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发表于 2012-5-17 17:06 |只看该作者 |倒序浏览 |打印

Real-time quantification of microRNAs by! ?3 f2 C: Y2 Y) t' T( `9 f+ Q
stem–loop RT–PCR
Caifu Chen*, Dana A. Ridzon, Adam J. Broomer, Zhaohui Zhou, Danny H. Lee,
Julie T. Nguyen, Maura Barbisin, Nan Lan Xu, Vikram R. Mahuvakar, Mark R. Andersen,8 E8 L4 }. l- X# L7 `3 V6 P% |0 J; N
Kai Qin Lao, Kenneth J. Livak and Karl J. Guegler
Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404, USA) f9 h- W3 {3 @3 C  R
Received May 24, 2005; Revised July 8, 2005; Accepted October 25, 2005" A: _6 e9 i; d  M6 l
ABSTRACT0 I5 t6 L+ o$ c* X1 `1 R0 c% X
A novel microRNA (miRNA) quantification method
has been developed using stem–loop RT followed
by TaqMan PCR analysis. Stem–loop RT primers are) m, e1 n7 ~% L; x
better than conventional ones in terms of RT efficiency
and specificity. TaqMan miRNA assays are specific
for mature miRNAs and discriminate among+ V6 D. V+ E1 ?" C
related miRNAs that differ by as little as one nucleotide.# s1 x8 u. c- c6 H5 c) o, I
Furthermore, they are not affected by genomic
DNA contamination. Precise quantification is
achieved routinely with as little as 25 pg of total
RNA for most miRNAs. In fact, the high sensitivity,
specificity and precision of this method allows for
direct analysis of a single cell without nucleic acid( v  S" @5 Y5 R& }' c! n
purification. Like standard TaqMan gene expression, y( s' V% M) P
assays, TaqMan miRNA assays exhibit a dynamic
range of seven orders of magnitude. Quantification
of five miRNAs in seven mouse tissues showed variation4 v1 U! R' H4 ?7 z- c$ U6 B/ q7 f
from less than 10 to more than 30 000 copies per" A; T% d# t( c% {  D" E& W
cell. This method enables fast, accurate and sensitive1 Z: n+ C: R8 V
miRNA expression profiling and can identify and3 ]4 ]: `  H/ }' u
monitor potential biomarkers specific to tissues or' z7 P% d0 \* x; q
diseases. Stem–loop RT–PCR can be used for the. U! v3 w7 v5 U0 ^+ k1 S9 n
quantification of other small RNA molecules such
as short interfering RNAs (siRNAs). Furthermore, the
concept of stem–loop RT primer design could be: ?5 t5 O/ t8 n
applied in small RNA cloning and multiplex assays
for better specificity and efficiency.
INTRODUCTION
MicroRNAs (miRNAs) are naturally occurring, highly conserved: Z! }# X0 v8 U3 N
families of transcripts (18–25 nt in length) that are# i" P8 q: y$ V. Q7 B, T* l9 }
processed from larger hairpin precursors (1,2). miRNAs are
found in the genomes of animals (3–9) and plants (10–12). To6 j7 l0 c, ?5 a
date, there are 1000 unique transcripts, including 326 human
miRNAs in the Sanger Center miRNA registry (13).+ q. V# Q4 p4 s. ^* y$ A
miRNAs regulate gene expression by catalyzing the cleavage6 r" O) W5 r0 X& {
of messenger RNA (mRNA) (14–19) or repressing mRNA
translation (19–21). They are believed to be critical in cell
development, differentiation and communication (2). Specific
roles include the regulation of cell proliferation and metabolism. x. o, f+ Z0 {! P2 H: C- A1 X, u9 _
(22), developmental timing (23,24), cell death (25),
haematopoiesis (26), neuron development (27), human$ B) `, n5 I6 ]4 r$ s+ [" l& V4 V
tumorigenesis (28) and DNA methylation and chromatin6 {9 L# O* }. M7 V, l( H7 F
modification (29).  k1 H& b8 \/ m6 I
Although miRNAs represent a relatively abundant class of
transcripts, their expression levels vary greatly among species
and tissues (30). Less abundant miRNAs routinely escape
detection with technologies such as cloning, northern hybridization* |' Y: b+ u1 d- y0 }
(31) and microarray analysis (32,33). Here, we present
a novel real-time quantification method for accurate and sensitive
detection of miRNAs and other small RNAs. This method
expands the real-time PCR technology for detecting gene
expression changes from macromolecules (e.g. mRNAs) to
micro molecules (e.g. miRNAs).
