DNA and RNA extraction and quantification
Nucleic acid extraction is performed on a variety of sample types including fresh and frozen whole blood, dried blood spots, purified cells, and cell-free body fluids. Manual extraction and enrichment using commercial kits or in-house developed protocols are available. For example, we have optimized protocols for extraction of Trypanosoma cruzi parasite DNA from blood using guanidine HCl lysis, HemoBind™ buffer and target capture; Babesia microti parasite DNA from blood using freeze-thaw lysis and differential centrifugation; and genomic DNA from leukocytes using proteinase K lysis. Automated nucleic acid extraction is available using the Promega Maxwell® 16 System. Quantification and quality control can be done by spectrophotometry using NanoDrop™, by electrophoresis using Agilent 2100 BioAnalyzer, by fluorescence using PicoGreen®, or by qPCR using in-house validated assays.
One-step real-time RT-PCR is available using commercial kits. For two-step real-time RT-PCR, we have developed protocols for cDNA synthesis followed by amplification using in-house optimized buffers. We perform qPCR in 96-well or 384-well formats using two LightCycler 480 Systems (Roche) and one 7500 Real-Time PCR System (Applied BioSystems). The Molecular Transfusion Core has designed hundreds of SYBR Green and TaqMan probe based assays for viral, parasitic, human, murine, and primate gene targets. Custom primer/probe assay design can also be performed for gene expression, SNP genotyping, and multiplex detection.
Results are reported based on raw Ct values, quantification relative to standard curves, or relative quantification based on 2^-dCt method. We provide assistance with generating figures and slides and writing text for manuscripts and presentations.