Characterization

Description(s):

Newly discovered microRNAs (miRNAs) are important to the regulation of gene expression, and up to 30% of mammalian genes may be regulated by miRNAs. So far, more than 400 miRNAs have been identified in the human genome and many of them are only different in one or a few nucleotides. The expression of mature miRNAs is tissue-specific and the abundance of miRNAs varies over several orders of magnitude. More importantly, mis-regulation of miRNA expression may contribute to human cancers. Systematic profiling of miRNA expression plays the key role in the research of a number of cancers.

Signosis' Human miRNA array I allows you to profile 60 of the most popular miRNAs and their isoforms.

Applicable Grid:

List of Applicable miRNAs

let-7a-5p	let-7b-5p	let-7c-5p	let-7d-5p	let-7e-5p	let-7f-5p	let-7g-5p	let-7i-5p	miR-1	miR-7-5p
miR-9-5p	miR-10a-5p	miR-15a-5p	miR-15b-5p	miR-16-5p	miR-17-5p	miR-18a-5p	miR-18b-5p	miR-19a-3p	miR-19b-3p
miR-20a-5p	miR-21-5p	miR-25-3p	miR-28-5p	miR-34a-5p	miR-99a-5p	miR-122a-5p	miR-124a-3p	miR-125a-5p	miR-125b-5p
miR-126-3p	miR-131	miR-133a-3p	miR-133b	miR-143-3p	miR-145-5p	miR-146a-5p	miR-146a-5p	miR-148a-3p	miR-155-5p
miR-181a-5p	miR-181b-5p	miR-181c-5p	miR-182-5p	miR-192-5p	miR-194-5p	miR-195-5p	miR-199a-5p	miR-199a-5p	miR-199a*-3p
miR-200a-3p	miR-200c-3p	miR-204-5p	miR-206	miR-216-5p	miR-223-3p	miR-224-5p	miR-342-3p	mir-368	miR-375

Principle

miRNA is different from large messenger RNA in three aspects; (1) miRNAs are small size molecules with quite a big difference in abundance, (2) mature miRNAs co-exist with their precursor pre-miRNA and pri-miRNA, which only differ in length, and (3) many miRNAs are very closely related in sequences, such as isoforms, only differing by one or a few nucleotides. Therefore, the conventional mircoarray technologies cannot directly be applied to analyzing these molecules. A number of miRNA microarray products are commercially available, but they are either tedious in requiring pre-isolation of microRNA, lack the discriminative power to differentiate between isoforms, or are not sensitive enough to monitor low abundant miRNAs.

In our array assay, each miRNA molecule is targeted by two oligos that hybridize with the target miRNA to form a RNA/DNA duplex. When the sequences are perfectly matched, the oligos are aligned with the miRNA and the joint can be ligated by DNA ligase (figure 1). A single nucleotide difference among miRNAs will block either the hybridization or the ligation; Thus, miRNA isoforms can be differentiated. Due to the small size of miRNA, the hybrid might not be stable; Therefore, we introduce the stacking sequences. By extending these two oligos along with their complementary oligos, the stability is increased. Once the pair of oligos is ligated, the ligated molecules are subjected to linear amplification via T7 transcription into RNA in the presence of biotin-UTP, which are used as probes for array hybridization. To differentiate each isoform, we assigned unique tag sequences to the ligation oligos, so that single nucleotide differences are converted into unique tag sequences. In this way, each isoform can be easily distinguished by array hybridization.

The assay procedure includes three steps: (1) mix the miRNA with provided oligos to form miRNA/oligo hybrids; (2) select the hybrids and remove free oligos, and ligate miRNA-directed pairing of oligos to become a single DNA; and (3) amplify the ligated DNA with T7 transcription.

Data

miRNA analysis of miRNA expression in human brain and liver. 
5µg RNA was incubated and annealed with DNA oligo mix. DNA-RNA hybrids were selected and ligated. The ligated molecules were amplified and labeled with T7 RNA polymerase in the presence of biotin-UTP. The labeled RNAs were hybridized with miRNA array, and detected with chemiluminescence imaging system.