MATERIALS AND METHODS4 J4 [" [! J+ Q( X8 r* X
Targets, primers and probes (Supplementary Data)
Seventeen miRNA genes were selected from the Sanger2 W% c, `$ u2 G* H, e
Center miRNA Registry at http://www.sanger.ac.uk/Software/
Rfam/mirna/index.shtml. All TaqMan miRNA assays are
available through Applied Biosystems (P/N: 4365409). Standard
TaqMan assays for pri-miRNA precursors, pri-let-7a-3% o* f1 a. j! D, d- h4 C9 F- n: n
and pri-miR-26b and pre-miRNA precursor pre-miR-30a were( r* ^1 n/ P& b- p
designed using PrimerExpress software (Applied Biosystems,  v9 p% M+ Z  l! f* q9 J
Foster City, CA). All sequences are available in the  a3 \' m- Q% D2 @& G, a
section of the Supplementary Data. Synthetic miRNA oligonucleotides
were purchased from Integrated DNA Technologies3 T9 a# c# ~8 r$ u) x
Tissue RNA samples, cells, cell lysates and/ {. a5 ^. x7 r, K1 `
total RNA preparation! [) p+ R, J3 R' j7 F1 |
Mouse total RNA samples from brain, heart, liver, lung,
thymus, ovary and embryo at day 10–12 were purchased1 N7 O! P$ a8 T
from Ambion (P/N: 7810, 7812, 7814, 7816, 7818, 7824,% A: u; E1 ?! o7 N2 r
7826 and 7968). Ambion’s mouse total RNAs are derived
from Swiss Webster mice. All RNA samples were normalized3 C; L+ Z) M3 L  w% G! E
based on the TaqMan Gene Expression Assays for human or# E4 F& y. k' v
mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
endogenous controls (P/N: 4310884E and 4352339E, Applied
Biosystems).8 j" w$ I$ ]7 x; Z# o. q. E+ e( A
Two cell lines, HepG2 and OP9, were cultured using
Gibco MEM (P/N: 12492–021, Invitrogen, Carlsbad, CA)$ x1 ^- l) v" w0 J
supplemented with 10% fetal bovine serum (FBS) (P/N:& @" e+ K/ l% o
SH30070.01, HyClone, Logan, UT). Trypsinized cells were% S' s4 g' @- ~, {
counted with a hemocytometer. Approximately 2.8 · 106
suspended cells were pelleted by centrifugation (Allegra 6,
Beckman Coulter, Fullerton, CA) at 1500 r.p.m. for 5 min,: ^0 J2 l* Q5 y
washed with 1 ml Dulbecco’s phosphate-buffered saline (PBS)
without MgCl2 and CaCl2 (P/N: 14190078, Invitrogen, Carlsbad,
CA). The cell pellets were re-suspended in 140 ml PBS/ S: l% c4 T- z' ?
