Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Summary

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Abstract

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)¹. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)². Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers³. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation⁴ and pathologies such as inflammation⁵. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages⁶ and microglia⁷. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Protocol

1. Isolation of Microglia from Normal CNS

Isolation of microglia is performed as was described previously with modifications³.

1. Prepare instruments for dissection and perfusion: scissors, forceps, 10 ml syringe with 27g needle, 15 ml Dounce homogenizer (Teflon/glass), 40 μm nylon cell strainer, 40% and 70% Percoll solutions, 1X PBS.
2. Euthanize mice by quick asphyxiation (deprivation of oxygen) in CO₂ camera and immediately transfer them to laboratory bench for surgical manipulations and perfusion.
3. Perform surgical manipulations (use scissors and forceps and 70% ethanol as an antiseptic): o....

Discussion

Recently significant interest was given to microglia and involvement of these cells in many pathologies associated with neuroinflammation and neurodegeneration. In addition, the role of microRNAs in the differentiation of immune and cancer cells has dramatically grown over the last several years^5,10. We have previously reported the role of miR-124 in the maintenance of non-activated or resting phenotype of microglia in normal CNS⁷, but the role of the other types of microRNAs in microglia phenotype .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1X PBS	GIBCO	14190	Can be prepared from dry components in the laboratory, pH: 7.2-7.4
10X PBS	Thermo Scientific	SH30258.02	Can be prepared from dry components in the laboratory, pH: 7.2-7.4
DMEM; FBS	GBCO	11965; 10438
miRVana kit	Invitrogen	AM1561
Percoll	Sigma	P4937-500 ml	100 ml of 100% Percoll is prepared by mixing 10 ml of 10X PBS and 90 ml of Percoll from Sigma. 50 ml of 70% Percoll is prepared by mixing of 35 ml of 100% Percoll and 15 ml of DMEM 50 ml of 40% Percoll is prepared by mixing 20 ml of 100% Percoll and 30 ml of 1X PBS
MOG35-55 peptide	AnaSpec Inc	60130-5
CFA	DIFCO	263810
Pertussis Toxin	Sigma	P7208
FcR blocking antibodies	BD Biosciences	553142	Clone 2.4G2
Anti-CD11b-PE mAb	BD Biosciences	553311	Can be used with different fluorophores (e.g. AF488)
Anti-CD45-APC-Cy7 mABs	Biolegend	103116	Can be used with different fluorophores (e.g. APC)
Paraformaldehyde (16%)	EMS Inc	15710	Dilute 1:15 in 1X PBS
15% TBE-Urea Gel	Life Technologies Inc.	EC68855BOX
TaqMan MicroRNA Reverse Transcription Kit	Life Technologies Inc	4366596
TaqMan Universal PCR Master Mix	Life Technologies Inc	4304437
TaqMan MicroRNA Assays mmu-miR-124a	Life Technologies Inc	4427975 ID 001182
TaqMan MicroRNA Assays snoRNA-55	Life Technologies Inc	4427975 ID 001228
Nuclease-free water	Life Technologies Inc	AM9937	Nuclease-free water
384-well clear optical reaction plate	Life Technologies Inc.	4309849	Can be substituted with other plates (e.g. 96 well plate) in different real-time PCR instruments
Dounce homogenizer (15 ml)	Wheaton Science Products.	358044	Teflon/glass
40 μ nylon cell strainer	Falcon	352340
Big centrifuge	Sorval	RT 6000B	Rotor diameter 18.5 cm
Small centrifuge	Eppendorf	5415 R	Rotor diameter 6.5 cm
Real-time PCR Instrument	Life Technologies Inc.	AB 7900 HT	Can be substituted with other instruments
Flow cytometer	BD Biosciencess	LSR II	Can be substituted with other instruments
FACS	BD Biosciences	FACSAria	Can be substituted with other instruments
Nanodrop spectrophotometer	Thermo Scientific	Nanodrop 1000