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

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.

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.