Project description:Goal: Determine differences in gene expression profile between microglia and macrophages isoalted from the brains of mice during viral-induced neuroinflammation. Methods: microglia and macrophages were obtained by FACS cells sorting (based on CD45 and CD11b level of expression) from brains of C57BL/6J mice, 6 days post i.c infection with TMEV or injection with PBS. RNA was extracted with TRYzol and precipitated using glycogen. polyA mRNA was enriched using oligo d(T)25 magnetic beads Smarter Stranded RNA-Seq Kit was then utilized to prepare cDNA library for RNA-seq. Results: Using RNA-seq and later RT-qPCR to validate our candidates, we found genes that are speifically expressed by activated microglia or infiltrating macrophages during viral-induced neuroinflamamtion
Project description:Transcription profiling of brains from mice fed with or without creatine supplementation to study the molecular mechanism of creatine improving health and life span in mice
Project description:CETP transfers lipids and cholesteryl esters between lipoproteins. This leads to elevated LDL-cholesterol levels. We found elevated cholesterol levels in the brains CETP transgenic animals. We wanted to test whether this is due to increased cholesterol synthesis by astrocytes 2 animals that were genotyped as wild type showed CETP transcription in our RT-qPCR (WT_1 & WT_5) and were excluded
Project description:Arabidopsis thaliana has been used regularly as a model plant in gene expression studies on transcriptional reprogramming upon pathogen infection, such as that by Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), or when subjected to stress hormone treatments including jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA). RT-qPCR has been extensively employed to quantitate these gene expression changes. However, the accuracy of the quantitation is largely dependent on the stability of the expressions of reference genes used for normalization. Recently, RNA-seq has been widely used to mine stably expressed genes for use as references in RT-qPCR. However, the amplification step in RNA-seq creates an intrinsic bias against those genes with relatively low expression levels, and therefore does not provide an accurate quantification of all expressed genes. In this study, we employed mass spectrometry-based label-free quantification (LFQ) in proteomic analyses to identify those proteins with abundances unaffected by Pst DC3000 infection. We verified, using RT-qPCR, that the levels of their corresponding mRNAs were also unaffected by Pst DC3000 infection. In addition, using RT-qPCR, we verified that the mRNAs were stably expressed upon stress hormone treatments including JA, SA, and ABA. Results indicated that the candidate genes identified here had stable expressions upon these stresses and are suitable to be used as reference genes for RT-qPCR. Among the 18 candidate reference genes reported in this study, many of them had greater expression stability than the commonly used reference genes, such as ACT7, in previous studies. Here, besides proposing more appropriate reference genes for Arabidopsis expression studies, we also demonstrated the capacity of mass spectrometry-based LFQ to quantify protein abundance and the possibility to extend protein expression studies to the transcript level.
Project description:Transcriptomic profiling of the brains of WNV and CHIKV infection-induced encephalitis in young mice after 2, 3 or 5 days post infection