Two simplified methods of identifying messenger ribonucleic acid (mRNA), and compact, low-power apparatuses to implement the methods, are at the proof-of-concept stage of development. These methods are related to traditional methods based on hybridization of nucleic acid, but whereas the traditional methods must be practiced in laboratory settings, these methods could be practiced in field settings.
Hybridization of nucleic acid is a powerful technique for detection of specific complementary nucleic acid sequences, and is increasingly being used for detection of changes in gene expression in microarrays containing thousands of gene probes. A traditional microarray study entails at least the following six steps:
- Purification of cellular RNA,
- Amplification of complementary deoxyribonucleic acid [cDNA] by polymerase chain reaction (PCR),
- Labeling of cDNA with fluorophores of Cy3 (a green cyanine dye) and Cy5 (a red cyanine dye),
- Hybridization to a microarray chip,
- Fluorescence scanning the array(s) with dual excitation wavelengths, and
- Analysis of the resulting images.
This six-step procedure must be performed in a laboratory because it requires bulky equipment.
The present developmental methods require fewer steps and are not restricted to laboratory settings because they do not require bulky equipment. In principle, they could be implemented by means of low-power, portable, lightweight units having sizes of the order of a cubic foot (≈0.03m3). These methods could be used to perform field studies as precursors to full laboratory gene-expression analyses and can be used for detecting rare and little- expressed mRNA samples. This present method does not require the PCR-amplification, fluorescent-labeling, and scanning steps (steps 2, 3, and 5 listed above).
The steps involved in the method are depicted schematically in the figure. In this method, the initial mRNA from cell or tissue lysates is purified in one step, using oligo dT beads, and is then directly labeled by cross-linking to a reporter enzyme [horseradish peroxidase (HRP)]. The HRP-linked mRNA is then hybridized to a cDNA gene array printed on a nylon membrane, the membrane is incubated with a chemiluminescence substrate, and the resulting chemiluminescence from the affected area of the membrane is detected by contact digital imaging. The whole procedure takes less than five hours. This method is useful for identifying rare genes without much processing, and for diagnostic genomic screening for biomarkers. The apparatus for implementing this method can be miniaturized for rapid screening for stem-cell research or analyzing rare cell samples from tissues.
This work was done by Eduardo Almeida of Ames Research Center and Geeta Kadambi of National Space Grant Foundation. For further information, access the Technical Support Package (TSP) free online at www.techbriefs.com/tsp under the Bio-Medical category.
This invention is owned by NASA and a patent application has been filed. Inquiries concerning rights for the commercial use of this invention should be addressed to the Ames Technology Partnerships Division at (650) 604-2954. Refer to ARC-15177-1.