Monitoring RNA for Diagnosis and Prognosis: Visualization of RNA in Tissues and Monitoring RNA in Circulation

Abstracts

Development of quantitative multiplex RNA in situ hybridization for diagnostic applications
Pavol Cekan, PhD, The Rockefeller University

We recently developed a multicolor miRNA-FISH method with the following innovations: (1) a novel carbodiimide-containing fixative for crosslinking the miRNA 5'-phosphate end to surrounding protein, (2) elimination of tissue permeabilization and enhanced antibody-based fluorescence signal amplification through the use of long linkers between the hapten and probe (3) prevention of RNA mishybridization through the use of short LNA-modified DNA (LNA/DNA) probes, and (4) directly-labeled fluorescent rRNA and poly(A) probes for assessing RNA retention and normalizing miRNA signals. We validated our method using two histologically similar skin tumors, namely basal cell carcinoma (BCC) and Merkel cell carcinoma (MCC), which respectively express miR-205 and miR-375. After designing relevant probes, amplified miRNA signals were normalized against directly-labeled rRNA signals to correctly identify 16 BCC and MCC tumors. We are also optimizing mRNA-FISH, especially RNA retention and hybridization steps. We eliminated conventional signal amplification using multiple, short fluorescently-labeled LNA/DNA probes targeting different mRNA segments. We selected probe sequences to avoid RNA mishybridization and the length of each member of the probe pool was adjusted according to melting temperature. This approach requires 40-60 short probes per target mRNA, each carrying multiple fluorophores. mRNA-FISH signals are normalized using probes targeting poly(A), nuclear-encoded rRNA and mitochondrial rRNA; these probe sets also monitor RNA retention, integrity and enable subcellular localization. To date, we have successfully developed specific probes for ERBB2 in a breast cancer diagnostic system, where HER2+ (HCC1954), HER2- (MDA-MB231) paraffin-embedded tumor cell line pellets along with HER2+/HER2- mixed pellet were mounted on the same slide, processed and scanned in parallel.
 

Deep Sequence Profiles of Circulating MicroRNA
Iddo Ben-Dov, MD, PhD, The Rockefeller University

We profiled miRNA in cell-free serum and plasma samples from human volunteers using barcoded deep sequencing of small RNA cDNA libraries. By introducing calibrator synthetic oligonucleotides during library preparation we were able to calculate the total as well as specific concentrations of circulating miRNA. Studying trios of samples from newborn babies and their parents we were able to show placental-specific miRNA in maternal and newborn circulation. Furthermore, sequence variation in the miRNA profiles could be traced to the specific placenta of origin. These deep sequencing profiles, which may serve as a model for tumor or disease detection, along with additional studies of patients with kidney or heart disease, allow us to define the repertoire of miRNA abundance in the circulation and to discuss potential biomarker opportunities.
 

RNA Mimics of Green Fluorescent Protein
Samie R. Jaffrey, MD, PhD, Weill Medical College of Cornell University

Green fluorescent protein (GFP) and its derivatives have transformed the use and analysis of proteins for diverse applications. Like proteins, RNA has complex roles in cellular function and is increasingly used for various in vitro and in vivo applications, but a comparably robust and simple approach for fluorescently tagging RNA is lacking. We now describe the generation of RNA aptamers that bind fluorophores resembling the fluorophore in GFP. These RNAs activate the fluorescence of these fluorophores, resulting in a palette of RNA-fluorophore complexes that span the visible spectrum. An RNA-fluorophore complex resembling enhanced GFP (EGFP), termed Spinach, emits a green fluorescence comparable in brightness to fluorescent proteins. Spinach is markedly resistant to photobleaching, and Spinach fusion RNAs can be imaged in living cells. These RNA mimics of GFP provide an approach to genetically encode fluorescent RNAs.
 

