1. Invitrogen Corporation 1600 Faraday Ave. Carlsbad, CA 92008 USA Tel: 760 603 7200 FAX: 760 602 6500 Toll Free Tel: 800 955 6288 E-mail: tech_service@invitrogen.com www.invitrogen.com A Novel method to profile miRNA expression in different organisms: Sensitive and specific miRNA qRT-PCR with the NCode™ SYBR® Green miRNA qRT-PCR Kit Sam An, Mark Landers, Mason Brooks, Pete Joszi, Ranjan J. Perera, and Christopher Adams Invitrogen Corporation, Carlsbad California MicroRNAs (miRNAs) are short 22-24 nucleotide RNAs that play a vital roles in regulating gene expression. These single stranded RNA molecules are incorporated into the RISC (RNA-inducing silencing complex) and regulate gene expression through various methods including translational inhibition, transcriptional cleavage and transcriptional gene silencing. Recent interest in these non-coding RNAs has been explosive because of its implication in cellular differentiation and disease genesis. Therefore, there is an urgent need to identify and validate miRNA expression in cell lines, tissues, and organs. qRT-PCR has become the standard for microarray data validation, as well as an invaluable tool for quantifying individual or subsets of miRNAs with the greatest sensitivity and accuracy. Most commercially available miRNA qRT- PCR systems employ proprietary, pre-designed miRNA-specific primers for cDNA synthesis by reverse transcription. Unfortunately, this approach requires that the sequence of the miRNA is publicly available and a commercial qRT-PCR assay has been developed for that specific sequence, limiting the availability of qRT-PCR assays for many model organisms as well as recently discovered miRNAs or proprietary miRNAs. Here, we describe a novel, universal, flexible and user-friendly qRT-PCR method, that was developed to measure and characterize miRNA expression in almost all organisms, starting from either total RNA and/or enriched miRNA. The NCode™ miRNA SYBR® Green qRT-PCR protocol is based on carefully optimized polyadenylation reaction with the reverse transcriptase, SuperScript™ III RT, in a “universal” 1 st strand cDNA synthesis reaction.. The miRNA specific amplification occurs during the PCR reaction where the sequence of the miRNA of interest is used as the target-specific PCR primer. Platinum® SYBR® qPCR Supermix combines Platinum® Taq DNA polymerase with SYBR® Green fluorescent dye, delivers excellent sensitivity in the quantification of target sequences. This protocol offers 7 logs of dynamic range for maximum sensitivity (detection down to 100 copies) and single nucleotide discrimination for distinguishing between closely related miRNA families. Abstract Figure 1– NCode TM miRNA qRT-PCR Overview Figure 2– Assay Performance (Dynamic Range/Sensitivity) Summary Figure 3– Closely Related miRNA Family Discrimination Figure 4– Validation of Microarray Data Through qRT-PCR miRNA target polyA tailing Hybridization of tagged primer Standard qPCR w/SYBR Green Highly Flexible System Only 1 primer needed No special probes Quick assay design Permits analysis of other small RNAs Accuracy over a broad dynamic range of samples Slope = -3.51 Y-int = 38.12 R2 = 0.995 miRNASequence Let – 7aUGAGGUAGUAGGUUGUAUAGUU Let – 7bUGAGGUAGUAGGUUGUGUGGUU Let – 7cUGAGGUAGUAGGUUGUAUGGUU Let – 7dAGAGGUAGUAGGUUGCAUAGU- Let – 7eUGAGGUAGGAGGUUGUAUAGU- The NCode™ SYBR® Green miRNA qRT-PCR Kit was used to profile synthetic let-7a-e miRNA templates from 100,000 copies using the let-7 A sequence as the primer. Using the annealing temperature of 65°C, the assay clearly discriminates the closely related family members. The closest mismatch (Let 7c) is approximately 10.5 C T s behind, which correlates to over 1000 fold difference in expression level. PrimersRelative Expression in BrainRelative Expression in Liver Mir-124A HIGH Ct = 23.55 LOW Ct = 31.79 Mir-128AHIGH Ct = 25.15 LOW Ct = 33.89 Mir-122A LOW Ct = 35.56 HIGH Ct = 21.55 Mir-194LOW Ct = 31.89 HIGH Ct = 25.99 Published microarray data was verified through miRNA qRT-PCR. 4 pairs of miRNA primers, 2 of which illustrated high expression level in either brain or liver tissue were used to perform the qPCR. The qPCR results correlated identically with the microarray data, as exhibited by C T values. Amplification plot of a synthetic miRNA using the NCode™ SYBR® Green qRT-PCR Kit. The assay exhibits a broad dynamic range up to seven logs (100M to 100 copies) and shows an excellent linear correlation between copy number and C T value.  The NCode TM miRNA qRT-PCR Assay offers unparalleled flexibility along with a simple protocol to detect/quantitate miRNAs. No proprietary primers are needed and can be used with any species on various small RNAs (incl. siRNAs, snoRNAs, etc.)  The NCode™ miRNA qRT-PCR Assay provides 7 logs of dynamic range and single nucleotide discrimination for maximum performance.