelines for RT-qPCR (Bustin et
al. , 2010). The most stable three reference genes were chosen from a pool of seven candidate genes
deriving from independent pathways and tested for intergroup stability in our skin and nerve
samples using geNorm algorithm (Vandesompele et al. , 2002). Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), succinate dehydrogenase complex, subunit A, flavoprotein (SDHA), and
TATA box binding protein (TBP) reached a coefficient of stability (M value) lower than the cut-off
value, and they were therefore selected for the RT-qPCR experiments.
Ct data (cycle thresold) were calculated with ΔCt method using as internal calibrator the sample
with lowest Ct and resulting values were analysed by geNorm algorithm for normalization. Finally,
the variability of each sample has been calculated to verify the reproducibility of our measurements.
All PCR reactions were carried out in duplicate and averaged.
Statistical analysis
Reproducibility and repeatability of splicing variants measurements were inspected by the BlandAltman plot and analyzed with the Friedman’s repeated measures ANOVA on ranks.
References
Bustin SA, Beaulieu JF, Huggett J, Jaggi R, Kibenge FS, Olsvik PA, et al. MIQE precis: Practical
implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR
experiments. BMC Mol Biol 2010; 11: 74.
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate
normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal
control genes. Genome Biol 2002; 3: RESEARCH0034.