Detection of Lowly Expressed Genes using T7 RNA polymerase based Linear Amplification and RLS Imaging on a Microarray Platform
Introduction
A vast majority of medical and biological research projects focus on monitoring gene expression. They include disease classification, understanding of basic biological processes, and identification of new biomarkers and drug targets. The advent of cDNA microarray technology has accelerated the discovery of pharmacological targets for many cancer types and other diseases. Although, microarray technology exists since the early 90’s, current microarrays for gene expression analysis have major limitations. They rely on relatively high inputs of RNA (ug) that cannot be extracted from needle biopsies or micro-dissected tumor tissues. Also, their hybridization detection techniques, based on fluorescent labeling have limitations. There exists a limit on the detection sensitivity of scanners (0.1 fluorophores/um2) and also fluorophores are prone to quenching and photobleaching.
Current protocols used for amplification of DNA/RNA are based either on polymerase chain reaction (PCR) or transcription of DNA. The former is biased amplification while the latter gives linear amplification of RNA. Different promoters for in vitro transcription are SP6, T3 and T7 and they are unique to their respective polymerases, thus avoiding any crosstalk (Appendix C). Presently, T7 based amplification using (dT)n-T7 oligonucleotide primer is used for reverse transcription of input RNA for subsequent T7 RNAP amplification of cDNA [1, 2]. We propose to replace this primer by a library of sequence specific combinatorial oligonucleotides synthesized on-chips in our lab [3]. This will allow better discrimination between closely related gene sequences by having the 3’-end of the designed oligonucleotide target sequences non-conserved between genes, since only primers perfectly hybridized at their 3’-end can be extended during RT. In addition, our primer design insures RT of short tag cDNA rather than full length, resulting in enhanced amplification yield and linearity.
Recently, a new technology based on resonance light scattering (RLS) has
been developed for highly sensitive imaging of DNA microarrays [4, 5]. It is
based on the scattering of a specific light wavelength (color) when
colloidal metal particles (gold, silver; 40-120 nm) are illuminated by white
light. The light emitting power of a single RLS particle is approx. 6 orders
of magnitude greater than a single fluorescent label, thus enhancing the
detection sensitivity to about 0.003 RLS particles/um2 with no
quenching or photobleaching effect [6, 7]. We intend to use RLS for
detection of hybridization on an array format using an incident white light
source and CCD camera as described by Yguerabide et al [6].
Experimental Design
Our experimental design is represented on figure 1. The first step involves the synthesis of a library of oligos. Structurally, the oligonucleotide is divided into three regions, which are a gene specific sequence, a shared sequence (promoter region, flag) and a unique sequence (see (1), fig 1). The 5’ end consists of a 20-25 mer sequence used as a unique tag, specific to a molecular entity (mRNA, exon), targeting a single spot, followed by a flag sequence which will be the same for all oligonucleotides. The flag (5 mer) is for specific labeling on chips. This will then be followed by a T7 RNAP promoter (19 mer, complementary) for transcription of flag and tag sequences. Finally, the 3’ end is a 20-25 mer sequence specific to cellular transcript. The design of the Oligos will be done using OligoArray 2.1 software developed by Dr. Jean-Marie in our lab [8]. Special care has been taken in the software to prevent the addition of rUTP residue in the first 8 positions of transcribed RNA [9]. Addition of rUTP at these positions favors dissociation of T7 polymerase rather than elongation to produce full length RNA. Also addition of 3 or more rGTP leads to slippage of RNA polymerase and generates variable length transcripts (RNA)
Figure 1. Experimental Design
The second step (see (2), fig 1) involves the hybridization of this oligonucleotide library to total RNA extracted from a biological source, followed by reverse transcription using biotinylated dNTP’s to synthesis biotinylated 1st Strand cDNA. This is followed by purification step using streptavidin-coated magnetic beads (see (3), fig 1), which minimizes loss of material that occurs during other purification steps (precipitation & columns). Then, 2nd cDNA strand will be synthesized using T7 sequence primer. Only, the flag and tag sequence are synthesized (see (3)).
Next, the cDNA will be amplified into aRNA by in vitro transcription using T7 polymerase. Two different strategies will be used for transcription using either modified rNTP’s (biotin, FAM, see (4), fig 1) or regular rNTP’s (see (5), fig 1). This will help us to compare amplification yields. Prior labeling might reduce amplification yields, but it avoids the need for subsequent probe labeling. An amplification yield between 200X-2000X has been reported starting from 1ng-1pg DNA, although the length of transcript (RNA) produced is unknown [1, 2]. If necessary, we might perform a 2nd amplification step, after radioactive quantification of aRNA to achieve our goal of 104 fold amplification.
Labeled aRNA will be hybridized to a microarray of sequences complementary to the tag sequence on the aRNA (see (6), fig 1). This microarray will be fabricated using parallel light directed oligonucleotide synthesis. Synthesis of oligonucleotide probes is performed on silicon based arrays using phosphoramidite chemistry and photogenerated acid in the 3’ to 5’ direction. The synthesis protocol will be reversed to perform 5’ to 3’ synthesis to have an active 3’-OH if necessary. Unlabeled aRNA will also be hybridized to a similar array platform, followed by a RT carried out on chip using modified dNTP’s (biotin, FAM, see (7), fig 1). Only a few bases corresponding to the flag sequence need to be incorporated. Thus, by using a homopolymer as a flag per input condition (control vs. cancerous cells) and two different modified dNTP, we can perform dual labeling on a single spot.
To realize our goal of high detection sensitivity, we will adapt the RLS technology (see (8), (9); fig1; Appendix C) to our microfluidic chips and use a CCD camera with appropriate color filters for detection. Commercially available gold particles coupled to anti-biotin and silver particles coupled to anti-FAM will be used to directly label on chips, either modified aRNA (see (6)) or elongated probe (see (7)).
References
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