Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens

Background: Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffinembedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens. Principal Findings: For effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll TM Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and-RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and –RNA tha

Herd immunity against rubella according to a survey of the population in Medellin, Colombia

Objective. Calculate the critical proportion (P-c) for achieving herd immunity based on a 2009 population study conducted in Medellin, Colombia, by age, globally and disaggregated by sex, location, and socioeconomic stratum. Methods. A survey of seroprevalence in the population was conducted by means of a random sample of 2 124 individuals aged 6 to 64 that was representative of age, sex, and location. The basic reproduction number was estimated using a quadratic regression of the average IgG titers for rubella by age in unvaccinated individuals with titers greater than or equal to 15 IU/ml. The effective reproduction number (R-e) was calculated with the data on the weighted proportion of protection by age, sex, location, and socioeconomic stratum. Results. Overall, the P-c was 90.0% (95% CI, 88.6-95.2%) and the R-e was 0.95 (95% CI, 0.8-1.8), for a weighted proportion of protection of 89.4% (95% CI, 86.891.6%). Protection was lower than the expected P-c in both sexes, in high and low socioeconomic strata, and in the rural area. In the urban area, protection was greater than the P-c (89.4%, with a 95% CI, 86.6-91.7%, compared to 87.4% and a 95% CI, 85.2-87.8%). Conclusions. The urban area has made progress toward herd immunity, but the overall proportion of protection in women, the rural area, and the high socioeconomic strata must be increased. The effective number may be greater than one, indicating the potential for the spread of the disease