Simultaneously extracting DNA, RNA, and protein using kits: is sample quantity or quality prejudiced?
Interdisciplinary "omics" research and the stringent quality requirements of array-based technologies require the simultaneous yet efficient extraction of DNA, RNA, and protein from the same tissue block. However, the few commercially available simultaneous extraction kits have not been evaluated. We compare the TriplePrep (GE Healthcare) and AllPrep (Qiagen) kits using good, intermediate, and poor quality tissue with specialist single-extract methods: Puregene (DNA), RNeasy (RNA), and homogenizations into buffer (protein). The following parameters were evaluated: DNA-yield (total DNA and double-stranded), purity (260:280 and 260:230), and integrity (gel electrophoresis); RNA-yield, purity, and integrity (RNA integrity numbers [RINs] and quantitative reverse transcription polymerase chain reaction [Q-RT-PCR]); protein-yield and quality (two-dimensional difference gel electrophoresis [2D-DIGE]). Puregene DNA yields were 183% and 506% those of TriplePrep and AllPrep, respectively. For RNA, AllPrep and RNeasy were indistinguishable, but their yields were 412% to 588% those of TriplePrep (depending on block condition) and their between-sample variability was better. TriplePrep protein yields were 57% those of the control, and 6.9% of the gel spots were more than 2-fold altered. However, AllPrep yields were 20% of the control, with 11% of the gel spots being more than 2-fold altered. Therefore, TriplePrep outperformed AllPrep in DNA and protein extractions, the reverse was true for RNA, but neither kit achieved optimal efficiency because both yield and quality were compromised.
Department of Surgery and Cancer, Imperial College London, London W12 0NN, UK. bill.mathieson@imperial.ac.uk
William
W
Mathieson
Gerry A
GA
Thomas
D000818:Animals; D002021:Buffers; D005591:Chemical Fractionation / Q000379:methods*; D019295:Computational Biology; D004247:DNA / Q000302:isolation & purification*; D006801:Humans; D008099:Liver / Q000737:chemistry; D061986:MCF-7 Cells; D011506:Proteins / Q000302:isolation & purification*; D011786:Quality Control; D012313:RNA / Q000302:isolation & purification*; D051381:Rats; D012015:Reference Standards; D013997:Time Factors
D002021:Buffers; D011506:Proteins; D012313:RNA; D004247:DNA