A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries.

RNA sequencing (RNA-seq)-based whole transcriptome analysis (WTA) using ever-evolving next-generation sequencing technologies has become a primary tool for coding and/or noncoding transcriptome profiling. As WTA requires RNA-seq data for both coding and noncoding RNAs, one key step for obtaining high-quality RNA-seq data is to remove ribosomal RNAs, which can be accomplished by using various commercial kits. Nonetheless, an ideal rRNA removal method should be efficient, user-friendly and cost-effective so it can be adapted for homemade RNA-seq library construction. Here, we developed a novel reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) method. We demonstrated that RTR2D was simple and efficient, and depleted human or mouse rRNAs with high specificity without affecting coding and noncoding transcripts. RNA-seq data analysis indicated that RTR2D yielded highly correlative transcriptome landscape with that of NEBNext rRNA Depletion Kit at both mRNA and lncRNA levels. In a proof-of-principle study, we found that RNA-seq dataset from RTR2D-depleted rRNA samples identified more differentially expressed mRNAs and lncRNAs regulated by Nutlin3A in human osteosarcoma cells than that from NEBNext rRNA Depletion samples, suggesting that RTR2D may have lower off-target depletion of non-rRNA transcripts. Collectively, our results have demonstrated that the RTR2D methodology should be a valuable tool for rRNA depletion.

Influence of comorbidity on self-assessment instrument scores of patients with idiopathic adhesive capsulitis.

BACKGROUND: The purpose of this study was to determine whether comorbid factors influence the results of self-assessment instruments completed by patients with idiopathic adhesive capsulitis of the shoulder. We hypothesized that an increased number of comorbidities would be correlated with greater pain and worse function as measured by general and shoulder-specific outcome tools. METHODS: One hundred consecutive patients with phase-II adhesive capsulitis were prospectively evaluated with use of a general health questionnaire, a visual analog pain scale, the Simple Shoulder Test, the Disabilities of the Arm, Shoulder and Hand questionnaire, and the Short Form-36. There were seventy-one women and twenty-nine men, with a mean age of fifty-two years (range, thirty-six to eighty-two years). Comorbidities included medical factors (e.g., diabetes and heart disease) and social factors (e.g., tobacco use and pending litigation). Linear regression analysis was performed to determine correlations between the number of comorbidities and the results of the questionnaires. RESULTS: Patients with more comorbidities had significantly lower scores on the Disabilities of the Arm, Shoulder and Hand Questionnaire (p = 0.0005) and the Short Form-36 subscale of physical function (p = 0.0009) as well as poorer scores on the Simple Shoulder Test and the Short Form-36 subscales of physical role, social function, emotional role, and mental health. Although there was no correlation between increased comorbidity and pain as measured on the visual analog scale, the comfort/pain subscale of the Short Form-36 showed a significant correlation with increased comorbidity (p = 0.004). CONCLUSIONS: Idiopathic adhesive capsulitis is a debilitating condition. Comorbid factors have a significant effect on the pain and dysfunction (as measured on shoulder-specific and general health instruments) experienced by patients with this disorder. These findings can be applied to the evaluation of these patients and may help to identify patients who potentially require a longer treatment course or those whose outcome will be less satisfactory.