Literature

View user manual

Citations

1. MicroRNA-21 is Induced by Rapamycin in a Model of Tuberous Sclerosis (TSC) and Lymphangioleiomyomatosis(LAM). Trindade AJ, Medvetz DA, Neuman NA, Myachina F, Yu J, Priolo C, Henske EP. PLoS One. 2013;8(3):e60014. 

2. Prevention of Chlamydia-induced infertility by inhibition of local caspaseactivity.Igietseme JU, Omosun Y, Partin J, Goldstein J, He Q, Joseph K, Ellerson D, Ansari U, Eko FO, Bandea C,Zhong G, Black CM. J Infect Dis. 2013 Apr; 207(7):1095-104. doi: 10.1093/infdis/jit009. Epub 2013 Jan 9.

3. Induction of MiR-17-3p and MiR-160a by TNFα and LPS. Jiang X, Li N. Cell Biochem Funct. 2011 Mar;29(2):164-70.

4. Downregulation of Inflammatory MicroRNAs by Ig-like Transcript 3 Is Essential for the Differentiation of Human CD8+ T Suppressor Cells. Chang CC, Zhang QY, Liu Z, Clynes RA, Suciu-Foca N, Vlad G. J Immunol., 2012 Apr;188(7):3042-52.

5. Molecular basis of cardiac endothelial-to-mesenchymal transition (EndMT): Differential expression of microRNAs during EndMT.Ghosh AK, Nagpal V, Covington JW, Michaels MA, Vaughan DE. Cell Signal. 2012 May;24(5):1031-6.

6. Microrna Expression Is Dysregulated In Lymphangioleiomyomatosis (LAM). Trindade AJ, Myachina F, Medvetz D, Neuman N, Henske EP. Am. J. Respir. Crit. Care Med., 2011 May; 183: A3660.

7. MicroRNA27a Regulates Cardiomyocytic Apoptosis During Cardioplegia-Induced Cardiac Arrest by TargetingInterleukin 10-Related Pathways. Yeh CH, Chen TP, Wang YC, Lin YM, Fang SW. Shock. 2012 Dec;38(6):607-14.

8. MTORC1-Dependent Dysregulated Expression Of MicroRNA Contributes To The Pathogenesis Of Lymphangioleiomyomatosis (LAM) Trindade AJ, Medvetz DA,  Neuman NA, Myachina F, Zhang E, Yu J, Henske EP. Am. J. Respir. Crit. Care Med., 2012 May; 185: A6821.

9. Curcumin inhibits the proliferation and invasion of human osteosarcoma cell line MG-63 by regulating miR-138, D Yu, F An, X He, X Cao. International Journal of Clinical and Experimental  2015. Int J Clin Exp Pathol. 2015; 8(11): 14946–14952. Published online 2015 Nov 1.

10. Molecular basis of cardiac endothelial-to-mesenchymal transition (EndMT): Differential expression of microRNAs during EndMT. Ghosh AK, Nagpal V, Covington JW, Michaels MA, Vaughan DE. Cell Signal. 2012 May;24(5):1031-6.

11. ST Depression, Arrhythmia, Vagal Dominance, and Reduced Cardiac Micro-RNA in Particulate-Exposed Rats. Farraj AK, Hazari MS, Haykal-Coates N, Lamb C, Winsett DW, Ge Y, Ledbetter AD, Carll AP, Bruno M, Ghio A, Costa DL. Am J Respir Cell Mol Biol. 2011 Feb;44(2):185-96.

12. Differential profiling of human red blood cells during storage for 52 selected microRNAs.Meganathan Kannan, Chintamani Atreya.Kannan M, Atreya C. Transfusion. 2010 Jul;50(7):1581-8.

13. Circulating Blood MicroRNAs In The Diagnosis Of Lung Cancer.Le W, Wang W, Ghani M, Upadhyay D.Am. J. Respir. Crit. Care Med., May 2010; 181: A3482.

14. Membrane array–based differential profiling of platelets during storage for 52 miRNAs associated with apoptosis.Kannan M, Mohan KV, Kulkarni S, Atreya C.Transfusion. 2009 Jul;49(7):1443-50