Improved Methodology for Assessment of mRNA Levels in Blood of Patients with FMR1 Related Disorders

<p>Abstract</p> <p>Background</p> <p>Elevated levels of FMR1 mRNA in blood have been implicated in RNA toxicity associated with a number of clinical conditions. Due to the extensive inter-sample variation in the time lapse between the blood collection and RNA extraction in clinical practice, the resulting variation in mRNA quality significantly confounds mRNA analysis by real-time PCR.</p> <p>Methods</p> <p>Here, we developed an improved method to normalize for mRNA degradation in a sample set with large variation in rRNA quality, without sample omission. Initially, RNA samples were artificially degraded, and analyzed using capillary electrophoresis and real-time PCR standard curve method, with the aim of defining the best predictors of total RNA and mRNA degradation.</p> <p>Results</p> <p>We found that: (i) the 28S:18S ratio and RNA quality indicator (RQI) were good predictors of severe total RNA degradation, however, the greatest changes in the quantity of different mRNAs (<it>FMR1, DNMT1, GUS, B2M </it>and <it>GAPDH</it>) occurred during the early to moderate stages of degradation; (ii) chromatographic features for the <it>18S, 28S </it>and the inter-peak region were the most reliable predictors of total RNA degradation, however their use for target gene normalization was inferior to internal control genes, of which <it>GUS </it>was the most appropriate. Using <it>GUS </it>for normalization, we examined in the whole blood the relationship between the <it>FMR1 </it>mRNA and CGG expansion in a non-coding portion of this gene, in a sample set (n = 30) with the large variation in rRNA quality. By combining <it>FMR1 </it>3' and 5' mRNA analyses the confounding impact of mRNA degradation on the correlation between <it>FMR1 </it>expression and CGG size was minimized, and the biological significance increased from p = 0.046 for the 5' <it>FMR1 </it>assay, to p = 0.018 for the combined <it>FMR1 </it>3' and 5' mRNA analysis.</p> <p>Conclusion</p> <p>Our observations demonstrate that, through the use of an appropriate internal control and the direct analysis of multiple sites of target mRNA, samples that do not conform to the conventional rRNA criteria can still be utilized to obtain biologically/clinically relevant data. Although, this strategy clearly has application for improved assessment of <it>FMR1 </it>mRNA toxicity in blood, it may also have more general implications for gene expression studies in fresh and archival tissues.</p

Tissue- and age-specific DNA replication patterns at the CTG/CAG-expanded human myotonic dystrophy type 1 locus

International audienceMyotonic dystrophy, caused by DM1 CTG/CAG repeat expansions, shows varying instability levels between tissues and across ages within patients. We determined DNA replication profiles at the DM1 locus in patient fibroblasts and tissues from DM1 transgenic mice of various ages showing different instability. In patient cells, the repeat is flanked by two replication origins demarcated by CTCF sites, with replication diminished at the expansion. In mice, the expansion replicated from only the downstream origin (CAG as lagging template). In testes from mice of three different ages, replication toward the repeat paused at the earliest age and was relieved at later ages-coinciding with increased instability. Brain, pancreas and thymus replication varied with CpG methylation at DM1 CTCF sites. CTCF sites between progressing forks and repeats reduced replication depending on chromatin. Thus, varying replication progression may affect tissue- and age-specific repeat instability