Biology Reference
In-Depth Information
MicroRNA expression levels can be studied using several
different methods, such as microarray analysis, northern blots,
in situ hybridization, or real-time PCR with TaqMan or SYBR
Green chemistries. While each of these techniques can successfully
be used to investigate miRNA expression, this chapter focuses on
the use of real-time PCR with TaqMan as it is a specifi c, sensitive,
and accurate method for assessing miRNA expression, allowing
direct analysis in a single cell without the need for nucleic acid
purifi cation (
5
).
Higuchi et al. (
6, 7
) pioneered the use of a “real-time” system
to analyze PCR kinetics using ethidium bromide as a fl uorescent
reporter. Real-time PCR is able to monitor the progress of the
PCR as it occurs (i.e., in real time) as opposed to the PCR endpoint
detection (commonly known as reverse transcription (RT) PCR, or
RT-PCR, not to be confused with “real-time PCR”). This completely
revolutionizes the approach to PCR-based quantitation of DNA
and RNA. In real-time PCR, reactions are characterized from the
point in time during thermocycling when amplifi cation of a target
is fi rst detected. This is in contrast to traditional PCR in which a
single measurement of target product is made after a fi xed number
of thermocycles.
So far, two types of commercialized real-time PCR fl uorescent
reporters have been developed, TaqMan and SYBR Green. The
primary difference in the two chemistries is that TaqMan-based
real-time PCR offers specifi c hybridization between probe and
target, whereas the SYBR Green-based assays detect any double-
stranded DNA. Since no specifi c probes are required in SYBR
Green assays, they are generally more simple to set up and are run
at a lower cost, but the lack of target specifi city can lead to false
positive signals. The specifi city of TaqMan probes in real-time PCR
is high enough to discriminate among related miRNAs that differ
by as little as one nucleotide (
5
). Furthermore, TaqMan real-time
PCR assays are not affected by genomic DNA contamination (
5
).
Accurate quantifi cation is achieved routinely with as little as 25 pg
of total RNA for most miRNA (
5
).
In this chapter, we describe the use of TaqMan miRNA arrays
from Applied Biosystems to detect and measure miRNA in the
blood and brain of rats. Each microfl uidics PCR card includes 384
wells pre-plated with primers and probes specifi c for one miRNA in
each well. In addition, TaqMan real-time PCR can also be used for
individual assay of miRNA species. The general procedure we
describe for the real-time PCR of TaqMan miRNA arrays can be
applied to individual TaqMan real-time PCR assays in which the
appropriate primers and probes are used.
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