Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 Sep;16(3):239-47.

Optimization of in vitro transcription and full-length cDNA synthesis using the T4 bacteriophage gene 32 protein

Affiliations

Optimization of in vitro transcription and full-length cDNA synthesis using the T4 bacteriophage gene 32 protein

Caroline Piché et al. J Biomol Tech. 2005 Sep.

Abstract

We evaluated the effect of the T4 bacteriophage gene 32 protein (T4gp32) on in vitro transcription and reverse transcription. T4gp32 doubled the yield of in vitro transcripts obtained with T7 RNA polymerase and increased the yield of cDNA synthesis when used in combination with an RNaseH-deficient Moloney murine leukemia virus [Au: ok] reverse transcriptase. The positive effect could be correlated with the RNA chaperone activity of T4gp32. T4gp32 stimulated the synthesis of long cDNAs, particularly species longer than 7 kb. By comparison, thermal activation of reverse transcriptase with trehalose only boosted the production of shorter cDNAs. For the construction of an Arabidopsis thaliana cDNA library, where the average cDNA size is 1.2 kbp, both the presence of T4gp32 under standard reaction conditions as well as thermal activation resulted in similarly high percentages of full-length cDNA. However, the inclusion of T4gp32 in a standard reverse transcription reaction resulted in the highest cDNA yield. We conclude that the addition of T4gp32 in standard reverse transcription reactions can increase the quality and yield of full-length cDNA libraries.

PubMed Disclaimer

Figures

FIGURE 1
FIGURE 1
T4gp32 binds and denatures RNA. A: 1.89-kb radiolabeled in vitro transcript, run on a 4% acrylamide: bisacrylamide (30:1)/7 M urea gel. B: Native polyacrylamide gel electrophoresis of the same in vitro transcript, free (lane 1), incubated with bovine serum albumin (lane 2) and increasing amounts of T4gp32 (0.04 μM in lane 3, 0.2 μM in lane 4, 1 μM in lane 5 μM in lane 6 ). The RNA:protein complexes are resolved on a 4% acrylamide:bisacrylamide gel.
FIGURE 2
FIGURE 2
T4gp32 activates both in vitro transcription and reverse transcription. A: 1.89-kb radiolabeled in vitro transcript synthesized in the presence of increasing amounts of T4gp32. The transcripts are resolved on a 4% acrylamide/7M urea gel. B: Alkaline agarose gel electrophoresis of a 2.4 kb cDNA band synthesized with Superscript II reverse transcriptase in the presence of increasing amounts of T4gp32. C: Phosphorimage quantification of the gels shown in A and B. The in vitro transcription products are plotted as black squares, the reverse transcription products as white lozenges.
FIGURE 3
FIGURE 3
cDNA synthesis using five different RT conditions. A: Radiolabeled first strand cDNA synthesized from a 0.24–9.5 kb RNA ladder in five different conditions: incubation at 45°C in standard RT buffer (lane 1); with 4.5 μM T4gp32 (lane 2); or incubation at high cycling temperature (50–60°C) in standard RT buffer (lane 3); with 4.5 μM T4gp32 (lane 4); or with 23% trehalose (lane 5). First strand cDNA is run on a 1% alkaline agarose gel and visualized on a Phosphor Imager. The size of each cDNA product is indicated at the right. B: Each band from the gel shown in A was quantified using the ImageQuant 5.2 software and plotted on a graph showing the intensity of each cDNA band in each condition.
FIGURE 3
FIGURE 3
cDNA synthesis using five different RT conditions. A: Radiolabeled first strand cDNA synthesized from a 0.24–9.5 kb RNA ladder in five different conditions: incubation at 45°C in standard RT buffer (lane 1); with 4.5 μM T4gp32 (lane 2); or incubation at high cycling temperature (50–60°C) in standard RT buffer (lane 3); with 4.5 μM T4gp32 (lane 4); or with 23% trehalose (lane 5). First strand cDNA is run on a 1% alkaline agarose gel and visualized on a Phosphor Imager. The size of each cDNA product is indicated at the right. B: Each band from the gel shown in A was quantified using the ImageQuant 5.2 software and plotted on a graph showing the intensity of each cDNA band in each condition.
FIGURE 4
FIGURE 4
A. thaliana full-length cDNA libraries made under different RT conditions. A: CAP trapping of 6 first-strand cDNA libraries: RT1 and RT2 were done with Superscript II at 45°C, RT3 and RT4 with trehalose thermostabilized Superscript II, and RT5 and RT6 with Thermoscript at 50–60°C. RT2, 4, and 6 contained 4.5 μM T4gp32. The CAP trapped positive control (C+) is a capped 1.3-kb in vitro transcript, reverse transcribed with Superscript II RT. For each RT, the nontrapped supernatant (−) and the CAP trapped, eluted fractions (+) were loaded in parallel on a 1% alkaline agarose gel. The marker (M) is a radiolabeled first strand cDNA obtained from the 0.24–9.5-kb RNA ladder. The size of each band in kilobases is indicated at the left. B: For each RT condition, the black bar indicates the efficiency of the RT (cDNA synthesized/RNA template, quantified by scintillation counting and expressed in percentage). The gray bar indicates the proportion of cDNA that was CAP trapped as evaluated by volume analysis using the ImageQuant 5.2 software (Amersham Bioscience) and expressed in percentage (%). RTase SS (Superscript II); RTase TS (Thermoscript).
FIGURE 4
FIGURE 4
A. thaliana full-length cDNA libraries made under different RT conditions. A: CAP trapping of 6 first-strand cDNA libraries: RT1 and RT2 were done with Superscript II at 45°C, RT3 and RT4 with trehalose thermostabilized Superscript II, and RT5 and RT6 with Thermoscript at 50–60°C. RT2, 4, and 6 contained 4.5 μM T4gp32. The CAP trapped positive control (C+) is a capped 1.3-kb in vitro transcript, reverse transcribed with Superscript II RT. For each RT, the nontrapped supernatant (−) and the CAP trapped, eluted fractions (+) were loaded in parallel on a 1% alkaline agarose gel. The marker (M) is a radiolabeled first strand cDNA obtained from the 0.24–9.5-kb RNA ladder. The size of each band in kilobases is indicated at the left. B: For each RT condition, the black bar indicates the efficiency of the RT (cDNA synthesized/RNA template, quantified by scintillation counting and expressed in percentage). The gray bar indicates the proportion of cDNA that was CAP trapped as evaluated by volume analysis using the ImageQuant 5.2 software (Amersham Bioscience) and expressed in percentage (%). RTase SS (Superscript II); RTase TS (Thermoscript).

Similar articles

Cited by

References

    1. Sugahara Y, Carninci P, Itoh M, et al. Comparative evaluation of 5′-end-sequence quality of clones in CAP trapper and other full-length-cDNA libraries. Gene 2001; 263:93–102. - PubMed
    1. Sambrook J, Russel DW. Molecular Cloning, a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2001.
    1. Carninci P, Nishiyama Y, Westover A, et al. Thermostabilization and thermoactivation of thermolabile enzymes by trehalose and its application for the synthesis of full length cDNA. Proc Natl Acad Sci USA 1998;95:520–524. - PMC - PubMed
    1. Carninci P, Shiraki T, Mizuno Y, Muramatsu M, Hayashizaki Y. Extra-long first-strand cDNA synthesis. BioTechniques 2002;32:984–985. - PubMed
    1. Demeke T, Morris F. Molecular characterization of wheat polyphenol oxidase (PPO). Theor Appl Genet 2002;104:813–818. - PubMed

Publication types

MeSH terms

LinkOut - more resources