RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies

Abstract Background The development of protocols for RNA extraction from paraffin-embedded samples facilitates gene expression studies on archival samples with known clinical outcome. Older samples are particularly valuable because they are associated with longer clinical follow up. RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue is problematic due to chemical modifications and continued degradation over time. We compared quantity and quality of RNA extracted by four different protocols from 14 ten year old and 14 recently archived (three to ten months old) FFPE breast cancer tissues. Using three spin column purification-based protocols and one magnetic bead-based protocol, total RNA was extracted in triplicate, generating 336 RNA extraction experiments. RNA fragment size was assayed by reverse transcription-polymerase chain reaction (RT-PCR) for the housekeeping gene glucose-6-phosphate dehydrogenase (G6PD), testing primer sets designed to target RNA fragment sizes of 67 bp, 151 bp, and 242 bp. Results Biologically useful RNA (minimum RNA integrity number, RIN, 1.4) was extracted in at least one of three attempts of each protocol in 86–100% of older and 100% of recently archived ("months old") samples. Short RNA fragments up to 151 bp were assayable by RT-PCR for G6PD in all ten year old and months old tissues tested, but none of the ten year old and only 43% of months old samples showed amplification if the targeted fragment was 242 bp. Conclusion All protocols extracted RNA from ten year old FFPE samples with a minimum RIN of 1.4. Gene expression of G6PD could be measured in all samples, old and recent, using RT-PCR primers designed for RNA fragments up to 151 bp. RNA quality from ten year old FFPE samples was similar to that extracted from months old samples, but quantity and success rate were generally higher for the months old group. We preferred the magnetic bead-based protocol because of its speed and higher quantity of extracted RNA, although it produced similar quality RNA to other protocols. If a chosen protocol fails to extract biologically useful RNA from a given sample in a first attempt, another attempt and then another protocol should be tried before excluding the case from molecular analysis.

Computational simulation of the flow-induced vibration of a circular cylinder subjected to wake interference

In this work, we considered the flow around two circular cylinders of equal diameter placed in tandem with respect to the incident uniform flow. The upstream cylinder was fixed and the downstream cylinder was completely free to move in the cross-stream direction, with no spring or damper attached to it. The centre-to-centre distance between the cylinders was four diameters, and the Reynolds number was varied from 100 to 645. We performed two- and three-dimensional simulations of this flow using a Spectral/hp element method to discretise the flow equations, coupled to a simple Newmark integration routine that solves the equation of the dynamics of the cylinder. The differences of the behaviours observed in the two- and three-dimensional simulations are highlighted and the data is analysed under the light of previously published experimental results obtained for higher Reynolds numbers.

Avaliação comparativa entre custos dos processos MBBR/IFAS e lodo ativado para o tratamento de esgoto sanitário

O processo MBBR/IFAS, Moving Bed Biofilm Reactor/Integrated Fixed Film Activated Sludge, é uma tecnologia recente para o tratamento de esgoto que incorpora tanto biomassa em suspensão como aderida. Seus parâmetros de controle são os mesmos aplicados ao processo de lodo ativado, observadas algumas especificidades. Como objetivo principal deste trabalho, apresenta-se uma análise comparativa entre os custos de implantação de uma unidade IFAS e de lodo ativado operando sob alta taxa, na faixa convencional e com aeração prolongada, para populações de 50.000 e 500.000 habitantes. Os resultados mostraram que, sob as hipóteses assumidas para a implantação dos reatores, o custo do processo de lodo ativado foi de 36% até 100% do custo do processo IFAS, dependendo da carga orgânica aplicada, do preço do terreno ou do custo do meio suporte.