For some patients, electrocardiography (ECG) and endocrinological evaluation were further performed. To confirm the DM2 diagnosis, PCR amplification across the DM2 CCTG repeat was performed as previously described. R894X, the most frequent ClC1 mutation in German patients with the recessive inherited type of myotonia, was screened for as described (21). ClC1 splice variants. antiangiogenic Quadriceps Inhibitors,research,lifescience,medical muscle was taken
from 3 inhibitor moderately affected and 1 mildly affected patient (CCTG carriers without R894X mutation) with their informed consent. Control muscle was taken from 5 individuals negative in a malignant hyperthermia testing protocol. Total cellular RNA was extracted from homogenized skeletal muscle tissues and cultured cells using Trizol R (Gibco BRL, USA). Eight pairs of primers were designed Inhibitors,research,lifescience,medical to cover the whole cDNA of CLCN1 (Table 1). RT-PCR amplification was carried out in a final volume of 50 μl, using equal amounts (1-2 μg) of total RNA, and 50 pmol upstream and downstream primers with an one
step RT-PCR kit (Qiagen, Hilden, Germany). After first strand cDNA synthesis (50°C for 30 min), 35 cycles of amplification were performed, each consisting of 60 sec at 94°C, 60 s at 55°C and 60 s at 72°C, followed by a final 10 min extension at 72°C. The products of amplification were electrophoretically resolved on 2 % agarose gels stained with ethidium bromide (0.5 μg/ml). All variants Inhibitors,research,lifescience,medical were confirmed by sequencing. Percent of splicing variant was calculated as (cpm variant band)/(cpm variant band
+cmp normal band) x 100 using Scion Image software. Table 1. Primer pairs used for RT-PCR. Plasmid Inhibitors,research,lifescience,medical construction. A 519 bp product, with exons 6 and 7 missing, was amplified from DM2 Inhibitors,research,lifescience,medical affected persons RNA, using CLCN2F as forward primer and CLCN3R as reverse primer. PCR product was digested with BstEII and the resulting 225 bp fragment used to replace the corresponding fragment into the human skeletal muscle choride channel mammalian expression construct pRc-CMV-hClC1 (22). To obtain a GFP-ClC1 wt and variant fusion constructs, the pRc-CMV-hClC1 wt and variant were digested to completion with KpnI. The coding fragments were gel-purified and subcloned into pEGFP-C1 (Clontech). The sequence of the resulting inframe pEGFP-ClC1 fusion constructs were verified by restriction digestion and sequence analysis. AV-951 Functional study. The pRcCMV constructs containing the full length or the truncated form of the ClC1 channel were transfected into tsA201 cells. Successful expression of the resulting prematurely truncated protein ClC1236X (stop at codon 236) was confirmed by Western blots of total protein of transfected tsA201 cells using rabbit polyclonal primary antibody for ClC1 (1:1,000) and goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:5,000), both from Santa Cruz, Biotechnology.