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Mirna Research Guide Mirna Guide Cover Final.Qxd 9/28/05 10:24 AM Page 4 miRNA_guide_cover_final.qxd 9/28/05 10:24 AM Page 3 miRNA Research Guide miRNA_guide_cover_final.qxd 9/28/05 10:24 AM Page 4 Contents Introduction to microRNAs and Experimental Overview Introduction to microRNAs . .1 miRNA Experimental Overview . .2 microRNA Isolation and Enrichment miRNA Isolation . .3 mirVana™ miRNA Isolation Kits . .3 “miRNA Certified” FirstChoice® Total RNA . .3 RecoverAll™ Total Nucleic Acid Isolation Kit . .3 miRNA Enrichment . .4 flashPAGE™ Fractionator System . .4 Global microRNA Expression Profiling miRNA Expression Profiling . .5 Overview of the mirVana™ Array System . .6 mirVana™ miRNA Labeling Kit . .7 mirVana™ miRNA Probe Set . .7 mirVana™ miRNA Bioarrays . .8 Detection and Quantification of Specific microRNAs mirVana™ miRNA Detection Kit . .9 mirVana™ miRNA Probe and Market Kit . .9 mirVana™ miRNA Probe Construction Kit . .10 mirVana™ qRT-PCR miRNA Detection Kit . .11 microRNA Functional Analysis miRNA Functional Analysis . .12 Anti-miR™ miRNA Inhibitors . .12 Pre-miR™ miRNA Precursor Molecules . .13 siPORT™ NeoFX™ Transfection Agent . .13 pMIR-REPORT™ miRNA Expression Reporter Vector . .13 microRNA Information Resources miRNA Resource . .14 miRNA Database . .14 miRNA Array Resource . .14 Introduction to microRNAs . .14 miRNA Application Guide . .15 Technical miRNA Seminars . .15 Highly Trained miRNA Technical Support Scientists . .15 miRNA e-Updates . .15 TechNotes Newsletter . .15 Reference Relative miRNA Expression Among Common Cell Types . .16 miRNA_guide_guts_final.qxd 9/28/05 10:26 AM Page 1 Introduction to microRNAs and Experimental Overview Introduction to microRNAs Overview of microRNA processing Small regulators with global impact miRNAs are transcribed as regions of longer RNA molecules that can be as long as 1000 nt (Figure 3). Description of microRNAs MicroRNAs (miRNAs) are evolutionarily conserved, small, noncoding RNA mol- The longer RNA molecules are processed in the nucleus into hairpin RNAs of ecules that regulate gene expression at the level of translation (Figure 1). 70–100 nt by the dsRNA-specific ribonuclease Drosha (Figure 4). The hairpin RNAs are transported to the cytoplasm via an exportin-5 dependent mechanism Although the first miRNA was described in 1993, only in the last few years has the where they are digested by a second dsRNA specific ribonuclease called Dicer. breadth and diversity of this gene class been uncovered (Figure 2). Vertebrate The resulting 19–23 mer miRNA is bound by a complex that is similar to the genomes are predicted to encode as many as 1000 unique miRNAs [1], which are RNA-Induced Silencing Complex (RISC) that participates in RNA interference predicted to regulate expression of at least 30% of genes [2]. (RNAi). In animals, the complex-bound, single-stranded miRNA binds specific mRNAs through sequences that are significantly, though not completely, com- miRNAs have been reported to be: plementary to the mRNA. By a mechanism that is not fully characterized—but • Critical in the development of organisms [3,4], which apparently does not usually involve mRNA degradation as in RNAi—the • Differentially expressed in tissues [5], bound mRNA remains untranslated, resulting in reduced expression of the corre- • Involved in viral infection processes [6], and sponding gene. • Associated with oncogenesis [7,8]. mRNA degradation Translational regulation Transcriptional regulation mRNA mRNA Active chromatin 5' Cap AAAAAAAAAAAAAA 3' 5'Cap AAAAAAAAAAAAAA 3' 3' 5' 3' 5' 3' 5' 3' 5' RISC* RISC Histone methylation miR-34 miR-124 * miR-34 ****** **** 5' Cap 5' Cap AAAAAAAAAAAAAA 3' 3' 5' AAAAAAAAAAAAAA 3' RISC Silent chromatin 3' 5' RISC* miR-39 Common in yeast, plants, Common in plants Common in animals and possibly animals miR-37 miR-38 Figure 1. Small RNAs—Key Regulators of Gene Expression. miR-36 miR-40 miR-35 350 5' 3' miR-41 5' 3' 5' 3' 300 Figure 3. Transcription of miRNAs. Approximately 60% of miRNAs are expressed independently, 15% 250 of miRNAs are expressed in clusters, and 25% are in introns. 200 150 100 Number of miRNA Publications 50 0 Pre-miRNA 1993-1999 2000 2001 2002 2003 2004 2005 NUCLEUS Year of Publication Drosha Exportin 5-Induced processing Figure 2. Exponential Growth of miRNA Publications. nuclear export Transcription * 5' 3' CYTOPLASM REFERENCES Pri-miRNA 1. Berezikov E, Guryev V, van de Belt J, Wienholds E, Plasterk RHA, Cuppen E (2005) Phylogenetic Shadowing and Computational Identification of Human microRNA Genes. Cell 120:21−24. 5' * 3' Dicer 2. Lewis BP, Burge CB, Bartel DP (2005) Conserved Seed Pairing, Often Flanked by Adenosines, Indicates RNA that Thousands of Human Genes are MicroRNA Targets. Cell 120: 15–20. processing *3' 5' 3. Ambros V (2003) MicroRNA pathways in flies and worms: growth, death, fat, stress, and timing. Cell Mature miRNA 113:673–676. within RISC 4. Chen CZ, Li L, Lodish HF, Bartel DP (2004) MicroRNAs modulate hematopoietic lineage differentiation. RISC Science 303:83–86. 