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RNA interference (RNAi) is a mechanism in which the introduction of small interfering RNAs (siRNAs) into a diverse range of organisms and cell types causes degradation of the complementary mRNA. Applications of RNAi include gene function and pathway analysis, target identification and validation, and therapeutics. There is a need to develop reliable and easy-to-use assays to evaluate siRNA delivery efficiency and distribution, study pathways, and stability of siRNAs in cells (posttransfection) and in animals (postinjection). We have leveraged the Applied Biosystems TaqMan®-based stem-loop RT-PCR technology, originally developed for quantification of endogenous microRNAs in cells, to fulfill these needs. The application protocols developed enable robust quantification of siRNA, including chemically modified siRNA molecules, in vitro and in vivo.

MicroRNAs have been increasingly implicated in human cancer and interest has grown about the potential to use microRNAs to combat cancer. Lung cancer is the most prevalent form of cancer worldwide and lacks effective therapies. Here we have used both in vitro and in vivo approaches to show that the let-7 microRNA directly represses cancer growth in the lung. We find that let-7 inhibits the growth of multiple human lung cancer cell lines in culture, as well as the growth of lung cancer cell xenografts in immunodeficient mice. Using an established orthotopic mouse lung cancer model, we show that intranasal let-7 administration reduces tumor formation in vivo in the lungs of animals expressing a G12D activating mutation for the K-ras oncogene. These findings provide direct evidence that let-7 acts as a tumor suppressor gene in the lung and indicate that this miRNA may be useful as a novel therapeutic agent in lung cancer.

SummaryMicroRNAs (miRNAs) are regulatory RNAs found in multicellular eukaryotes, including humans, where they are implicated in cancer. The let-7 miRNA times seam cell terminal differentiation in C. elegans. Here we show that the let-7 family negatively regulates let-60/RAS. Loss of let-60/RAS suppresses let-7, and the let-60/RAS 3′UTR contains multiple let-7 complementary sites (LCSs), restricting reporter gene expression in a let-7-dependent manner. mir-84, a let-7 family member, is largely absent in vulval precursor cell P6.p at the time that let-60/RAS specifies the 1° vulval fate in that cell, and mir-84 overexpression suppresses the multivulva phenotype of activating let-60/RAS mutations. The 3′UTRs of the human RAS genes contain multiple LCSs, allowing let-7 to regulate RAS expression. let-7 expression is lower in lung tumors than in normal lung tissue, while RAS protein is significantly higher in lung tumors, providing a possible mechanism for let-7 in cancer.

Although the majority of gene function studies center themselves around protein-encoding RNAs, the study of non-protein-encoding RNAs is becoming more widespread because of the discovery of hundreds of small RNA termed micro (mi) RNA that have regulator functions within cells. Currently, over 470 human miRNA genes are predicted to exist and are annotated within the "miRBase" public miRNA database ( http://microrna.sanger.ac.uk/ ). There is no denying that short interfering (si) and short hairpin (sh) RNAs have revolutionized how scientists approach understanding gene function; however, si and shRNAs are not effective for analyzing the function of miRNAs given that miRNAs are typically short (17-24 bases). In turn, new sets of agents that allow for the expression of miRNA above endogenous levels and inhibition of miRNAs have become a valuable technology for the study of these small regulatory RNAs. In this chapter, we provide step-by-step methods on how to utilize synthetic precursor and antisense inhibitor molecules for understanding miRNA function.

Knockdown of cellular RNA using short interfering RNA has enabled researchers to perform loss-of-function (LOF) experiments in a wide variety of cell types and model systems. RNA interference techniques and reagents have made possible experiments that test everything from the analysis of function of single genes to screening for genes that are involved in critical biological pathways on a genome-wide scale. Although siRNA experiments are generally common practice in research laboratories, it is still important to keep in mind that many factors can influence efficacy of knockdown. A properly designed siRNA, optimized protocols of siRNA delivery, and an appropriate and well-optimized readout are all critical parameters for ensuring the success of your experiment. In this chapter, we provide step-by-step procedures for performing an siRNA knockdown experiment from cell culture to analysis of knockdown using quantitative real-time PCR.