and processed with three different sample preparation methods.5 ~7 e( @9 V$ i
With the first method, a 50 ml sample (106 cells) was
mixed with an equal amount of Nucleic Acid Purification3 {" f  J* a9 ?8 s5 j3 w
Lysis Solution (P/N: 4305895; Applied Biosystems) by pipetting7 C& k7 @9 j: X( [* ~# o( o2 j
up and down 10 times, and then spun briefly. The lysate6 |8 E4 C" W; g& r8 C
was diluted 1/10 with 1 U/ml RNase inhibitor solution (P/N:
N8080119; Applied Biosystems) before adding the solution to
an RT reaction. In the second method, a 50 ml sample (106
cells) was used to purify total RNA using the mirVana
miRNA Isolation Kit (P/N: 1560, Ambion, Austin, TX)
according to the manufacturer’s protocol. Purified total
RNA was eluted in 100 ml of elution buffer. The third method* i3 n* p3 ^& u' S0 s3 `( h
involved diluting cells 1/2 with 1· PBS, heating at 95C for 5
min, and immediately chilling on ice before aliquotting directly% P: W% I( Q0 z- X& n, O2 B
into RT reactions.
miRNA detection using mirVana miRNA$ \$ v2 b' b# |# U0 L& h9 F! P
detection kit8 J; s# z! P! y# Z7 e( G2 _. ]
Solution hybridization-based miRNA analysis was carried out
using the mirVana miRNA Detection Kit (Cat. #: 1552,
Ambion) according to the manufacturer’s protocol. RNA* U; U" O6 G0 Z7 `, I0 q
probes were synthesized by IDT. The radioisotope labeled* j" Z3 l5 c1 ]
RNA fragments were detected and quantitated with a Cyclone
Storage Phosphor System (PerkinElmer, Boston, MA).- O" |0 c  a8 F5 ?  a! F
Reverse transcriptase reactions! H) P& l" a2 I' d/ Y  @
Reverse transcriptase reactions contained RNA samples6 A1 ~- _! z. P# V) ?5 f7 [
including purified total RNA, cell lysate, or heat-treated/ P# C' b; O# A9 @
cells, 50 nM stem–loop RT primer (P/N: 4365386 and3 t: u( U) ?6 |& A" @/ G. s$ l3 u# r
4365387, Applied Biosystems), 1· RT buffer (P/N:
4319981, Applied Biosystems), 0.25 mM each of dNTPs,
3.33 U/ml MultiScribe reverse transcriptase (P/N: 4319983,
Applied Biosystems) and 0.25 U/ml RNase inhibitor (P/N:
N8080119; Applied Biosystems). The 7.5 ml reactions were+ D7 J" Y( t$ o0 `! Y) F% S: ?  I
incubated in an Applied Biosystems 9700 Thermocycler in a
96- or 384-well plate for 30 min at 16C, 30 min at 42C, 5 min
at 85C and then held at 4C. All Reverse transcriptase reactions," j& `% f% c3 T0 t# ~$ R
including no-template controls and RT minus controls,2 O4 v7 c# R8 P+ y, x1 u" v# ]
were run in duplicate.- b2 ^' }6 f% O4 U
PCR" N9 x/ f' |* m/ _. d
Real-time PCR was performed using a standard TaqMan, l: p- n; j; P& O* ]
PCR kit protocol on an Applied Biosystems 7900HT Sequence
Detection System (P/N: 4329002, Applied Biosystems). The
10 ml PCR included 0.67 ml RT product, 1· TaqMan Universal" _& _. X# _" r& m
PCR Master Mix (P/N: 4324018, Applied Biosystems),4 d) S3 ]: k! D3 @" g
0.2 mM TaqMan probe, 1.5 mM forward primer and 0.7 mM
reverse primer. The reactions were incubated in a 384-well  {* L7 v: H" G3 [
plate at 95C for 10 min, followed by 40 cycles of 95C for 15 s/ U) ^1 ?# `0 h) J5 u4 ^
and 60C for 1 min. All reactions were run in triplicate. The
threshold cycle (CT) is defined as the fractional cycle number
at which the fluorescence passes the fixed threshold. TaqMan
CT values were converted into absolute copy numbers using a
standard curve from synthetic lin-4 miRNA.+ l: I. k0 Z$ u" F7 [
The method for real-time quantification of pri-miRNA# i, n; o( O# q7 `8 h/ v
precursors, let-7a-3 and miR-26b, and pre-miRNA precursor0 S7 T8 h& E# ?* p9 g  T
miR-30a was described elsewhere (34).