5' 3' 5. Xu P, Vernooy SY, Guo M, Hay BA (2003) The Drosophila microRNA Mir-14 suppresses cell death and is Translationally repressed mRNA * required for normal fat metabolism. Curr Biol 13:790–795. 5' AAAAAAAAAAAAAA3' 6. Pfeffer S, Zavolan M, Grasser FA, Chien M, Russo JJ, Ju J, John B, Enright AJ, Marks D, Sander C, Tuschl 3' 5' T (2004) Identification of virus-encoded microRNAs. Science 304:734–736. Represents RISC* the mature 7. Calin GA, Sevignani C, Dumitru CD, Hyslop T, Noch E, Yendamuri S, Shimizu M, Rattan S, Bullrich F, Negrini miRNA M, Croce CM. (2004) Human microRNA genes are frequently located at fragile sites and genomic regions * involved in cancers. Proc Natl Acad Sci USA 101:2999–3004. 8. Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, Noch E, Aldler H, Rattan S, Keating M, Rai K, Rassenti L, Kipps T, Negrini M, Bullrich F, Croce CM. (2002) Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci USA Figure 4. The miRNA Processing Pathway. 99(24):15524-15529. www.ambion.com/miRNA 1 miRNA_guide_guts_final.qxd 9/28/05 10:26 AM Page 2 Introduction to microRNAs and Experimental Overview miRNA Experimental Overview As a unique class of small RNA molecules, miRNAs require special tools for accurate and sensitive analysis. Ambion’s scientists have developed a portfolio of products that provide a complete solution to accelerate identification and characterization of this gene class. These tools will help you uncover the relationship between mRNA and protein expression, potentially leading to a new segment of targets for diagnostics and therapeutics. Isolation and Enrichment Isolation of Small RNAs PRODUCTS Th e fi rst step in the analysis of miRNAs is purifi cation from a biological sample. •mirVana™ miRNA Isolation Kit Most RNA isolation kits were developed to recover messenger RNA, while ignor- ing smaller molecules such as miRNAs. Th e mirVana™ miRNA isolation products •“miRNA Certifi ed” FirstChoice® Total RNA are optimized to isolate all RNA species, including small RNA (less than 200 nt). •mirVana™ PARIS™ Kit Th e fl ashPAGE™ Fractionator can then be used to isolate RNA 10–40 nt in size, •fl ashPAGE™ Fractionator including mature miRNA. •RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE Detection & Quantifi cation Global miRNA Expression* Specifi c miRNA Expression miRNA Microarray Profi ling Sensitive Detection of Specifi c miRNAs Th e expression levels of miRNAs vary between tissues and developmental stag- Specialized tools provide sensitive, quantitative detection of miRNAs. Th ese es, and several miRNAs appear to be down-regulated in patients with chronic detection products provide highly accurate analysis of specifi c miRNAs, which lymphocytic leukemia, colonic adenocarcinoma, and Burkitt’s lymphoma. Eval- is critical for the verifi cation of data from microarray profi ling for in-depth uation of the global expression patterns of miRNAs provides key opportunities analysis of a small number of specifi c miRNAs. to identify regulation points for many diff erent biological processes. PRODUCTS PRODUCTS •mirVana™ qRT-PCR Detection Kit •mirVana™ miRNA Bioarrays •mirVana™ miRNA Detection Kit •mirVana™ miRNA Array Probe Set •mirVana™ Probe and Marker Kit •mirVana™ miRNA Array Labeling Kit •mirVana™ miRNA Probe Construction Kit Functional Analysis Functional Studies PRODUCTS miRNA functional analysis can be performed with protocols that are similar to standard genes. Up-regulation of the miRNAs can be conducted to identify •Pre-miR™ miRNA Precursor Molecules gain-of-function phenotypes; down-regulation or inhibition can be conducted •Anti-miR™ miRNA Inhibitors to identify loss-of-function phenotypes. Th e combination of up- and down- regulation can be used to identify genes that are regulated by specifi c miRNAs as •siPORT™ NeoFX™ Transfection Agent well as to identify cellular processes that are aff ected by specifi c miRNAs. •pMIR-REPORT™ miRNA Expression Reporter Vector *REFERENCES 1. Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, Noch E, Aldler H, Rattan S, Keating M, Rai K, Rassenti L, Kipps T, Negrini M, Bullrich F, Croce CM. (2002) Frequent dele- tions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci USA 99(24):15524–15529. 2. Michael MZ, O’Connor SM, van Holst Pellekaan NG, Young GP, James RJ (2003) Reduced accumulation of specifi c microRNAs in colorectal neoplasia. Molec Cancer Res 1:882–891. 3. Metzler M, Wilda M, Busch K, Viehmann S, Borkhardt A. (2003) High Expression of precursor microRNA-155/BIC RNA in children with Burkitt lymphoma. Genes Chrom Cancer 39:167–169. 4. Calin GA, Sevignani C, Dumitru CD, Hyslop T, Noch E, Yendamuri S, Shimizu M, Rattan S, Bullrich F, Negrini M, Croce CM. (2004) Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci USA 101:2999–3004. 5. Johnson, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A, Labourier E, Reinert KL, Brown D, Slack FJ.(2005) RAS is regulated by the let-7 microRNA family.
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