MicroRNA Expression Profi ling of Rat Blood and Brain Tissues: TaqMan Real-Time PCR MicroRNA Assays - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 162

4. Other
Considerations for
Using TaqMan
Real-Time PCR for
miRNA Analysis

Studies have demonstrated that the TaqMan real-time PCR method
can be used to detect transcriptional differences as early as 3 h and
as late as several months after stroke ( 1-4 ).

4.1. Timing for
Detection of miRNA
Expression with
Real-Time PCR After
Stroke

An miRNA expression profi le can be generated with larger amounts
of RNA sample (350-1,000 ng); however, if the isolated total RNA
from a sample is in the range of 1-350 ng, a PA reaction (described
in Sects. 2.5 / 3.5 of this chapter) is required.

4.2. Quantity of Total
RNA Sample

4.3. DNA Contamination
of RNA Sample

Though DNA contamination would not affect TaqMan miRNA
array studies, it is recommended to remove the trace DNA using
on column RNase-Free DNase Set (Cat. #: 79254, Qiagen), fol-
lowed with RNeasy MinElute Cleanup Kit (Cat. #: 74204, Qiagen).
Since total RNA is isolated, the researcher has the opportunity to
investigate both miRNA and mRNA (gene) expression from the
same RNA sample ( 10 ). Microarray analysis (the most common
method of the latter assay) requires DNA-free RNA input.

References

1. Jeyaseelan K, Lim KY, Armugam A (2008)
MicroRNA expression in the blood and brain
of rats subjected to transient focal ischemia by
middle cerebral artery occlusion. Stroke
39:959-966
2. Dharap A, Bowen K, Place R et al (2009)
Transient focal ischemia induces extensive tem-
poral changes in rat cerebral microRNAome.
J Cereb Blood Flow Metab 29:675-687
3. Liu DZ, Tian Y, Ander BP et al (2010) Brain
and blood microRNA expression profi ling of
ischemic stroke, intracerebral hemorrhage,
and kainate seizures. J Cereb Blood Flow
Metab 30:92-101
4. Tan KS, Armugam A, Sepramaniam S et al
(2009) Expression profi le of MicroRNAs in
young stroke patients. PLoS One 4:e7689
5. Chen C, Ridzon DA, Broomer AJ et al (2005)
Real-time quantifi cation of microRNAs by stem-
loop RT-PCR. Nucleic Acids Res 33:e179

6. Higuchi R, Dollinger G, Walsh PS, Griffi th R
(1992) Simultaneous amplifi cation and detec-
tion of specifi c DNA sequences. Biotechnology
(NY) 10:413-417
7. Higuchi R, Fockler C, Dollinger G, Watson R
(1993) Kinetic PCR analysis: real-time monitor-
ing of DNA amplifi cation reactions.

Biotechnology (NY) 11:1026-1030
8. Schmittgen TD, Lee EJ, Jiang J et al (2008)
Real-time PCR quantifi cation of precursor and
mature microRNA. Methods 44:31-38
9. Gusev Y (2008) Computational methods for
analysis of cellular functions and pathways col-
lectively targeted by differentially expressed
microRNA. Methods 44:61-72
10. Liu DZ, Ander BP, Tian Y et al (2012)
Integrated analysis of mRNA and microRNA
expression in mature neurons, neural progeni-
tor cells and neuroblastoma cells. Gene
495:120-127