RESULTS6 U' z+ I% U2 u: ]+ z; i
We proposed a new real-time RT–PCR scheme for miRNA
quantification (Figure 1). It included two steps: RT and realtime, d. h. H3 L' N. I" {3 J- `
PCR. First, the stem–loop RT primer is hybridized to a! f- R* ?0 r$ [2 X
miRNA molecule and then reverse transcribed with a Multi-
Scribe reverse transcriptase. Next, the RT products are quantified/ w% o3 |( |# H" i/ J2 \' |
using conventional TaqMan PCR.
Figure 1. Schematic description of TaqMan miRNA assays, TaqMan-based, f$ l% V7 O1 y3 p% A( E
real-time quantification of miRNAs includes two steps, stem–loop RT and realtime
PCR. Stem–loop RT primers bind to at the 30 portion of miRNA molecules9 \3 k4 w4 d  j( A
and are reverse transcribed with reverse transcriptase. Then, the RT product is1 c! S1 e  r7 z
quantified using conventional TaqMan PCR that includes miRNA-specific
forward primer, reverse primer and a dye-labeled TaqMan probes. The purpose
of tailed forward primer at 50 is to increase its melting temperature (Tm). [, Q7 G8 `+ n
depending on the sequence composition of miRNA molecules.The dynamic range and sensitivity of the miRNA quantification
scheme were first evaluated using a synthetic cel-lin-4
target. Synthetic RNA was quantified based on the A260 value
and diluted over seven orders of magnitude. The cel-lin-48 D3 E6 V6 R" [$ x( K6 O
TaqMan miRNA assay showed excellent linearity between& U8 s9 C) B" m+ B$ f- p
the log of target input and CT value, demonstrating that the
assay has a dynamic range of at least 7 logs and is capable of5 D% ]. g' k% O; \! \
detecting as few as seven copies in the PCR reaction (Figure 2).
Eight additional miRNA assays were also validated using
mouse lung total RNA. The RNA input ranged from 0.025 to& p7 m, v9 B8 ]# z
250 ng (Figure 3). The CT values correlated to the RNA input% {2 y5 B! r7 x" {4 L( Z8 X
(R2 > 0.994) over four orders of magnitude. A negative control
assay, cel-miR-2, did not give a detectable signal, even in9 Z9 e& H4 L# e
reactions with 250 ng mouse total RNA.
The expression profile of five miRNAs was determined in
seven different mouse tissues to create a miRNA expression+ b; x' @9 @9 ~
map. The copy number per cell was calculated based on the
input total RNA (assuming 15 pg/cell) and the standard curve
of synthetic lin-4 target. Several interesting observations were# E2 O8 w/ [/ T! i  H" x
made from this expression map. First, miRNAs are very( W9 G! X: U) ?- l7 P& X
abundant, averaging 2390 copies per cell in these tissues.4 X& [3 M/ q' j# J
The level of expression ranged from less than 10 to 32 0904 n9 i1 O' p6 c9 A- ^+ Q1 L
copies per cell. Of the 12 miRNAs, miR-16 and miR-323 were
the most and least abundant miRNAs, respectively, across all
tissues. In addition, each tissue had a distinctive signature of
miRNA expression. The overall level of miRNA expression
was highest in mouse lung and lowest in embryos. Finally, the
dynamic range of miRNA expression varied greatly from less7 r3 M/ c, S! w/ G  _6 x7 R
than 5-fold (let-7a) to more than 2000-fold (miR-323) among
these seven tissues (Table 1).
