Levels of 5' RNA tags in plasma and buffy coat from EDTA blood increase with time
1CIGMR, Manchester Interdisciplinary Biocentre (MIB), The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK.
2Analytical Sciences, ISAS, Bunsen Kirchhoff Strasse 11, 44139 Dortmund, Germany.
3Centre for Integrated Genomic Medical Research, (CIGMR), The University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK.
4UK Biobank, Units 1&2 Spectrum Way, Adswood, Stockport, Cheshire SK3 0SA, UK.
* Corresponding author. E-mail: tim.peakman{at}ukbiobank.ac.uk
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Abstract |
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Background For biological sample banking it is important to precisely document sample treatment prior to extraction and storage. A major variable is the interval between blood sampling and subsequent processing and storage. We have determined the relationship between this time interval and frequency of 5' transcript tags. This study was designed to establish guidelines for collecting RNA from blood in prospective studies and ensure maximum availability of RNA analytes.
Methods Venous blood was collected from 40 healthy volunteers. Samples were processed immediately, 12, 24 and 36 h post collection and buffy coat and/or plasma removed. Total RNA was extracted and reverse transcribed, assays were optimized and levels of 5' RNA tags quantified by qPCR.
Results Stably expressed reference genes were selected to examine 5' tags in plasma and buffy coat blood fractions. Whole blood was processed at various time points post collection to determine the affect on the presence and stability of 5' RNA tags. A significant increase (P < 0.05 to P < 0.001) in 5' RNA tags was observed at 12 h and up to 36 h in plasma and buffy coat samples isolated from EDTA blood which was maintained at 4°C prior to processing when compared with plasma and buffy coat isolated from EDTA blood processed immediately.
Conclusions Over time 5' RNA tags increase in both plasma and buffy coat samples. It has been previously shown that removing cells from their normal environment produces cellular activation and up-regulation of pathways resulting in increased transcript expression. Positive correlation was observed between the time interval from sample collection to storage and amount of 5' transcript tags present. This increase could be due to white blood cells undergoing necrosis and lysis, or from RNA protected within apoptotic bodies. As 5' RNA tags were targeted using random primers for reverse transcription, even RNA partly degraded by RNases would have been detected.
Keywords Plasma, buffy coat, RNA, gene expression, real-time PCR
Accepted 10 December 2007
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