Focus: Probe Labeling
Using ImProm-II™ Reverse Transcription System to Generate Fluorescently Labeled
Probes
In this
report, the new ImProm-II™ Reverse Transcription System was
tested against SuperScript™ II in generating Cy™3 and Cy™5-dCTP(dUTP)
fluorescent probes. The ImProm-II™ System from Promega is comparable to
SuperScript™ II. |
By Randy Hoffman, B.S., Bing Hui Lin and
Katharine Miller, B.S.
Promega Corporation
Introduction
Reverse transcription in vitro is a powerful technique for converting RNA into a
complementary DNA (cDNA) sequence. Such cDNA can be used for second-strand synthesis (for
cDNA library construction), cloning, probe generation and RT-PCR, to name a few
applications. For many applications, full-length cDNA is critical, but achieving it can
prove difficult, depending on the length and potential secondary structure in a particular
target RNA. Special reverse transcriptases have been developed to combat these problems;
some allow for full-length cDNA synthesis of long or difficult templates. We conducted
experiments that demonstrate the ability of the ImProm-II™ Reverse Transcription
System (Cat.#
A3800) to reverse transcribe RNA templates of 1.2 and 7.5kb into full-length,
radioactively labeled cDNA. Parallel first-strand cDNA reactions were performed comparing
SuperScript™ II first-strand synthesis system with Promega's ImProm-II™ Reverse
Transcription System for oligo(dT)-primed incorporation of 32P-labeled dNTPs.
Another important property of reverse transcriptases is their ability to incorporate
fluorescently labeled deoxynucleotides. In cDNA microarray research, fluorescent cDNA
probes are used to analyze PCR products spotted onto glass slides. One approach is to
incorporate the fluorescent dyes, Cy™3 and Cy™5 coupled to dCTP or dUTP, using a
reverse transcriptase. Currently, SuperScript™ II is used predominantly to generate
such probes. We tested ImProm-II™ RT directly against SuperScript™ II for
producing fluorescent probes.
Results
Figure 1 demonstrates that the
ImProm-II™ Reverse Transcription System efficiently produced high yields of
full-length 1.2 and 7.5kb cDNA, comparable to that of the SuperScript™ II system.
To test the ability of ImProm-II™ System to incorporate Cy™3 and
Cy™5-dCTP into first-strand cDNA, the 1.2kb kanamycin transcript was reverse
transcribed in the presence of either form of labeled dCTP using the ImProm-II™ and
SuperScript™ systems.
Note that the values are higher for ImProm-II™ System for both Cy™3 and
Cy™5 probes. While this is only one experiment, it seems that ImProm-II™ System
performs as well as, if not better than, SuperScript™ II for this application.
Figure 2 demonstrates
fluorescent nucleotide incorporation using a 1.2kb kanamycin transcript as template. The
large “bands” at the bottom of each panel correspond to unincorporated
fluorescent nucleotides. In both instances, Cy™3-labeled dCTP showed better
incorporation than Cy™5-labeled dCTP. Figure 3 shows background-corrected
volume units (in Relative Fluorescent Units) for both Cy™3 and Cy™5 probes
produced using either SuperScript™ II or the ImProm-II™ System.
Conclusions
The ImProm-II™ Reverse Transcription System is an efficient tool for first-strand
cDNA synthesis up to 7.5kb. The ImProm-II™ System incorporates fluorescent
nucleotides as well as SuperScript™ II. Moreover, the system is able to synthesize
full-length of multiple sizes as demonstrated by incorporation of 32P-labeled
dNTPs into 1.2 and 7.5kb cDNA in mass quantities competitive with SuperScript™ II
first-strand synthesis system. The optimized reaction conditions generate high yields of
full-length cDNA for many downstream applications.
Methods
The ImProm-II™ Reverse Transcription System (Cat.# A3800)
and the supplied protocol (Technical Manual #TM236)
were used unless indicated otherwise.
Synthesis of Radiolabeled cDNA
First-strand cDNA synthesis reactions were performed using the SuperScript™ II
first-strand synthesis system or ImProm-II™ Reverse Transcription System.
1. Add the following reagents together in a thin-walled, nuclease-free tube:
| |
ImProm-II™ System or SuperScript™ II |
Oligo(dT) |
0.5µg |
Poly(A)+ RNA (0.5µg/ml) |
2.0µl |
2. Anneal at 70°C for 5 minutes followed by quick chill on
ice.
3. Add to the above the following reagents (per 20µl
reaction):
| Buffer |
1X |
MgCl2 |
3mM |
dNTPs, each |
0.5mM |
[32P]dCTP |
0.5µl |
4. Preheat the tube at 37°C for 2 minutes and add 1µl of Reverse Transcriptase.
5. Incubate at 37°C for 60 minutes.
6. Terminate reactions by addition of 2.5µl EDTA (200mM).
7. A 10µl aliquot of each reaction was added to 10µl of alkaline loading dye (NaOH,
20mM; glycerol, 20%; bromophenol blue, 0.04%) and resolved by alkaline gel
electrophoresis.
8. Perform autoradiography at –70°C for 2 hours.
Incorporation of Cy™3- and Cy™5-dCTP into Kanamycin cDNA
1. Add the following reagents together in a 1.5ml tube:
| |
SuperScript™
II |
ImProm-II™ System |
Oligo(dT) |
1.0µl |
1.0µl |
Kanamycin RNA (0.5µg/ml) |
2.0µl |
2.0µl |
Water |
7.0µl |
7.6µl |
dNTP mix (10mM)* |
1.0µl |
-- |
*10mM dNTP mix: 2.0µl each 100mM dATP,
dGTP, dTTP and 1.12µl dCTP in 20µl total. |
2. Denature at 70°C for 5 minutes, then chill on ice for 5 minutes.
3. Add the following reagents to each tube:
| |
SuperScript™ II |
ImProm-II™
System |
5X Buffer |
4.0µl |
4.0µl |
dNTP mix |
1.0µl |
1.0µl |
Cy™3- or Cy™5-dCTP,
1mM |
1.0µl |
1.0µl |
RT |
1.0µl |
1.0µl |
Ribonuclease
Inhibitor |
1.0µl |
-- |
DTT, 100mM |
2.0µl |
-- |
MgCl2, 25mM
|
-- |
2.4µl
(3mM final) |
Total |
20µl |
20µl |
4. Incubate at room temperature (25°C) for 5 minutes. Incubate at 42°C, 60 minutes.
5. Incubate at 70°C, 15 minutes, to denature RNA:DNA hybrids.
6. Add 1µl RNase H to each tube and incubate at 37°C for 20 minutes.
7. Analyze 1.0µl on a 2.0% agarose gel.
8. Scan gels at 585nm (Cy™3) or 650nm (Cy™5) on an FMBIO® II
(Hitachi) fluorimaging system.
9. Relative volume data was calculated using the quantification settings on the FMBIO®
system.
ImProm-II is a trademark of Promega Corporation.
Cy is a trademark of Amersham Pharmacia Biotech. FMBIO is a registered trademark of
Hitachi Software Engineering Company. SuperScript is a trademark of Life Technologies,
Inc.
*Products may be covered by pending or issued patents. Please visit
our patent and trademark web page for more information.
**For Research Only.
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