To assess the need for RNA isolation, we added cell lysates
directly to miRNA assays. The equivalent of 2.5–2500 cells
were added directly to 7.5 ml RT reactions. When detected,
the CT values correlated (R2 > 0.998) to the number of cells inthe RT reactions over at least three orders of magnitude
(Figure 4).
The effect of non-specific genomic DNA on TaqMan
miRNA assays was also tested for 12 assays. Results showed5 [8 x" i' ^* \3 J6 f) M
no difference in CT values in the presence or absence of 5 ng of5 C+ f% I- L. v0 |4 Y
human genomic DNA added to the RT reactions, suggesting$ b3 r4 u/ A4 Y1 G5 Y
that the assays are highly specific for RNA targets (data not$ I/ L) i! W$ a5 c
shown). Based on this observation, we added heat-treated cells
directly to miRNA quantification assays. Figure 5 illustrates& X2 g4 c/ x& L0 j1 u0 x$ N8 q& E
the comparison of miRNA quantification using purified totalRNA, cell lysates and heat-treated cells derived from an equal  E- v0 O& V, m4 _
number of HepG2 cells. Adding heat-treated cells directly to3 N- i$ J" j' M+ |) t& m$ @' [1 J; M
the miRNA assays produced the lowest CT values, and good
concordance was observed among all three different sample' H( A1 z: `0 B
preparation methods.  D# \! b2 x' b$ A0 B; D1 T
The reproducibility of TaqMan miRNA assays was
examined by performing12 miRNA assays with 16 replicates
performed by two independent operators (data not shown).) ~; e; J6 y3 [8 i
The standard deviation of the CTs averaged 0.1, demonstrating# r' X# R! E% ]7 U+ O
the high precision of the assays.0 @1 b; o$ d( |7 P/ t: `
Solution hybridization-based miRNA northern analysis was# \" `/ B. G" ?+ O
used as an independent technology to compare with TaqMan
miRNA assays (Figure 6). We observed that hybridizationbased
miRNA analyses were less reproducible and that concordance( I1 s6 v5 M3 f9 \
with TaqMan assays varied from target to target.$ w  y9 X- S8 ~
There was a general concordance between the two methods
(R2 ¼ 0.916) for miR-16 across five mouse tissue samples.
However, correlations were relatively low for less abundant
miRNAs, such as miR-30 (R2 ¼ 0.751).
Hybridization methods can lack specificity for the mature
miRNAs. We investigated the ability of the TaqMan miRNA
assays to differentiate between the mature miRNAs and their
longer precursors, using synthetic targets for pri-miRNA precursors,# C. ]* z  h# C( i  |
pri-miR-26b and pri-let-7a and pre-miRNA precursor* |% C) t" W/ L/ j% M9 \: D' A
pre-miR-30a (Table 2). TaqMan assays designed to detect
either precursors or mature miRNAs were tested with synthetic
targets averaging 1.5 · 108 copies per RT reaction (1.3 · 107: H* l  s6 ]: p3 O- F* K9 K! q
copies per PCR). TaqMan miRNA analyses with only primiRNA* J1 p& K8 q) s
precursor molecules produced CT values at least( U1 \! a% d) U' }4 U; c
11 cycles higher than analyses with mature miRNA ones.
This result implies that if mature miRNA and precursor+ h# N! n( i: C; v
were at an equal concentration, the latter would contribute5 W0 V/ y( s6 N5 }1 ~+ ?
<0.05% background signal to the assay of mature target.
For pre-miR-30a where the mature miRNA miR-30a-3p is
located at the 30 end of the pre-miR-30a sequence, a differenceof 8.4 CT was observed. The results showed that TaqMan
miRNA assays are specific to mature miRNAs. However,
the assay specificity is better if the miRNA is located at the
50 strand of the pre-miRNA precursor. Experiments analyzing0 H6 ?( t9 |7 V4 U  V
total RNA instead of synthetic targets indicated that the precursors) b: n$ S. G* R2 ?* p! f
are at least two orders of magnitude less abundant than5 ^! \1 C4 C% ]+ \/ P- e
mature miRNAs, based on CT differences of 7 or more for5 K1 ]- e% M2 z; A) J6 Z* R& }
miR-26b-1 and let-7a-2 precursors. Considered together, these
results suggest that the TaqMan miRNA assays are highly4 C" C  f$ E' ~- A# _
specific for the mature miRNAs.
