A window into live cell RNA

Detecting intracellular gene expression is vital when performing knockdown studies – it is also difficult. Here, Don Weldon and Grace Johnston demonstrate a way of examining RNA levels in live cells

To study gene function and biological pathways, researchers will modulate gene expression using methods such as RNAi¹. When performing RNAi-mediated gene knockdown studies, the ability to detect intracellular gene expression in individual, live cells is crucial in order to determine the efficiency of the gene knockdown experiments. An ideal RNA detection agent provides a non-invasive approach to interrogating gene expression while enabling sorting of live cells that can be separated and directly used for downstream studies.

The most widely used detection methods, however, require cell samples to be destroyed with lysis or permeabilisation and fixation. Another disadvantage of traditional techniques is that their results only reflect the average expression of the gene across the collected cell population. One non-destructive option is the use of transfected reporter constructs. However, although the cells remain alive when researchers use this technique, the method can have negative effects on cell health and cannot reveal endogenous gene expression.

[caption id="attachment_36345" align="alignright" width="200"] Figure 1: Survivin gene knockdown in LNCaP and SCC12 cells was distinguishable by measuring relative SmartFlare signals. Histograms of SmartFlare signals corresponding to surviving expression in A. LNCaP cells and B. SCC12 cells, with or without siRNA knockdown of survivin.[/caption]

SmartFlare RNA detection probes help resolve these challenges with their ability to detect target mRNA and microRNA levels in live, intact cells. The probes enable users to quickly verify gene expression, and, in conjunction with cell sorting, isolate desired cell populations. In addition, these reagents require no sample preparation and leave cells intact after the detection event, allowing for downstream studies. Finally, they require no carrier agent to enter cells and have no toxic effect on cell health².

Methods

mRNA knockdown

LNCaP (human prostate adenocarcinoma) and SCC12 (human squamous cell carcinoma) cells were seeded at 10,000 cells/well in 96-well plates. Cells were transiently transfected with control or survivin-targeted siRNA 24 hours after seeding, and then incubated for 48 hours. After the incubation period, the medium was changed to remove the siRNA and transfection agent.

Modulation detection

After the siRNA treatment, the SCC12 and LNCaP cells were incubated with 1000x dilution of stock survivin-specific SmartFlare probe or control SmartFlare probe overnight in cell growth medium. The next morning, cells were trypsinised and analysed using the guava easyCyte 8HT flow cytometer. Concurrently, siRNA-treated cells not interrogated with SmartFlare probes were also harvested for comparative analysis by quantitative reverse transcription polymerase chain reaction (qRT-PCR).

qRT-PCR

Total RNA was extracted using the RNeasy kit (Qiagen) and added to the TaqMan RNA-to-Ct 1-step kit (Life Technologies). qRT-PCR was carried out using a LightCycler 480 system (Roche).

Human Survivin³

TaqMan probe: 5’-TGGTGCCACCAGCCTTCCTGTG-3’

Sense primer: 5’-GCACCACTTCCAGGGTTTATTC -3’

Antisense primer: 5’-TCTCCTTTCCTAAGACATTGCTAAGG-3’

Human GAPDH⁴

TaqMan probe: 5’-ACCACAGTCCATGCCATCACTGCCA-3’

Sense primer: 5’-CAAGGTCATCCATGACAACTTTG-3’

Antisense primer: 5’-GGCCATCCACAGTCTTCTGG-3’

Results

Using flow cytometry for mRNA detection

Survivin is an antiapoptotic gene that is highly upregulated in many cancer cell lines⁵. To detect gene knockdown, survivin-targeted probes were incubated with LNCaP and SCC12 cells that had been treated with survivin and control siRNAs. Differences in expression levels of the target gene were detectable using flow cytometry analysis of cells treated with target-specific probes (Figure 1).

Using qRT-PCR for mRNA detection

[caption id="attachment_36347" align="alignleft" width="200"] Figure 2: Survivin gene knockdown in LNCaP and SCC12 cells by RNAi are distinguishable by SmartFlare detection as well as by qRT-PCR.
A. Survivin expression in LNCaP cells with or without survivin knockdown as determined using SmartFlare technology and analysed by flow cytometry.
B. Confirmation of relative survivin expression levels with or without survivin knockdown in LNCaP cells by qRT-PCR.
C. Survivin expression in SCC12 cells with or without survivin knockdown as determined using SmartFlare technology and analysed by flow cytometry.
D. Confirmation of relative survivin expression levels with or without survivin knockdown in SCC12 cells by qRT-PCR.[/caption]

To detect gene knockdown using qRT-PCR, surviving targeted SmartFlare probes and control  probes were incubated with LNCaP and SCC12 cells that had been treated with survivin and control siRNAs. SmartFlare technology was used to determine relative gene expression levels, and the information was plotted as bar graphs and compared to qRT-PCR data. The comparison showed that both technologies produced qualitatively similar relative gene expression levels when measuring knockdown (Figure 2).

Additional information about the population distribution of the various treated cells was obtained because SmartFlare technology enabled expression analysis in individual, intact cells. Specifically, LNCaP cells treated with a survivin-specific SmartFlare probe displayed a unimodal distribution of surviving signal, while SCC12 cells exhibited a bimodal distribution.

This article has demonstrated the ability to detect intracellular gene expression in individual, live cells using SmartFlare technology. The probes enabled fast and accurate detection of target gene expression at single cell resolution.

This capability is critical when performing RNAi-mediated gene knockdown studies, in which it has been traditionally difficult to determine the cause of incomplete knockdown. Conventional methods of RNA measurement – which measure average RNA levels – cannot distinguish between inefficient knockdown due to ineffective entry of the siRNA into target cells, a poorly designed siRNA sequence, or larges differences in endogenous gene expression within the target cells.

Use of the probes reveals the degree of gene expression knockdown in individual cells. This provides information on cell-to-cell variation in expression, knockdown and efficiency of siRNA entry, and these details may greatly facilitate the interpretation of analyses performed subsequent to RNAi treatment. In addition, the ability to specifically detect RNA levels on a cell-by-cell basis provides new opportunities to link biological pathways and physiological processes to gene functions.

Finally, the new technology makes it possible to sort cells and return them to culture, enabling downstream analyses such as antibody staining, flow cytometry and qRT-PCR. As a result, it is now possible to measure physiological changes in the exact same cell samples assessed for gene knockdown, increasing the strength of observed correlations between gene expression and cell phenotype.

References

1. Hannon, G. J. Nature 2002; 418:244.

2. Seferos, D. S. et al. J. Am. Chem. Soc. 2007; 129:15477.

3. Carrasco RA. et al. Mol Cancer Ther. 2011 Feb;10(2):221-32.

4. Tang Y et al. Mol Cancer Res. 2004 Feb;2(2):73-80.

5. Ambrosini, G.et al. Nat Med 1997; 3:917.