Abstract
BackgroundElevated 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.MethodsHere, 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.ResultsWe 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 (FMR1, DNMT1, GUS, B2M and GAPDH) occurred during the early to moderate stages of degradation; (ii) chromatographic features for the 18S, 28S 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 GUS was the most appropriate. Using GUS for normalization, we examined in the whole blood the relationship between the FMR1 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 FMR1 3' and 5' mRNA analyses the confounding impact of mRNA degradation on the correlation between FMR1 expression and CGG size was minimized, and the biological significance increased from p = 0.046 for the 5' FMR1 assay, to p = 0.018 for the combined FMR1 3' and 5' mRNA analysis.ConclusionOur 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 FMR1 mRNA toxicity in blood, it may also have more general implications for gene expression studies in fresh and archival tissues.
Highlights
Elevated levels of FMR1 mRNA in blood have been implicated in RNA toxicity associated with a number of clinical conditions
We defined the best predictor of target gene mRNA degradation, utilizing it for normalization in the whole blood RNA with a high variability of rRNA quality. In this sample set we examined the relationship between the FMR1 mRNA and the CGG expansion in a non-coding portion of this gene, and found that by incorporating the 3' and 5' mRNA analysis data from patients with small to intermediate CGG expansions, we have significantly improved upon the current approach used to examine the FMR1 mRNA toxicity, demonstrating the clinical relevance of our strategy
Combining the analysis of 3' and 5' mRNA sites and GUS normalization minimizes the confounding impact of mRNA degradation in RNA samples from whole blood with highly variable rRNA quality We have previously demonstrated using freshly extracted RNA, that FMR1 expression was significantly elevated in carriers of CGG expansion, compared with normal controls of a similar age, and that the expression was proportional to the size of CGG expansions within the grey zone and lower premutation range [4]
Summary
Elevated levels of FMR1 mRNA in blood have been implicated in RNA toxicity associated with a number of clinical conditions. Fragile X Tremour Ataxia Syndrome (FXTAS) is the most prevalent disorder associated with PM alleles occurring in 50% of all older carrier males [5] It is progressive neurodegenerative disorder manifesting with ataxia and/or intension or other tremors, cognitive decline, psychiatric involvement and characteristic MRI and histopathological features [5,6,7,8]. In humans the level of mRNA is usually assessed in whole blood, there is a significant correlation between FXTAS associated with these levels and the typical neurological [12], and psychiatric [13] manifestations This implies that the assessments of FMR1 mRNA levels in carriers of small CGG expansions, apart from being essential in the mechanisms involved in RNA toxicity, may have a diagnostic and prognostic significance
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