RNA quantification using gold nanoprobes - application to cancer diagnostics
© Conde et al. 2010
Received: 23 November 2009
Accepted: 24 February 2010
Published: 24 February 2010
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© Conde et al. 2010
Received: 23 November 2009
Accepted: 24 February 2010
Published: 24 February 2010
Molecular nanodiagnostics applied to cancer may provide rapid and sensitive detection of cancer related molecular alterations, which would enable early detection even when those alterations occur only in a small percentage of cells. The use of gold nanoparticles derivatized with thiol modified oligonucleotides (Au-nanoprobes) for the detection of specific nucleic acid targets has been gaining momentum as an alternative to more traditional methodologies. Here, we present an Au-nanoparticles based approach for the molecular recognition and quantification of the BCR-ABL fusion transcript (mRNA), which is responsible for chronic myeloid leukemia (CML), and to the best of our knowledge it is the first time quantification of a specific mRNA directly in cancer cells is reported. This inexpensive and very easy to perform Au-nanoprobe based method allows quantification of unamplified total human RNA and specific detection of the oncogene transcript. The sensitivity settled by the Au-nanoprobes allows differential gene expression from 10 ng/μl of total RNA and takes less than 30 min to complete after total RNA extraction, minimizing RNA degradation. Also, at later stages, accumulation of malignant mutations may lead to resistance to chemotherapy and consequently poor outcome. Such a method, allowing for fast and direct detection and quantification of the chimeric BCR-ABL mRNA, could speed up diagnostics and, if appropriate, revision of therapy. This assay may constitute a promising tool in early diagnosis of CML and could easily be extended to further target genes with proven involvement in cancer development.
The National Cancer Institute envisions that over the next years, nanotechnology will result in significant advances in early detection, molecular imaging, targeted and multifunctional therapeutics, prevention and control of cancer . Nanodiagnostics is a burgeoning field as more and improved techniques are becoming available for clinical diagnostics with increased sensitivity at lower costs [2–10]. Due to their optical properties, gold nanoparticles (AuNPs) have been used for DNA/RNA screening approaches, namely via functionalization with thiolated oligonucleotides (Au-nanoprobes), capable of specifically hybridizing with a complementary oligonucleotide sequence .
Chronic myeloid leukemia (CML) is a clonal neoplastic disease of the hematopoietic stem cell, whose hallmark molecular event is the genetic t(9;22)(q34;q11) translocation known as the Philadelphia (Ph) chromosome [11, 12]. This translocation - ABL gene (chromosome 9) and BCR gene (chromosome 22) - originates a BCR-ABL fusion gene, leading to the expression of a chimeric BCR-ABL protein with tyrosine-kinase activity [13–15]. The most commonly used procedures for the initial diagnosis and management of CML patients are expensive and time consuming, e.g karyotype analysis, reverse transcriptase polymerase chain reaction analyses (RT-PCR) and fluorescence in-situ hybridization (FISH) [16–18]. Therefore, there is a need for molecular methods able to detect and quantify the BCR-ABL fusion transcripts, which is of paramount relevance when monitoring minimal residual disease and genetic recurrence in patients known to harbor the translocation [19, 20].
Here we present an Au-nanoprobe based approach for the molecular recognition and quantification of BCR-ABL b3a2 (e14a2) fusion for the early diagnosis of CML, which is inexpensive very easy to perform and uses total human RNA as target without reverse transcription and/or amplification.
The 13 nm gold nanoparticles were prepared by the citrate reduction method described by Lee and Meisel . The thiolated oligonucleotide was dissolved in 1 ml of 0.1 M DTT, extracted three times with ethyl acetate, and further purified through a desalting NAP-5 column (Pharmacia Biotech, Sweden) according to the manufacturer's instructions. The Au-nanoprobe was prepared as described in Baptista et al . Briefly, 500 μl of 10 μM thiol modified oligonucleotide was initially incubated with 6 ml of an aqueous solution of AuNPs (≈8.55 nM) for at least 16 h. After centrifugation (20 min at 14500 G), the oily precipitate was washed with 5 ml of 10 mM phosphate buffer (pH 8.0), 0.1 M NaCl, recentrifuged and redispersed in 5 ml of the same buffer to a final concentration in AuNPs of 8.5 nM. The resulting Au-nanoprobe was stored in the dark at 4°C.
K562 erythroleukemic cells (BCR-ABL positive cell line derived from CML patients in blast crisis) and HL-60 cell line, a human leukemic promyelocytic cell line (BCR-ABL negative) were cultured in 90% RPMI 1640 and 10% FBS at 37°C with 5% CO2. Saccharomyces cerevisae cells were grown in YPD medium at 30°C overnight. Human peripheral blood mononuclear cells (PBMC) from control individuals were separated from 3 ml of heparinized peripheral venous blood by Ficoll gradient (Histopaque®-1077, Sigma-Aldrich, St. Louis, USA) according to manufacturer's specifications. Isolation of total RNA was performed using a High Pure RNA Isolation Kit (Roche Applied Science) according to the manufacturer's protocol. RNA concentration was determined by UV photometry and the RNA was stored at -80°C until use. RNA integrity was evaluated on a 1% agarose gel stained by GelRed™.
