Gene Expression Analysis from Human Peripheral Blood Mononuclear Cells

Abstract

Transcriptomic analysis or gene expression profiling is gaining importance in molecular biology research. Transcriptomics deals with studying the transcriptome or complete set of RNA transcripts produced in the specific cell at a particular time under some specific condition using high-throughput (DNA microarray) and low through put screening methods. In cells, DNA is the hereditary material which not only transfers genetic information from one generation to another but also codes for major proteins required for cell survival and communication. DNA is transcribed to RNA (mRNA is produced after processing of nascent or hnRNA) and mRNA then translates into proteins. The process of transcription and translation are referred as central dogma. Thus, the analysis of expression of genes can be done at either the level of transcript (analyzing RNA) or at the level of protein (analysis of proteins). In multicellular organisms, expression of genes and thus of proteins is under spatial (different in different cells and tissues) and temporal (different expression at different times) regulation. In transcriptomic/transcript analysis it is important to choose correct source and nature of the product, the resolution of expression and the number of genes that analyst intend to analyze at one point of time. In this, usually crude RNA/cDNA is used as a source material. Expression in tissue section or cells can be assessed by tissue/cell in situ hybridization. Expression analysis in isolated RNA can be done by northern blot hybridization, RNA dot-blot hybridization, or real-time PCR/qPCR. These are low-through put expression screens which analyze expression of only one gene or a very small number of genes at a time. In this chapter, the readers would learn about isolation of total RNA from peripheral blood mononuclear cells, agarose gel electrophoresis for quality check of RNA, cDNA synthesis, and real-time PCR reaction. These experiments can be undertaken when the researcher needs to study gene expression in disease versus control or in response to stress/stimuli.