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Focus: RNA interference

RNAi in Drosophila S2 Cells: Effect of dsRNA Size, Concentration, and Exposure Time

We used the T7 RiboMAX™ Express RNAi System (Cat.# P1700) to create double-stranded RNA of varying sizes using the Erk-A gene as a template. We investigated the effect of RNA size, concentration, and exposure time on RNA interference of Erk-A gene expression in Drosophila S2 cells. 

By Natalie Betz, Ph.D.
Promega Corporation

Introduction

RNA interference is the phenomenon in which double-stranded RNA (dsRNA) can specifically suppress expression of a target gene, and was originally discovered in C. elegans (1). The purpose of this set of experiments was to determine the efficiency of different sized dsRNAs against Erk-A to reduce its protein levels in Drosophila S2 cells through RNA interference (RNAi). Erk-A is the Drosophila homolog of mitogen-activated protein kinase (MAPK) and part of the sevenless (Sev) signal transduction pathway (2). Previous work has demonstrated that Erk-A is a good target for RNAi in S2 cells (3–4). In general, dsRNA used in non-mammalian systems to induce RNAi is >400bp and usually encompasses the majority of the target mRNA sequence (5–6). However, the synthesis of shorter dsRNAs is usually more efficient, so it might be advantageous to use shorter dsRNAs if they are as effective as longer dsRNAs at inducing RNAi.

In addition, the duration of the RNAi effect was investigated by incubating S2 cells in the presence of dsRNA for up to 5 days. Earlier reports of the reduction of Erk-A protein levels by Erk-A dsRNA were measured following a 3 day incubation period with the dsRNA (3–4), but the duration of Erk-A inhibition was not investigated.

Methods and Results

Erk-A dsRNAs and a nonspecific control dsRNA for the Renilla luciferase gene (Rluc) were synthesized, purified, and quantitated as described in reference 4 using the T7 RiboMAX™ Express RNAi System (Cat. #P1700). The Erk-A dsRNAs were 180bp, 505bp, or 778bp. The Rluc negative control dsRNA was 500bp. Drosophila S2 cells were treated with increasing concentrations of each dsRNA (0, 9.5, 38 or 190nM) in triplicate for 3 days (4). The dsRNA concentration refers to the initial 1ml treatment. Replicate wells were pooled and a cell lysate prepared. The cell lysates were then subjected to Western blot analysis for Erk-A protein levels (4). The quantity of Erk-A protein in each sample was quantitated using enhanced chemifluorescent detection reagents (Amersham) and a Molecular Dynamics STORM® fluorescent scanner (blue mode).

The results are shown in Figure 1 and demonstrate that all three Erk-A dsRNAs were able to reduce Erk-A protein levels in the S2 cells following a 3-day incubation period, in a dose-responsive manner. As expected, the nonspecific Renilla dsRNA (negative control) did not significantly reduce Erk-A protein levels compared to the untreated control. 

To better visualize the efficiency of inhibition by the various Erk-A dsRNAs, the percentage decrease in the Erk-A protein band with the various concentrations of dsRNAs compared to the untreated control was calculated and is presented in Figure 2. Thus the Erk-A 180bp dsRNA was as effective at equimolar concentrations as the longer Erk-A dsRNAs of 505bp and 778bp.

To investigate how long the suppression in Erk-A protein levels lasts following treatment with Erk-A dsRNA, the following experiment was performed. S2 cells were incubated in triplicate in the presence of either 38nM Erk-A 180bp dsRNA or 38nM Rluc 500bp dsRNA for 1–5 days. At each time point the triplicate wells were pooled, a cell lysate prepared, and the lysates were evaluated for Erk-A protein levels as above. The results are shown in Figure 3 and demonstrate that maximal protein inhibition is observed at day 3, but Erk-A protein levels are still reduced at day 4 and day 5. Thus at least in S2 cells, the RNAi effect is rather long-lived.

Conclusions

Shorter dsRNAs apear be as effective as longer dsRNAs at inducing RNA interference in Drosophila S2 cells. In S2 cells, the RNAi effect appears to persist for at last 5 days. Similar longevity was observed by Clemens and coworkers for a different target mRNA and protein (3).

References

  1. Fire, A. et al. (1998) Nature 391, 806–11.
  2. Biggs, W.H. et al. (1994) EMBO J. 13, 1628–35.
  3. Clemens, J.C. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 6499–503.
  4. Betz, N. and Worzella, T. (2003) Promega Notes 84, 7–11.
  5. Elbashir, S.M. et al. (2001) Genes Dev. 15, 188–200.
  6. Yang, D., Lu, H., and Erickson, J.W. (2000) Curr. Biol. 10, 1191–1200.
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Figures for RNAi in Drosophila S2 Cells: Effect of dsRNA Size, Concentration, and Exposure Time

 

Figure 1

 

Figure 1. Effect of Erk- A dsRNA length and concentration on Erk-A protein levels in S2 cells. Erk-A dsRNAs and a nonspecific control dsRNA (Rluc) were synthesized, purified, and quantitated using the T7 RiboMAX™ Express RNAi System. The Erk-A dsRNAs were 180bp, 505bp, or 778bp. The Rluc negative control dsRNA was 500bp. Drosophila S2 cells were treated with increasing concentrations of each dsRNA (0, 9.5, 38 or 190nM) in triplicate for 3 days. The dsRNA concentration refers to the the initial 1ml treatment. Replicate wells were pooled and a cell lysate prepared. The cell lysates were then subjected to Western blot analysis for Erk-A protein levels (4). The quantity of Erk-A protein in each sample was quantitated using enhanced chemifluorescent detection reagents (Amersham) and a Molecular Dynamics STORM® fluorescent scanner (blue mode). The basal level of Erk-A in the 180bp and 505bp Erk-A samples is different than in the other two samples because these samples were processed on different blots.

 

Figure 1

 

Figure 2. Effect of Erk- A dsRNA length and concentration on Erk- A protein levels expressed as a percentage of the untreated control. The data from Figure 1 was calculated as a percentage of the untreated control and is expressed as percent inhibition.

 

Figure 1

 

Figure 3. Duration of suppression in Erk-A protein levels following treatment with Erk-A dsRNA. S2 cells were incubated in triplicate in the presence of either 38nM Erk-A 180bp dsRNA or 38nM Rluc 500bp dsRNA for 1–5 days. At each time point the triplicate wells were pooled, a cell lysate prepared, and the lysates were evaluated for Erk-A protein levels.
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