The ability of the TaqMan miRNA assays to discriminate; l7 \" f& E4 H$ z0 F1 W# Y
miRNAs that differ by as little as a single nucleotide was tested
with the five synthetic miRNAs of let-7a, let-7b, let-7c, let-7d& g5 k9 E, M! @$ ?9 O# g
and let-7e (Figure 7). Each miRNA assay was examined: R2 b& i. z( |2 a# ^# G# }" }4 D- N
against each miRNA. Relative detection efficiency was calculated; _4 V) C6 f. H! e' F7 W/ q
from CT differences between perfectly matched and
mismatched targets, assuming 100% efficiency for the perfect
match. Very low levels of non-specific signal were observed,8 w- s6 `3 t* B2 s0 [
ranging from zero to 0.3% for miRNAs with 2–3 mismatched
bases and only 0.1–3.7% for the miRNAs that differed by a
single nucleotide. Most cross-reactions resulted from G–T
mismatches during the RT reaction (let-7a assay versus let-
7c target etc.). Only the targeted miRNA was detected if more
than three mismatched bases between any two miRNAs were& |6 |) J! d3 _. D6 A* t
present.
We compared the discrimination ability of the TaqMan, h. G4 L# ~, w, d& T6 B4 p8 F/ G
miRNA assays to that of solution-based hybridization analysis
(Figure 8). In our hands, the hybridization method discriminated% K7 ]: c. D5 l- m3 l3 I
well between let-7a and let-7b. However, poor or no
discrimination was observed among let-7a, let-7c and let-! N9 Q2 D+ w' V
7d, which differ by 1–3 nt.$ F' x3 N+ @* a
We speculated that stem–loop primers might provide better
RT efficiency and specificity than linear ones. Base stacking of
the stemmight enhance the thermal stability of the RNA–DNA
heteroduplex. Furthermore, spatial constraint of the stem–loop
would likely improve the assay specificity in comparison to4 @1 P& @, U6 z, C4 [% {
conventional linear RT primers. We compared the sensitivity
and specificity of the stem–loop and linear RT primers using/ u9 _1 R8 P, B4 |
synthetic miRNAs for let-7a (Figure 9). We observed several! i8 }+ ~$ \5 x3 W9 H0 K1 y
advantages for the stem–loop RT. First, in the presence of the
synthetic let-7a target, the CT values between linear and stem–& s* z1 j9 o! [6 n' C
loop RT methods differed by 7, indicating that the efficiency of& u7 g' ?. m( s) n
stem–loop RT was at least 100 times higher. Secondly, stem–
loop RT discriminated better between miRNAs that differ by; t1 h1 x" g* k5 E0 c3 V1 ~* f
two bases based on DCT values. Finally, the stem–loop RT was+ c7 x) d& E% C! p! q
at least 100 times better able to discriminate between the
mature miRNA and its precursor, based on the DCT (precursor: \8 {- Q$ q1 T# u! K. H9 u
versus mature) of 7.$ q) H. p9 I! f$ f) J
DISCUSSION
Since the discovery of miRNAs, remarkable advances in the
characterization of these gene families have delineated the6 D2 H& m2 D* l& K% @6 f" \; O2 |
mechanism for their functions in gene regulation (35). As a
result, extensive surveys have begun to identify miRNA biomarkers, ?7 g: m  I0 m6 D& q# A
specific for tissue types or disease status. These studies
will benefit from methods that allow for both accurate! S0 m, M5 o- S: Q4 s
identification and quantification of miRNAs.
