Pulsed SILAC as a Approach for miRNA Targets Identification in Cell Culture
Pulsed stable isotope labeling by amino acids in cell culture (pSILAC) comprises a variation of the classical SILAC proteomic methodology that enables the identification of short-term proteomic responses such as those elicited by micro RNAs (miRNAs). Here
- PDF / 251,371 Bytes
- 11 Pages / 504.57 x 720 pts Page_size
- 10 Downloads / 170 Views
1
Introduction MicroRNAs (miRNAs) have been shown to play a key role in the control of gene expression, modulating the expression of many proteins in a wide range of biological processes [1]. These small noncoding RNA molecules can individually regulate multiple target genes by binding to target sites found within the 3′ untranslated region of the targeted mRNA, resulting in posttranscriptional gene silencing of gene networks. The posttranscriptional effect results from degradation of the targeted mRNA or repression of its translation [2, 3]. Most of the targets contain perfect complementary sites in their 3′-untranslated regions to the seed sequence of the miRNA. This conserved seed sequence of typically six to eight nucleotides is often used as the main feature for miRNA target site prediction [4]. Several computational algorithms such as TargetScan [5] and Diana-MicroT [6] have been developed to predict targets based on a combination of different parameters including these sequences. However, additional factors can impede these bioinformatic approaches, resulting in false positives and thus
Paul C. Guest (ed.), Multiplex Biomarker Techniques: Methods and Applications, Methods in Molecular Biology, vol. 1546, DOI 10.1007/978-1-4939-6730-8_11, © Springer Science+Business Media LLC 2017
149
150
Daniella E. Duque-Guimarães et al.
limiting their usefulness [7]. Experimental methods are therefore required for robust target predication. Approaches used to date include in vitro luciferase reporter assays for individual miRNAs and targets or high-throughput experimental methods such as next-generation sequencing following immunoprecipitation [8]. However, these have their limitations and the methodology is still challenging. There is no clear consensus that a functional miRNA target will be always identified. Stable Isotope Labeling by Amino acids in Cell culture (SILAC) is a proteomic approach that has been used to identify miRNA targets and investigate the consequences on protein levels [9, 10]. SILAC is a quantitative method based on whole proteome metabolic labeling with stable isotope-labeled amino acids in cell culture [11]. This method is based on the introduction of a mass difference between two proteomes, which creates two versions of every peptide (e.g.,12C versus13C) that can be distinguished in mass spectrometry (MS)-based analysis [11, 12]. The classical version of SILAC is based on growing cells in media with natural “light” or “heavy” amino acids for several days or for approximately five cell doublings. This allows virtually complete incorporation of the heavy amino acids into cellular proteomes. SILAC-based approaches have also been used to identify miRNA targets in many cell systems including HeLa cells [9], as well as in SW480 colon cancer [13] and MiaPaCa-2 pancreatic cancer [14] cells. In recent years, an increasing number of studies have adopted a variation of the classical SILAC method termed pulsed SILAC (pSILAC) to investigate miRNA proteomic effects [10, 15, 16]. The pSILAC method is based
Data Loading...