Chromosomal translocations and corresponding gene fusions play an important role in the initiation of tumorigenesis and these processes have been strongly associated with distinct tumor subtypes. The recent association of ALK, ROS and RET fusion transcripts as lung tumor therapy predictive biomarkers has increased the need of a technology that could detect these biomarkers starting from limited amount of material. Life Technologies and OncoNetwork consortium collaborated for the development of a lung fusion panel based on Ion AmpliSeq™ RNA chemistry. The OncoNetwork consortium is comprised of twelve -translational cancer research institutes with many years of experience in adopting the latest molecular techniques for lung therapy research. Material and Methods: Consortia's requirements for the panel development : 1) detect all variants of ALK, ROS1, or RET fusion transcripts described in COSMIC in a single reaction using 10 ng of total RNA 2) Include 5′ and 3′ ALK, ROS1, RET gene expression assays as an indicator of a translocation at this gene. 3) Include endogenous RNA assay controls to determine if the quality of the results could be affected by RNA quality. 4) Provide similar results on archived FFPE samples tested by FISH. Human lung adenocarcinoma cell lines H2228 (EML4-ALK positive), HCC78 (SLC4A2-ROS1 positive ) ,LC-2/ad (CCDC6-RET positive) and Ambion® FirstChoice® Human Brain Reference (HBR) RNA was used as positive and negative control for the study. Formalin fixed paraffin embedded (FFPE) tissue was isolated using different extraction methods. After amplification using Ion AmpliSeq™ RNA chemistry samples were sequenced on the Ion Torrent PGM™ sequencer using the Ion PGM™ 200 Sequencing Kit. The presence of the fusions was confirmed by custom TaqMan® gene expression assays when possible. Preliminary results of the panel on ALK or ROS1 or RET positive cell lines and FFPE archived cancer research samples gave good concordance with FISH results. Expected negative samples were confirmed negative. We found a KIF5B-RET fusion positive sample in a sample not previously tested for RET fusions. Two of the expected positive samples by FISH were found negative due to limited amount to tumor cells present in the sample. Cell line RNA dilutions were performed to determine the panel's limit of detection. We demonstrate a limit of detection of 1 % tumor RNA in the presence of 99 % normal RNA using the panel with 10 ng of RNA extracted by cell lines. Gene expression controls work well as an indicator of the RNA quality and of the translocation presence. The Ion AmpliSeq™ RNA lung cancer fusion panel workflow is easier and faster to perform in comparison to the FISH method. The results obtained to date are highly encouraging for panel to be used in the clinical research setting. More data needs to be analyzed before a final conclusion is made. Citation Format: Susan M. Magdaleno, Angie Cheng, Rosella Petraroli, Orla Sheils, Bastiaan Tops, Delphine Le Corre, Henriette Kurth, Helene Blons, Eliana Amato, Andrea Mafficini, Anna Maria Rachiglio, Anne Reimann, Christoph Noppen, Chrysanthi Ainali, Jin Katayama, Renato Franco, Harriet Feilotter, Jeoffrey Schageman, Ian Cree, Andrew Felton, Jose Luis Costa, Alain Rico, Aldo Scarpa, Jose Carlos Machado, Kazuto Nishio, Nicola Normanno, Marjolijn Ligtenberg, Cecily P. Vaughn, Ludovic Lacroix, Pierre Laurent-Puig. The OncoNetwork Consortium: A global collaborative research study on the development and verification of an Ion AmpliSeq RNA gene lung fusion panel. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3575. doi:10.1158/1538-7445.AM2014-3575

Gene expression profiling of cancer cells has become increasingly common over the past five years. Especially important is identifying changes in gene expression caused by drug treatment leading to side populations with altered pathways. These populations can escape drug treatment and lead to recurrence. Characterizing gene expression of proliferating cells requires a method of identifying proliferation while simultaneously maintaining high quality RNA. Typically, cells are screened in a drug panel in the presence of tritiated thymidine or BrdU to identify the drug resistance in the proliferating population. Proliferation detection with these methods is neither rapid nor does it yield high quality RNA for profiling. A new proliferation method based on click chemistry using an alkyne modified thymidine analog (EdU), which can be used to rapidly identify replicating cells appears ideally suited for this application; however, copper used for the catalysis of the click reaction causes undesired degradation of RNA. Recent improvements to the copper based click reaction are presented here whereby the copper is tightly controlled, resulting in higher quality RNA useful for sequencing. In this study we demonstrate gene expression changes in the proliferating population of a drug resistant cancer cell line compared to the parental cell lines. Proliferating cells were identified using an improved click reaction comprising of a copper(I) ligand combined with a picolyl modified dye azide detection reagent. Total RNA from these cells was isolated and gene expression was assessed by targeted RNA sequencing using an RNA Apoptosis panel and an RNA Cancer panel using proton based sequencing technology. Use of an improved copper “safe” click reaction for detection of nascent DNA, allows for detection of incorporated click analog for measuring proliferation and subsequent isolation of high quality RNA for use in sequence analysis. Citation Format: Scott T. Clarke, Angie Cheng, Aleksey Rukavishnikov, Michelle Yan, Upinder Singh, Kyle Gee, Susan Magdaleno. Gene expression of EdU proliferating cells made possible with improved click chemistry. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4033. doi:10.1158/1538-7445.AM2013-4033