Over the past few years high-throughput platforms for real-time quantitative PCR have become widely available. The cost of RNA extraction from a large number of samples are, however, quite notable. One method that stands out with respect to free up- or downscaling of sample size and reliability is the isolation of mRNA using oligodeoxythymidylate [oligo(dT)25]-coated magnetic particles. In combining this magnetic separation of mRNA with real-time reverse transcription PCR (RT-PCR), we have achieved a highly reproducible, economic, and fast way of analyzing large sample numbers. One difficulty that has so far prevented the fusion of these techniques relates to accurate mRNA quantification. We present a solution to this problem that enables excellent adjustment of cDNA amounts prior to the real-time PCR. Furthermore, as the mRNA is rapidly isolated from crude plant extracts, our method is widely applicable to herbaceous plant species and various tissue types without cumbersome adjustments. Although designed and tested here for plants, we anticipate that the principles should be applicable to gene expression studies in any other organism. Lastly, due to its flexibility, the method presented here can easily be adapted to specific requirements of various users and has great potential for further automation.
Transcriptional profiling has become a standard technique in the analysis of gene expression with conventional quantification methods such as Northem blot analyses, RNase protection assays, and competitive reverse transcription PCR (RT-PCR) being increasingly replaced by large-scale approaches. High-density DNA oligonucleotide or cDNA arrays are now available for a range of organisms and species, allowing for gene expression studies at the whole genome scale. Microarray technology is mostly used to compare RNA populations from limited sample numbers (1,2) so that the extraction of the template is not a limiting factor. High-fidelity cDNA amplification protocols (3,4,5) allow for comparison of small amounts of tissues and thereby information on tissue specificity. A complementary approach is the expression analysis of a limited number of genes of interest on large sample sets using real-time quantitative RT-PCR (6,7). Such sample sets may arise from sampling different genotypes (e.g., transgenic lines or ecotypes), differently treated source material (e.g., different growth conditions), or from serial sampling of different tissue types at several developmental stages. In these cases, numerous RNA extractions and reverse transcription reactions have to be performed. With the high-throughput capacity of quantitative PCR platforms, these initial steps are quickly becoming a real bottleneck for large scale expression analyses.
A key factor with respect to RNA extraction is the quality of the isolated mRNA (8,9). We developed a high-throughput technique for gene expression profiling from small amounts of plant tissues using the combination of mRNA extraction by oligodeoxythymidylate [oligo(dT)25]-coated magnetic beads and quantitative RT-PCR, with bead-bound cDNA as template. While the high quality, robustness, and sensitivity of mRNA isolations via magnetic beads (as well as their ability to isolate mRNA from a wide range of tissues and even single cells) has long been known (10,11,12,13), this technique has so far not been routinely used in quantitative RT-PCRs (14,15). One reason for this could be the difficulty to determine the cDNA concentration of the resulting solid-phase cDNA library, which prevents a reliable adjustment of cDNA concentrations among samples prior to the real-time PCR run. In this report we offer a solution to this problem of even sample loading, which makes magnetic quantitative RT-PCR applicable to a broad range of biological samples and adaptable to a wide range of platforms and scales of investigation.Materials and Methods Plant Material
Arabidopsis, wheat, and canola plants were grown in controlled growth chambers under 10, 13, and 12 h photoperiod, respectively, irradiance of 130, 350, and 600 µE m-2 s-1, day and night temperatures of 21°/19°, 23°/21°, and 26°/22°C, and 60% relative humidity.
Rice plants were grown in a glasshouse at 25°C (day) and 22°C (night) and 65%–75% relative humidity under a 14-h light period (natural day length extended whenever necessary by supplemental lighting of approximately 300 µE m-2s-1).Buffers and Solutions
For lysis buffer: 100 mM Tris, pH 8.0, 500 mM LiCl, 10 mM EDTA, 1% LiDS, 5 mM dithiothreitol (DTT); for wash buffer 1: 10 mM Tris, pH 8.0, 150 mM LiCl, 1 mM EDTA, 0.1% LiDS; for wash buffer 2: 10 mM Tris, pH 8.0, 150 mM LiCl, 1 mM EDTA; for 5× RT buffer: 250 mM Tris, pH 8.3, 250 mM KCl, 50 mM MgCl2, 0.5 mM DTT; for elution buffer: 2 mM EDTA, pH 8.0. All solutions were prepared with diethylpyrocarbonate (DEPC)-treated Milli-Q® water (Millipore, North Rye, New South Wales, Australia).RNA Extraction and First-Strand Synthesis
Plant material was ground to a fine powder under liquid nitrogen and stored at -80°C until use. All washing/elution steps were performed by relocation and magnetic separation on a 96-well magnetic platform (Dynal MPC®-9600; Invitrogen, Mount Waverley, Victoria, Australia). Prior to use, 10 µL magnetic bead solution [Dynabeads® Oligo(dT)25; Invitrogen] were aliquoted to each well and washed twice with 200 µL lysis buffer. Thirty milligrams of tissue powder were incubated in 300 µL lysis buffer for 10 min at 1800 rpm and room temperature on an orbital shaker. Extracts were centrifuged twice at 10,500× g and 15°C for 10 min, and 230 µL of the final supernatant were transferred to the washed magnetic beads. After a 10-min mRNA annealing phase to the oligo(dT)25 primer at room temperature and subsequent magnetic separation, the beads were washed twice in 200 µL wash buffer 1, once in wash buffer 2, twice in ice-cold 1× RT buffer, and finally resuspended in 10 µL DEPC-treated Milli-Q water. After incubation at 70°C for 5 min and chilling on ice, first-strand cDNA synthesis was carried out by adding 10 µL RT master mix containing 4 µL 5× RT buffer, 0.25 µL 100 mM DTT, 1 µL 100 mM dNTPs, 40 U RNasin® RNase Inhibitor, 50 U Moloney murine leukemia virus (MMLV) reverse transcriptase (all from Promega, Hawthorn, Victoria, Australia), and 4.25 µL DEPC-treated Milli-Q water and incubating for 1 h at 42°C. After two washes with 200 µL 1× RT buffer, the mRNA was eluted from the bead-bound cDNA at a melting temperature of 95°C for 10 min. The concentration of mRNA in the chilled supernatant was determined using a NanoDrop® ND-1000 UV-Vis spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Beads were washed with 100 µL Milli-Q water, the cDNA concentration adjusted to 2 ng/µL with sterile Milli-Q water and stored at -80°C. For the subsequent quantitative PCR, samples were diluted 1:10 in Milli-Q water, unless stated otherwise.