Quantitative PCR methods for RNA and DNA in marine sediments: maximizing yield while overcoming inhibition.
For accurate quantification of DNA and RNA from environmental samples, yield loss during nucleic acid purification has to be minimized. Quantitative PCR (qPCR) and reverse transcription (RT)-qPCR require a trade-off between maximizing yield and removing inhibitors. We compared DNA and RNA yield and suitability for quantitative SYBR Green PCR and RT-PCR using the UltraClean and PowerSoil extraction kits and a bead-beating protocol with phenol/chloroform extraction steps. Purification methods included silica-column-based procedures from the MoBio kits, RNeasy MinElute, WizardPlus miniprep columns, and an acrylamide gel extraction. DNA and RNA purification with WizardPlus and RNeasy, respectively, led to significant losses of nucleic acids and archaeal 16S rRNA or 16S rRNA gene, as measured with RiboGreen or PicoGreen, and RT-qPCR or qPCR. Extraction and purification of DNA with the MoBio DNA UltraClean and DNA PowerSoil kits also decreased the yields slightly, relative to gel purification, in all sediments, except those from the deep sea in the Gulf of Mexico. Organic matter in humic-rich sediments may bind to these silica columns, reducing their nucleic acid-loading capacity. Purification with gel extraction cleans up organic-rich sediment samples sufficiently for quantitative analysis while avoiding the yield loss associated with commonly used silica columns.
Lloyd
Karen G
KG
Department of Marine Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. karen.lloyd@biology.au.dk
Macgregor
Barbara J
BJ
Teske
Andreas
A
D004247:DNA / Q000032:analysis*; D019641:DNA, Archaeal / Q000032:analysis; D004269:DNA, Bacterial / Q000032:analysis; D016521:Electrophoresis, Gel, Pulsed-Field; D020459:Genes, rRNA; D019015:Geologic Sediments / Q000382:microbiology*; D019800:Phenol; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000032:analysis*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D019641:DNA, Archaeal; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D019800:Phenol; D012313:RNA; D004247:DNA