Liposome-mediated RNA transfection should be used with caution

Abstract

Liposome-mediated RNA transfection appears to present a number of advantages for studying the metabolism of reporter mRNAs in mammalian cells. This method is also widely used to transfect siRNAs. Here we describe results indicating that reporter mRNAs introduced into HeLa cells by liposomes do not present the expected behaviors. Namely, the stability of reporter mRNAs was independent of the presence or absence of an AUUUA instability element, a poly(A) tail, or even a 5' methylated cap. Confocal microscopy showed that fluorescent RNAs introduced by liposome-mediated transfection were present in discrete particles. These observations imply that a number of control experiments are required when using liposome to mediated RNA transfection, and the possible consequences are discussed.

FIGURE 1.

Aberrant stability of transfected RNAs. (A) The various capped and ³²P-labeled mRNAs, indicated on the left, were transcribed in vitro from the appropriate plasmids linearized by restriction with EcoRV and using the mMessage mMachine (Ambion) kit with the T7 polymerase: GbORF pA⁺ and pGbORF (Audic et al. 1997); GbORF-AUUUA pA⁺, pGbORF-AUUUA, GbORF-junARE pA⁺, and pGbORF-jun (Paillard et al. 2002); or the SP6 polymerase Cat-EDEN pA⁺, pCESA 14, Cat-EDENas pA⁺, and pCEASA 3 (Ezzeddine et al. 2002). The in vitro transcripts were purified and verified by PAGE before use. HeLa cells (12-well culture dish seeded at 10⁵ cells per well the day before use) were transfected using the DMRIE-C lipofectant (Invitrogen; 2 mg/mL) according to the manufacture's instructions. Briefly, 400 μL of opti-MEM supplemented with 10 μL of lipofectant and 2–3 pmol of RNA were added to each well, and the cells were cultured at 37°C/5% CO₂ for 2 h. The lipofectant was then removed by washing with PBS and the culture continued at 37°C/5% CO₂ after adding 1 mL/well of DMEM supplemented with 10% fetal calf serum. At the indicated times, the medium was removed and total RNA was extracted by the addition of 400 μL of Tri-Reagent (Medical Research Center Inc.) per well. The extracted RNAs were separated by electrophoresis on a 2% agarose MOPS/formaldehyde gel and transferred onto a Hybond N⁺ membrane (Amersham), and the fixed membrane was exposed to a phosphoimager screen. The radioactive signals were revealed using a Storm phosphoimager and then quantified by the ImageQuant software. (B) Hela cells were transfected, as described in A, by GbORF-AUUUA mRNA devoid of a poly(A) tail (GbORF-AUUUA pA⁻ (upper panel) or either uncapped or capped (respectively, No cap and Cap; bottom panel). The poly(A)⁻ mRNA was synthesized using pGbORF-AUUUA restricted with BamHI. To synthesize uncapped mRNA, the cap analog was omitted from the transcription reaction. The radioactive capped and uncapped mRNAs (10 fmol) were either added alone to the lipofectant (w/o carrier) or supplemented with 2.5 pmol of nonradioactive GbORF or GbORF-AUUUA pA⁺ as appropriate (with carrier). The transfected cells were cultured and the total RNA extracted and analyzed as described in A. (C) HeLa cells were transfected, as described in A, by GbORF pA⁺ and GbORF-AUUUA pA⁺ mRNAs as indicated. (Upper panel) After removal of the culture medium, the cells were washed with 1 mL of PBS and then detached from the dish with 100 μL of Trypsin-EDTA solution (Invitrogen). After an incubation for 2 min at 37°C, 900 μL of DMEM supplemented with 10% fetal calf serum was added and the cells collected by centrifugation (1500g, 5 min, room temperature). Total RNA was extracted from the cell pellet with 400 μL of Tri-reagent (lane Cells). To determine the amount of ³²P-labeled RNA remaining in wells after removal of the cells, 400 μL of Tri-reagent and untransfected cells treated with trypsin (acting as carrier for RNA extraction) were added after removal of the transfected cells (lane Well). The total RNA extracted from the various samples was processed and the ³²P-labeled RNA visualized as described in A. (Lower panel) After transfection the cells were cultured for the indicated times. The cells were then detached from the culture dish with trypsin, processed and analyzed as described for the upper panel.

FIGURE 2.

Confocal microscopy of transfected cells. HeLa cells (seeded in a 12-well culture dish at 10⁵ per well containing a glass cover slip the day before use) were transfected as described in the legend to Figure 1A using Cyanine 3-labeled RNAs (Human Universal Reference, Stratagene). After removal of the transfection solution, the cells were either fixed (3.7% formaldehyde in PBS, 10 min, 20°C) or cultured for a further 3 h at 37°C/5% CO₂ in DMEM supplemented with 10% fetal calf serum and then fixed. The fixed cells were washed twice with PBS. To reveal the plasma membranes, the fixed cells were incubated (10 min, 20°C) with a solution of Alexa Fluor 488 labeled wheat germ agglutinin (5 μg/mL) (Molecular Probes, 5 μg/mL) and then washed with PBS. The cells were then labeled with DAPI, and the cover slips were mounted on microscope slides. Cover slips were examined using a Leica SP2 confocal inverted microscope equipped with HCX PL APO 63×/1.40 oil immersion objective. Sequential excitations using the blue diode laser 405 nm, the 488-nm argon, and 561-nm diode lasers were carried out to prevent cross-contamination between fluorochromes. Confocal optical sections were taken in the x, y, z mode to make z series of images (10–20 planes) at 0.3-μm increments. The images were acquired and processed using the Leica Microsystems computer software. (Left column) Green indicates Alexa Fluor 488 labeled wheat germ agglutinin (membranes); blue, DAPI (DNA) fluorescence. (Middle column) Red indicates cyanine 3 (RNA); blue, DAPI (DNA) fluorescence. (Right column) Superposition of the Alexa fluor 488, cyanine 3 and DAPI fluorescent images.