Quality Assessment of Biobanked Nucleic Acid Extracts for Downstream Molecular Analysis

Abstract

Sample quality is of major importance when conducting molecular analysis of nucleic acids, and factors such as degradation, presence of impurities, and enzymatic inhibitors may have a significant impact on the quality of data. Issues of quality assessment become more important as the increased use of biobanking means that whole blood samples are being stored for longer periods. A range of commercially available kits/methods have been specifically developed for extraction of high quality nucleic acids from a variety of clinical samples, including blood, but there is limited information on how best to quality assess the extracts to determine their fitness for purpose in specific downstream applications. In this study, we have performed nucleic acid extractions from stored blood using a number of different methods. The resulting extracts were analyzed by a panel of quantity and quality metrics including UV spectrophotometry, PicoGreen fluorescence, electrophoresis, and a PCR approach. To evaluate the relevance of different metrics, DNA samples were subsequently assessed for their performance in real time PCR and microarray experiments. Our findings demonstrate that the most suitable extraction technique and quality assessment approach depends on the required downstream analytical method.

The Critical Role of DNA Extraction for Detection of Mycobacteria in Tissues

“…DNA purity was determined by the ratios 260 nm/280 nm (indicator of organic matter residues) and 260/230 nm (indicator of organic solvent residues) [27]. DNA amount was estimated at 260 nm by spectrophotometer (ND-1000 Spectrophotometer, NanoDrop).…”

Reduction of Systematic Bias in Transcriptome Data from Human Peripheral Blood Mononuclear Cells for Transportation and Biobanking

“…Although genomic DNA is stable against pre-analytical operations such as freezing and transportation [5], [6], RNA is less stable to conformational changes than DNA because RNA is subjected to spontaneous degradation at room temperature. To collect stabilized RNA from whole blood samples, the UK biobank (http://www.ukbiobank.ac.uk/) employs Tempus Blood RNA tubes (Life Technologies, Carlsbad, CA, USA) [7], while other biobank projects use the PAXgene RNA System (Becton Dickinson and Company, Franklin Lakes, NJ, USA) [8]. Whole blood components, such as plasma and heme-containing red blood cells, inhibit polymerase chain reaction (PCR) and reverse transcription [9], and high concentrations of globin mRNA in whole blood RNA interfere with accurate transcriptome analysis [10].…”

Aperfeiçoamento de uma técnica para extrair DNA de água do mar e comparação de métodos de extração de DNA na caracterização de uma comunidade bacteriana marinha.

“…Em alguns protocolos está presente mais uma etapa de purificação, com clorofórmio: álcool isoamílico. Esta também provocará a desnaturação das proteínas que permanecerão na fase inferior orgânica (AHMAD et al, 2004;URAKAWA;MARTENS-HABBENA;STAHL, 2010;WAHLBERG et al, 2012).…”

“…No OLSON, 1992;TEBBE;VAHJEN, 1993). Este teste para verificar a integridade do DNA tem sido utilizado em muitos trabalhos (ALEKSIC et al, 2012;BOSTRÖM et al, 2004;HURT et al, 2001;LEAR;DONG;LEWIS, 2010;ROGERS et al, 2007;WAHLBERG et al, 2012).…”

“…Assim como o fenol, o clorofórmio também pode ser usado para desnaturar as proteínas. Dessa forma, quando estas duas soluções estão juntas, a desproteinização se torna mais eficiente do que quando as soluções são usadas separadamente (SAMBROOK; FRITSCH; MANIATIS, 1989) e, por isso, muitos trabalhos utilizam esta mistura orgânica para purificar o DNA(AHMAD et al, 2004;URAKAWA;MARTENS- HABBENA;STAHL, 2010;WAHLBERG et al, 2012).Quando o fenol:clorofórmio é adicionado na amostra, as proteínas desnaturam e formam uma camada na interface da fase orgânica e fase aquosa. É extremamente importante que o pH esteja entre 7,8-8,0 para o DNA permanecer na fase aquosa, pois em pH menor que 7,8 o DNA pode ir para a interface, chegando a ir para a fase orgânica quando o pH está menor que 7,0(OLIVEIRA et al, 2007;SAMBROOK;FRITSCH;MANIATIS, 1989).…”