Massive Expansion of RNA Species in Tissues and in Circulation Revealed from RNA-Sequencing
Christopher E. Mason, PhD, Weill Medical College of Cornell University

Here we present some surprising findings from the ongoing work of three large-scale efforts in RNA-Seq and transcriptomics. To perfect the detection of expression-based molecular signatures using RNA-Seq and define the site-to-site variance in measurement, the FDA’s Sequencing Quality Control (SEQC) Consortium has performed sequencing on three platforms (Illumina, LifeTech, and 454) using the SEQC standardized samples with synthetic RNA spike-ins. The ABRF’s SEQC has also performed experiments on these standardized samples, but with additional testing across more platforms (IonTorrent, PacBio) of variables such as sample quality, input amount, and chemistry. Finally, the Non-Human Primate Reference Transcritpome (NHPRT) Resource is performing deep sequencing of 21 tissues across 15 primates to improve gene models, accelerate genome builds, and enable novel evolutionary comparisons. To date, we have also discovered tens of thousands of tissue-specific, species-specific and cross-species transcriptionally active regions (TARs) beyond existing gene annotations, which highlight the utility of using unbiased methods for transcriptome characterization.
 

A Transgenic Mouse Model for Imaging Single RNA Dynamics in Tissue
Hye Yoon Park, PhD, Albert Einstein College of Medicine

We have developed a novel mouse model for studying the dynamics of mRNA in live cells and tissues. The MS2-GFP labeling technique has been widely used to visualize single RNA in living cells. We extend the utility of the technique for single molecule imaging in the context of a whole-animal system. The MS2-GFP technique exploits the high affinity binding of the MS2 bacteriophage capsid protein (MCP) to the MS2 RNA binding site (MBS). MCP is fused with green fluorescent protein (GFP), and 24 repeats of MBS are inserted into the RNA of interest. In order to apply the technique for live tissue imaging, we genetically engineered two mouse models: MCP-GFP transgenic mouse, Tg(MCP-GFP), and Actb-MBS mouse. In the Actb-MBS mouse, MBS cassette is knocked into the 3' untranslated region (3' UTR) of the essential β-actin gene (Lionnet et al., Nature Methods, 8, 165, 2011). By crossing Tg(MCP-GFP) mouse and Actb-MBS mouse, we generated a hybrid mouse expressing fluorescent RNA in every cell and tissue where β-actin is present. The homozygous mouse is viable and exhibits no gross abnormalities despite the addition of 1.2 kbp MBS cassette and approximately 1.2 MDa of MCP-GFP to the essential β-actin mRNA. These results suggest that this technology could be applied to a wide range of genes with minimal perturbation. Using this mouse, we are investigating the dynamics of β-actin mRNA transport in acute brain slices using multiphoton microscopy. In addition, single-molecule tracking of mRNA is employed to understand the mechanism of mRNA localization in hippocampal neuron cultures. The mouse model combined with high-resolution imaging can provide important insights into the dynamic regulation of an endogenous gene in its native tissue environment.
 

Quantitation of tissue-specific target gene modulation using circulating RNA
Alfica Sehgal, Alnylam Pharmaceuticals, Inc., Cambridge, MA, USA

Pharmacologic target gene modulation is the primary objective for RNA antagonist strategies, including small interfering RNA (siRNA), microRNA (miRNA) therapeutics, and antisense oligonucleotide therapeutics. In clinical applications, monitoring tissue-specific target mRNA modulation requires tissue procurement from patients that is highly limited in availability, if even justified. Here we show that circulating RNAs encoding tissue-specific gene transcripts can be detected in biological fluids of humans and experimental animals. Surprisingly, we demonstrate that RNAi-mediated target gene silencing in the liver by systemic administration of siRNA results in quantitative reductions in serum mRNA levels which closely corroborate the degree and kinetics of tissue mRNA silencing, including proof of the RNAi mechanism of action. Further, administration of an anti-miRNA oligonucleotide directed against a liver-specific miRNA was found to result in decreased levels of the miRNA in circulation. This technique was extended to a different tissue, where silencing of a brain-expressed mRNA was monitored and quantified in cerebrospinal fluid following intraparenchymal infusion of a specific siRNA. This non-invasive method for monitoring tissue-specific RNA modulation could greatly advance clinical development of gene therapy and RNA-based therapeutics.
 
*Additional abstracts to come.