Nanotransporters for Delivery
Nanotransporters, ~30-nm-sized particles with particular surface characteristics, can deliver RNAi compounds to cells. Dr. Woolf notes that the use of nanotransporters for RNAi delivery offers several advantages. “These are small, organic compounds and not a mix of sizes that one gets using liposomes. Another advantage is that the siRNA fits on the outside of the particle, not on the interior. As a result, we have seen 90 percent inhibition in vivo using this strategy.”
Sarah Haigh, Ph.D., senior scientist at Molecular Cytomics (www.molecular-cytomics.com), says its scientists are using a different approach to tackle delivery problems. “Many cells are hard to transfect or transduce with siRNAs. We used a lentiviral system with blood cells and found less than 30% efficiency. Additionally, when looking at individual cells within this population, significant differences in expression were observed. Many cells exhibited little or no knockdown, while others were more pronounced. Unfortunately, this heterogeneity can mask potentially significant findings.”
To overcome this problem, the company uses their Optical LiveCell™ Array. “This is the first slide-based technology that arrays individual, living, adherent, and nonadherent cells into designated wells,” Dr. Haigh explains. “Numerous micron-sized wells populate the slide in a honeycomb pattern to hold individual cells. Vital for the study of blood cells, nonadherent cells can be imaged easily because the cells settle into the wells via gravity and stay in place even under flow. The LiveCell Array allows you to study each cell within a population individually, record real-time responses, and correlate data obtained from living cells with postfixation studies. Furthermore, you only need a standard upright or inverted microscope.”
Another player in RNA-directed gene expression is miRNA. The miRNAs impact a variety of important biological processes.
Similar to their coding cousins, mRNAs, the expression of the noncoding miRNAs vary among tissues and developmental states. To better understand global miRNA expression requires the keen eye of high-throughput technologies, such as microarrays, that give one the ability to perform simultaneous analysis of a large number of analytes per sample.
Scientists at Molecular Devices (www.moleculardevices.com) are developing tools for performing genome-wide miRNA profiling of frozen and formalin-fixed, paraffin-embedded tissues. Rajiv Raja, Ph.D., director of molecular biology R&D, explains, “With the new discovery that miRNAs are involved in transcriptional regulation, there is an increasing focus on finding key species important for disease correlations. To do that requires looking at this from a cellular level.”
Dr. Raja indicates that it is necessary to dissect out the pure-cell populations to obtain high-quality data from tissue biopsies and other clinical specimens without interference from other dissimilar cell types within them.
“Microdissection helps to achieve this,” Dr. Raja reports. “The challenge is that one needs miRNA-extracting technologies to profile different types of samples such as frozen or fixed. Unfortunately, the technology is not quite there yet to isolate miRNAs efficiently from microscopic samples. We are looking to optimize the process to get good-quality miRNA. The field is going in this direction, and once good miRNA samples can be isolated, microarray or quantitative PCR can be performed.”
While microarrays enable multiplexing samples to gather information for hundreds of miRNAs in a single study, an alternate technology, PCR, is useful for understanding smaller subset of miRNAs. Scientists at Qiagen (www.qiagen.com) have developed an efficient and accurate method for the transcriptome-wide quantification of miRNA using real-time PCR based upon a SYBR green detection system.
“We sought to improve miRNA quantification and make it more sensitive and specific,” says Eric Lader, Ph.D., director of Qiagen. “A problem with microarray quantification is that they have low-throughput, in that, you have one sample on an expensive array whose dynamic range is limited. We developed a modified real-time PCR assay called miScript. It has two aspects. For the reverse transcription part of the PCR, we have added a common tag to the 3´ end of the primer that adds a binding site. The next step adds a second primer that is RNA-specific. The end result is that researchers, with small amounts of input RNA, can simultaneously quantify miRNAs, as well as mRNAs, using the same cDNA preparation.
“This avoids the necessity to prepare multiple cDNA preparations to quantify multiple miRNA species from the same RNA sample. Other advantages are that the assay has a broad dynamic range from several picograms to a microgram of input RNA.”
According to Dr. Lader, both microarray and PCR technologies have strengths and weaknesses. “PCR is most applicable when one has many samples but a relatively small number of assays to perform on each. The strength of microarray analysis is that a single sample can be queried with a large number of assays simultaneously. At Qiagen, we view these as complementary technologies.”