ABCMdb

PMID: 21594783

Linde L, Kerem B
Nonsense-mediated mRNA decay and cystic fibrosis.
Methods Mol Biol. 2011;741:137-54., [PubMed]

Sentences

No. Mutations Sentence Comment

58 ABCC7 p.Trp1282* The template for these PCR reactions is DNA from HT-29 cells, carrying normal CFTR alleles, or DNA from KM cells, homozygous for the W1282X mutation. Login to comment 185 ABCC7 p.Trp1282* The DNA mix for the CFTR transfections contains 2 μg DNA per 6 well of WT or W1282X CFTR constructs together with 0.2 μg DNA of GFP. Login to comment 234 ABCC7 p.Trp1282* ABCC7 p.Gly542* 2. For quantification of CFTR W1282X transcript levels in HeLa and MCF7 cells, transfected with the CFTR constructs RNA is extracted according to the manufacturer`s protocol and RT reactions are performed to obtain cDNA. Real-time PCR is performed using the LightCycler with the Patients RelativeRNAlevel 0 0.1 0.2 0.3 0.4 0.5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 W /3849 W /G542X W /W W /ΔF508 W /ΔF508 W /W W /ΔF508W /ΔF508W /ΔF508W /ΔF508W /ΔF508W /ΔF508 W /ΔF508W /ΔF508W /ΔF508 Response to readthrough treatment No response Fig. 10.1. Login to comment 239 ABCC7 p.Trp1282* W - the W1282X mutation; 3849 - the 3849+10 kb C→T mutation. Login to comment 241 ABCC7 p.Trp1282* The level of RNA transcribed from the WT or W1282X CFTR constructs is normalized to the RNA level of GFP (which is used as a reference for transfection efficiency). Login to comment 248 ABCC7 p.Trp1282* Then, the level of the normalized transcripts following NMD Fig. 10.2. Effect of CHX treatment on the level of CFTR mRNA transcribed from constructs carrying the W1282X PTC. Login to comment 249 ABCC7 p.Trp1282* (a) A scheme of the wild-type (WT) (upper panel) and W1282X (lower panel) constructs, which contain all CFTR exons (marked in the boxes by numbers) and part of the intronic sequences between exons 20 and 22. Login to comment 252 ABCC7 p.Trp1282* The level of mRNA transcribed from CFTR constructs carrying either the normal sequence or the W1282X PTC was normalized to the mRNA level of GFP. Login to comment 254 ABCC7 p.Trp1282* The fold increase in the level of CFTR W1282X transcripts is shown as mean ± SEM (reproduced from (9)). Login to comment