Current methods for detection and quantification of miRNAs
are largely based on cloning, northern blotting (5), or' Y4 C& h4 \, ^$ I) C, v2 V6 r
primer extension (36). Although microarrays could improve' D+ x: u4 k) L1 S8 Q7 ]9 H
the throughput of miRNA profiling, the method is relatively
limited in terms of sensitivity and specificity (32,33). Low
sensitivity becomes a problem for miRNA quantification
because it is difficult to amplify these short RNA targets.
Furthermore, low specificity may lead to false positive signalfrom closely related miRNAs, precursors and genomic$ D& t; \2 R8 K4 l
sequences. More recently, a modified Invader assay has
been reported for the quantification of several miRNAs
(37). However, Invader assays have limited specificity and
sensitivity, requiring at least 50 ng total RNA, or 1000
lysed cells, per assay.
Real-time PCR is the gold standard for gene expression
quantification (38,39). It has been a long challenge for scientists
to design a conventional PCR assay from miRNAs averaging
22 nt in length. We developed a novel scheme to
design TaqMan PCR assays that specifically quantify; t6 _  d) ^' L& d
miRNA expression levels with superior performance over9 O  t8 a$ k! @/ r3 f
existing conventional detection methods. We have designed
and validated assays for 222 human miRNAs (Chen et al.,
unpublished data). These assays combine the power of PCR! d2 E5 B. ^+ `' v. C! U8 Y+ X3 H. N
for exquisite sensitivity, real-time monitoring for a large6 r; F* w  @+ [
dynamic range and TaqMan assay reporters to increase the
specificity. In our hands, miRNA precursors were at least 2000/ K: X4 }- G' k4 ~' G
times less effective targets than mature miRNAs (Table 2)." T% V: |+ s) k
Because these assays are insensitive to precursors or genomic# c+ r3 o( x3 F8 {2 N2 Q
DNA, we were able to add heat-treated cells directly to the/ h' c' [8 o. Y% N" I( {# v
assays, eliminating the need for sample preparation. For1 I" G! p( R, K8 l
applications where both mature miRNAs and their precursors
need to be assayed, conventional TaqMan assays can be used
in parallel to specifically detected precursors.! M9 A- R* A" u' P2 U5 F
We observed the better specificity and sensitivity of stem–
loop RT primers than conventional linear ones likely due to the
base stacking and spatial constraint of the stem–loop structure) C* y$ D6 [! {& _  \) q* n5 h; S
(Figure 9). The base stacking could improve the thermal stability% k$ _' ^* j; a8 t. N* o1 \9 t
and extend the effective footprint of RT primer/RNA8 \+ U: U& |' X2 Z
duplex that may be required for effective RT from relatively: |  ]- I1 T  a( x& C
shorter RT primers. The spatial constraint of the stem–loop
structure may prevent it from binding double-strand genomic
DNA molecules and, therefore, eliminate the need of TaqMan. n) c( j0 |9 l2 ?; }+ u
miRNA assays for RNA sample preparation. Stem–loop
RT primers can potentially be used for multiplex RT reactions
and small RNA cloning for possibly better efficiency and4 }- Z9 A) {% c  U. e
specificity.& A3 h/ Q  e, @9 c
There is an increasing need for sensitive and specific whole
miRNA profiling. The ability to effectively profile miRNAs
could lead to the discoveries of disease- or tissue-specific" x7 u. v! X; \* x
miRNA biomarkers, as well as contribute to the understanding0 j! `; [7 M. Y* Q7 O; ~& C
of how miRNAs regulate stem cell differentiation. Our stem–
loop RT–PCR method should provide a practical solution for
these studies. We are currently developing multiplex) B, t  M: P: ?$ c9 e3 g
approaches that should further increase the utility of this: t+ @2 z/ T, B2 J+ X
method.

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