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Promega Corporation

Using ImProm-II™ Reverse Transcription System to Generate Fluorescently Labeled Probes

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LabFact #4

ImProm-II™ Reverse Transcription reaction conditions support PCR amplification. No dilution of the cDNA is necessary. Add heat-inactivated reverse transcription products directly to the PCR mix. (from #TM236)
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Abstract

In this report, the 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.

Randy Hoffman, B.S., Bing Hui Lin and Katharine Miller, B.S.

Promega Corporation
Publication Date: 2001

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.

Reverse transcription of 1.2 and 7.5kb RNA using ImProm-II Reverse Transcription System.Figure 1. Reverse transcription of 1.2 and 7.5kb RNA using ImProm-II™ Reverse Transcription System.

Panel A: 1.2kb transcript. Lane 1, SuperScript II-generated cDNA; lane 2, ImProm-II™-generated cDNA. Panel B: 7.5kb transcript. Lane 1, ImProm-II™-generated cDNA; lane 2, SuperScript II-generated cDNA. Lanes M, Lambda DNA/HindIII Markers (Cat.# G1711).

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 of 200mM EDTA.
  7. A 10µl aliquot of each reaction was added to 10µl of alkaline loading dye (20mM NaOH, 20% glycerol, 0.04%bromophenol blue) 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 5minutes. 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.

How to Cite This Article

Hoffman, R., Lin, B.H. and Miller, K. Using ImProm-II™ Reverse Transcription System to Generate Fluorescently Labeled Probes . [Internet] 2001. [cited: year, month, date]. Available from: http://www.promega.com/resources/pubhub/enotes/using-impromii-reverse-transcription-system-to-generate-fluorescently-labeled-probes/

Hoffman, R., Lin, B.H. and Miller, K. Using ImProm-II™ Reverse Transcription System to Generate Fluorescently Labeled Probes . Promega Corporation Web site. http://www.promega.com/resources/pubhub/enotes/using-impromii-reverse-transcription-system-to-generate-fluorescently-labeled-probes/ Updated 2001. Accessed Month Day, Year.

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 or may have certain limitations. Please visit our Web site for more information.

Figures

Reverse transcription of 1.2 and 7.5kb RNA using ImProm-II Reverse Transcription System.Figure 1. Reverse transcription of 1.2 and 7.5kb RNA using ImProm-II™ Reverse Transcription System.

Panel A: 1.2kb transcript. Lane 1, SuperScript II-generated cDNA; lane 2, ImProm-II™-generated cDNA. Panel B: 7.5kb transcript. Lane 1, ImProm-II™-generated cDNA; lane 2, SuperScript II-generated cDNA. Lanes M, Lambda DNA/HindIII Markers (Cat.# G1711).

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