RNA Amplification – Preservation of a Precious Archive

Director, Bionomics Research and Technology Center (BRTC)
Environmental and Occupational Health Science Institute (EOHSI)
University of Medicine and Dentistry of New Jersey

Associate Director, Technology Development and Implementation
Rutgers University Cell and DNA Repository (RUCDR)
Rutgers University

Introduction
Understanding the molecular mechanisms of biological processes and disease is rapidly becoming a key integral component of academic and industrial research programs. As the promises of personalized molecular medicine and global health monitoring start to define new technological foci, it becomes clear that the quality and preservation of molecular by-products is of paramount importance for making prognostic and diagnostic assessments. Yet many of the applications employed today are poorly defined, and future biomarker discovery platforms are still to be developed. The approach being adopted today for sample acquisition, investigation and archiving involves the retooling of standard operating procedures for experimental and clinical samples alike.

All tissues are not created equally: Defining protocols for nucleic acid extraction, amplification, and quality assessment
Both high and low throughput gene expression interrogation requires exceptional quality of nucleic acid. Therefore, well defined RNA extraction protocols are critical when investigating technologies used to measure gene expression. Just as organs provide different functions in the human body, tissues from these organs may require independent protocols or modifications of existing protocols to maximize RNA quality during extraction. Currently there are several methods for total RNA extraction that are tissue dependant (1). When selecting a protocol for RNA extraction one must consider the downstream events or technologies that will be employed to interrogate the entire population and relative levels of messenger RNA. For an application such as a high density DNA microarray, the preservation of full length constructs is less important given the severe 3’ bias of most commercially available products; however, applications such as quantitative real time PCR (QPCR) often have no boundaries with respect to assay design, and targets validated or measured by QPCR are often stretched across a given gene to confirm the annotation and regulation of individual gene products. Such divergence in technological approaches for interrogating gene expression requires that RNA species be of the highest fidelity possible so the caveats associated with analysis are minimized. Investigators therefore need to: 1) choose extraction protocols that best suit the tissue types being analyzed; 2) establish a profile to measure quality between samples, and; 3) choose amplification protocols that enable and develop a resource for future utilization by a variety of down stream gene expression technologies.

Here today…gone tomorrow: Preservation of a precious, labile resource
Preserving and distributing RNA is becoming a routine procedure for biorepositories and small laboratories alike. The one commonality across both large and small laboratories is dealing with the labile nature of RNA and determining how to maximize its useful life. If we have learned anything with respect to gene expression analysis and the lack of utility of historical tissue sources, it is that we have been ill-equipped to preserve our ability to measure gene expression in an efficient and sensitive manner. If laboratories propose to extract RNA and store it for distribution, we may very well find ourselves facing similar problems many years from now when we learn that RNA storage and, most importantly, processing, delivers results too variable for making important measurements. To this end, many laboratories are establishing programs designed to minimize the variability associated with the processing of RNA (i.e. reverse transcription) and to remove the bias contributed by non-specific degradation of RNA over time. To accomplish this lofty goal, laboratories are moving towards archiving a nucleic acid species that is more stable than RNA and is used directly for measurements of gene expression. The species of choice is cDNA. Clearly the most “variable” component of any gene expression analysis is the reverse transcription of RNA into cDNA and many laboratories have seen variable results when comparing data generated on different days using the same RNA and enzymes. The variables during this process are too numerous to describe in this editorial but suffice it to say that data across platforms from the same cDNA has proven to be more stable and comparable than that obtained through repetitive processing of the same RNA (2). Additional advantages to this novel workflow include the preservation of the primary RNA resource as well as continuity with respect to data variability across laboratories that receive distributions of nucleic acid for their own research objectives. The last component of this operational schema entails “amplification” of cDNA at the time of reverse transcription to ensure that the resource is not easily depletable. To this end, we employ a number of linear nucleic acid amplification protocols to achieve this goal. Using NuGEN’s Ovation™ technology we utilize nominal (nanogram) amounts of total RNA to yield microgram quantities of cDNA, which is used for microarrays, quantitative PCR and solution based systems while populating a reagent archive for future investigations (3). Linear amplification coupled with a modular amplification and labeling approaches allows for a technology- independent and protracted gene expression investigation across as many laboratories as the archive curator wishes, all the while ensuring reproducible results by removing technological issues associate with RNA degradation and processing.