Total RNA extracted from K562 cells was subjected to RT with Revert-AidTM M-MuLV Reverse Transcriptase (Fermentas, Vilnius, Lithuania) according to the manufacturer's specifications, using 20 μM of BCR-ABLreverse primer, annealing at 42°C for 1 h and 70°C for 10 min to inactivate the reverse transcriptase. The reverse transcription reaction product, a 273-bp fragment of the human BCR-ABL fusion gene (b3a2 junction), was PCR amplified using primers BCR-ABLforward (18 nt): 5'-AGTCTCCGGGGCTCTATG-3' and BCR-ABLreverse (20 nt): 5'-GATTATAGCCTAAGACCCGG-3'. PCR amplification of the b3a2 region was carried out in 25 μl using 0.25 μM of primers, 0.2 mM dNTPs with 1 U Taq DNA polymerase (Amersham Biosciences, GE Healthcare, Europe, GmbH). The PCR reactions were performed in duplicate on a MyCycler Thermocycler (Bio-rad). Thermal cycling conditions consisted of denaturation at 95°C for 5 min and 30 cycles of amplification, each cycle consisting of denaturation of 95°C for 30 s, annealing at 52°C for 30 s, elongation was at 72°C for 30 s and final elongation at 72°C for 5 min and cooling at 4°C. The sequence of the PCR products was confirmed by sequencing.
The Real-Time PCR amplification was performed in a Corbett Research Rotor-Gene RG3000 using SYBR GreenER Real-Time PCR Kit (Invitrogen, Karlsbad, CA, USA) according to manufacturer's specifications in 50 μl reactions containing cDNA from K562 and HL-60 cell-lines, 1× SYBR Green SuperMix and 200 nM of BCR-ABLforward and BCR-ABLreverse. The amplification conditions consisted of 50°C for 2 min hold, 95°C during 10 min hold, followed by 40 cycles consisting of denaturation at 95°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 30 s, with a final extension step at 72°C for 5 min. All the results were originated from three independent experiments.
The Au-nanoprobe assay was performed in a total volume of 30 μl containing the Au-nanoprobe at a final concentration of 2.5 nM, the appropriate targets at a final concentration of 100 fmol/μl (100% complementary BCR-ABL target; 50% complementary BCR and ABL targets, and 100% non-complementary target) in 10 mM phosphate buffer (pH 8.0). Total RNA was used at a final concentration 10-60 ng/μl [100% complementary K562 cells RNA (BCR-ABL Positive); non-complementary HL-60 cells RNA (BCR-ABL Negative)]. Blank measurements were made in exactly the same conditions but replacing target or total RNA for an equivalent volume of 10 mM phosphate buffer (pH 8.0).
Following 5 min of denaturation at 95°C, the mixtures were allowed to stand for 30 min at 25°C and 0.3 M MgCl2 was added at a final concentration of 0.16 M. After 15 min at room temperature for color development, photographs were taken and assayed by UV-visible spectroscopic measurements of the SPR band. Absorption spectra were performed in a UNICAM, model UV2, UV-visible spectrophotometer with Ultra-Micro quartz cells (Hellma, Germany), using 10 mM phosphate buffer (pH 8.0), 0.1 M NaCl as reference. The areas under the curve (AUC500 nm-560 nm/AUC570 nm-630 nm) were calculated with the values for absorbance for 500 nm-600 nm/570 nm-630 nm using the trapezoidal rule.
The Au-nanoprobes were then used for the detection of the BCR-ABL b3a2 fusion mRNA in total RNA extracted from K562 cells (BCR-ABL positive cell line), HL-60 cells (BCR-ABL negative cell line), human peripheral blood mononuclear cells (PBMC) and S. cerevisiae cells - Figure 3B. Total RNA from HL-60 cell line and PBMC only express the normal BCR and ABL transcripts, which are 50% complementary to the probe sequence. Total RNA from an unrelated organism (S. cerevisae) was used to confirm specificity of the detection method. The results originate from a minimum of three individual parallel hybridization experiments. BCR-ABL fusion discrimination was observed only for samples containing the complementary RNA target (K562 cells). Samples containing the normal BCR and ABL genes showed a minor stabilization of the Au-nanoprobe, yet below the threshold for positive identification of the target (ratio <1).
In conclusion, we demonstrated the potential of an Au-nanoprobe based assay for the specific identification and quantification of aberrant expression of genes involved in cancer development. This Au-nanoprobe strategy allowed for detection of less than 100 fmol/μl of a specific RNA target, with the possibility of discriminating between a positive and negative from as little as 10 ng/μl of total RNA. As proof-of-concept we used the BCR-ABL fusion product that is of paramount importance in chronic myeloid leukemia, showing the application potential in cancer diagnosis. To our knowledge, this is the first report on quantification of human mRNA directly from total RNA without reverse transcription or amplification. The assay has a total work-up time of less than 45 minutes with comparable sensitivity to those demonstrated by traditional molecular biology methodologies.
Chronic myeloid leukemia
Surface plasmon resonance
Peripheral blood mononuclear cells
Area under the curve.
This work received the financial support of FCT/MCES through grants to CIGMH-FCT/UNL, PTDC/BIO/66514/2006 and PTDC/SAU-BEB/66511/2006. We thank Dr. A.S. Rodrigues for the human cell lines (K562 and HL-60) and M. Mateus for blood samples.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.