title: Measuring microRNA expression in mouse hematopoietic stem cells.
abstract: MicroRNAs (miRNAs) are important regulators of diverse biologic processes. In the hematopoietic system, miRNAs have been shown to regulate lineage fate decisions, mature immune effector cell function, apoptosis, and cell cycling, and a more limited number of miRNAs has been shown to regulate hematopoietic stem cell (HSC) self-renewal. Many of these miRNAs were initially identified as candidate regulators of HSC function by comparing miRNA expression in hematopoietic stem and progenitors cells (HSPCs) to their mature progeny. While the measurement of miRNA expression in rare cell populations such as HSCs poses practical challenges due to the low amount of RNA present, a number of techniques have been developed to measure miRNAs in small numbers of cells. Here, we describe our protocol for measuring miRNAs in purified mouse HSCs using a highly sensitive real-time quantitative PCR strategy that utilizes microfluidic array cards containing pre-spotted TaqMan probes that allows the detection of mature miRNAs in small reaction volumes. We also describe a simple data analysis method to evaluate miRNA expression profiling data using an open-source software package (HTqPCR) using mouse HSC miRNA profiling data generated in our lab.
authors: Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
authors: Wenhuo
authors: W
authors: Hu
authors: Christopher Y
authors: CY
authors: Park
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D005434:Flow Cytometry; D020869:Gene Expression Profiling / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D008297:Male; D051379:Mice; D035683:MicroRNAs / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D048348:Reverse Transcription; D012984:Software; D013223:Statistics as Topic; D019914:Taq Polymerase / Q000378:metabolism
chemical_list: D035683:MicroRNAs; D019914:Taq Polymerase