Nucleic acid amplification technologies: How small is small?
As gene expression analysis becomes a clinical reality, the “amount” of RNA that can be secured from clinical samples is a growing challenge. Often protocols are designed to minimize the invasive nature of tissue collection, which leads to low, and more importantly to variable, yields of nucleic acid from clinical samples. To address this from Good Laboratory Practice (GLP) and experimental perspectives, all clinical samples identified for gene expression analysis should be considered “small samples”. In addition, as cell isolation becomes more refined via laser capture microscopy (LCM) and fluorescent activated cell sorting (FACS), it becomes a necessity to define individual cell populations on a molecular level. Clearly then, it is desirable for gene expression analysis to standardize on a protocol that is insensitive to the amount of starting material, which will vary across the lifetime of a study or experimental procedure. Most amplification protocols require microgram amounts of starting materials and have neither the capability to generate products that can be used for multiple downstream applications nor the flexibility to look at the entire transcriptome in a regionally unbiased manner. Most laboratories treat precious, enriched and clinical samples as “small samples” to maximize the utility of specimens that are quantity limited. Our repository processes thousands of samples a year and employs NuGEN’s Ovation™ technology, which requires very little starting RNA to ensure that a sample is never excluded but more importantly, that a sample is never processed with a different protocol during the lifetime of any study. By using a single protocol that requires very small starting amounts of RNA, one can be assured that data generated over time and across studies is directly comparable at the level of raw data generated, irrespective of the technology used to measure gene expression output. Although “historical” data exists for a variety of gene expression technologies, the amount of data that will be generated over the next decade far outweighs what is in existence now. Adopting standard protocols for all samples will help ensure that gene expression data is preserved across experiments and over time; a fact that has been recognized most recently by the gene expression community as a critical issue.

The “gold” standard: Single modular protocols with endless applications
Due to the fact that the majority of variability in making gene expression measurements is a function of RNA quality and sample processing, steps need to be taken to preserve biological variability without increasing technological noise. Adopting a modular approach to generating and banking cDNA for gene expression measurements has been proposed among consortia and multidisciplinary programs given what we know about gene expression technologies and what their current limitations are. Retaining the flexibility to use any downstream technology for gene expression measurements from a single cDNA source alleviates major challenges in corroborating data across research programs. Today’s arsenal for RNA amplification includes a variety of T7 based approaches (which yield cRNA product), limited cycle PCR approaches (which use gene specific primers) and library based approaches (which employ a combination of PCR and enzymatic reactions). Without describing the details of the various approaches suffice it to say that when making a selection the following questions need to be addressed prior to amplifying, analyzing and preserving RNA for gene expression analysis:

1. Choose a technology that boasts superior linearity and reproducibility within and across samples. The importance of this metric will ultimately determine the sensitivity with which reliable measurements can be made for any downstream gene expression application
   
2. Choose a technology that has a “universal” potential for making expression measurements. By this we mean select an amplification technology that gives rise to a product that can be stored without fear of degradation and can be used as the direct input for a majority of gene expression technologies employed in your program. In the case of repository programs the storage and shipment of nucleic acid should also be taken into consideration. cDNA is the optimal format for most all applications and can be stored in a variety of cost effective formats.
   
3. Keep in mind that in addition to incremental cost associated with additional “rounds of amplification” the technological variability increases with every manipulation making each round of amplification less linear and less reproducible. In addition, this approach will make comparison of samples that are effectively processed using different protocols infinitely more difficult. Be sure to choose a technology that is more insensitive to the starting amounts of RNA needed for amplification. This will prove extremely useful when the amount of starting material becomes limited for any reason.
   
4. Most RNA amplification technologies result in a 3’ biased final product. For current high throughput gene expression queries this bias does not pose a problem due to the design of probes on most commercial microarray platforms, however, this bias does pose a problem for many quantitative PCR applications and emerging technologies such as exon arrays which are geared at studying splice variation. The ability to amplify the entire transcriptome without bias will prove to be critical for a variety of gene expression programs.
   
5. Genomics based technologies evolve at a rapid pace. In the gene expression arena reprocessing of RNA has become the norm due to different labeling approaches and assay designs. Once again there is an additional cost for operating in this manner but more importantly technological variability makes additional investigation a difficult task. Modularity for down stream applications which utilize the same amplified product is essential for maximizing the return on information and analysis that can be performed on a single sample.

Conclusion
At the end of the day a laboratory should select an amplification technology that addresses the issues above while taking into consideration the complexity of variability associated with making gene expression measurements over time. The desire to utilize fundamentally different platform technologies using the same amplified product should also weighed heavily in the decision to standardize against one protocol. Although the goal of every independent research program is different the means by which we measure gene expression transcends laboratories and the scientific questions we ask, however, as gene expression continues to develop into an approach that will be used in both basic and clinical science we must adopt technologies allowing us to get the most sensitive and reliable measures while preserving precious material for future investigation.

NuGEN Technologies, Inc. is the emerging leader of RNA Amplification technologies in the field of gene expression analysis. NuGEN develops, manufactures, and sells the Ovation™ System family of products for RNA amplification and labeling, powered by NuGEN’s proprietary core technology, Ribo-SPIA™. NuGEN’s approach is uniquely suited to performing fast and sensitive amplification, manually or through automation, of RNA isolated from very small or challenging samples. NuGEN’s proprietary technologies enable life scientists to procure and utilize specimens for gene expression profiling across the entire transcriptome, even when samples are limited or compromised. As gene expression analysis discoveries lead to new biomarkers for predicting disease risk and drug response, clinical diagnostic applications will expand rapidly. NuGEN’s products will offer clinical diagnostic laboratories robust, high fidelity RNA amplification from clinical specimens such as biopsies, laser capture microdissected tissue, cells sorted by flow cytometry, and whole blood samples. NuGEN with its core technologies is poised to significantly participate in the high growth potential of clinical RNA-based gene expression profiling.