Editing MicroRNA (section)

==Experimental detection and manipulation==
While researchers focused on miRNA expression in physiological and pathological processes, various technical variables related to microRNA isolation emerged. The stability of stored miRNA samples has been questioned.<ref name="Boeckel 616–617"/> microRNAs degrade much more easily than mRNAs, partly due to their length, but also because of ubiquitously present [[Ribonuclease|RNases]]. This makes it necessary to cool samples on ice and use [[Ribonuclease|RNase]]-free equipment.<ref name="pmid18388938">{{cite journal | vauthors = Liu CG, Calin GA, Volinia S, Croce CM | title = MicroRNA expression profiling using microarrays | journal = Nature Protocols | volume = 3 | issue = 4 | pages = 563–78 | year = 2008 | pmid = 18388938 | doi = 10.1038/nprot.2008.14 | s2cid = 2441105 }}</ref>

microRNA expression can be quantified in a two-step [[polymerase chain reaction]] process of modified [[RT-PCR]] followed by [[quantitative PCR]]. Variations of this method achieve absolute or relative quantification.<ref name="pmid16314309">{{cite journal | vauthors = Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL, Mahuvakar VR, Andersen MR, Lao KQ, Livak KJ, Guegler KJ | title = Real-time quantification of microRNAs by stem-loop RT-PCR | journal = Nucleic Acids Research | volume = 33 | issue = 20 | pages = e179 | date = November 2005 | pmid = 16314309 | pmc = 1292995 | doi = 10.1093/nar/gni178 }}</ref> miRNAs can also be hybridized to [[microarray]]s, slides or chips with probes to hundreds or thousands of miRNA targets, so that relative levels of miRNAs can be determined in different samples.<ref name="pmid16043497">{{cite journal | vauthors = Shingara J, Keiger K, Shelton J, Laosinchai-Wolf W, Powers P, Conrad R, Brown D, Labourier E | title = An optimized isolation and labeling platform for accurate microRNA expression profiling | journal = RNA | volume = 11 | issue = 9 | pages = 1461–70 | date = September 2005 | pmid = 16043497 | pmc = 1370829 | doi = 10.1261/rna.2610405 }}</ref> microRNAs can be both discovered and profiled by high-throughput sequencing methods ([[MicroRNA Sequencing|microRNA sequencing]]).<ref>{{cite journal | vauthors = Buermans HP, Ariyurek Y, van Ommen G, den Dunnen JT, 't Hoen PA | title = New methods for next generation sequencing based microRNA expression profiling | journal = BMC Genomics | volume = 11 | pages = 716 | date = December 2010 | pmid = 21171994 | pmc = 3022920 | doi = 10.1186/1471-2164-11-716 | doi-access = free }}</ref> The activity of an miRNA can be experimentally inhibited using a [[locked nucleic acid]] (LNA) [[oligonucleotide|oligo]], a [[Morpholino]] oligo<ref>{{cite journal | vauthors = Kloosterman WP, Wienholds E, Ketting RF, Plasterk RH | title = Substrate requirements for let-7 function in the developing zebrafish embryo | journal = Nucleic Acids Research | volume = 32 | issue = 21 | pages = 6284–91 | year = 2004 | pmid = 15585662 | pmc = 535676 | doi = 10.1093/nar/gkh968 }}</ref><ref>{{cite journal | vauthors = Flynt AS, Li N, Thatcher EJ, Solnica-Krezel L, Patton JG | title = Zebrafish miR-214 modulates Hedgehog signaling to specify muscle cell fate | journal = Nature Genetics | volume = 39 | issue = 2 | pages = 259–63 | date = February 2007 | pmid = 17220889 | doi = 10.1038/ng1953 | pmc = 3982799 }}</ref> or a 2'-O-methyl RNA oligo.<ref>{{cite journal | vauthors = Meister G, Landthaler M, Dorsett Y, Tuschl T | title = Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing | journal = RNA | volume = 10 | issue = 3 | pages = 544–50 | date = March 2004 | pmid = 14970398 | pmc = 1370948 | doi = 10.1261/rna.5235104 }}</ref> A specific miRNA can be silenced by a complementary [[antagomir]]. microRNA maturation can be inhibited at several points by steric-blocking oligos.<ref name="pmid24068553">{{cite journal | vauthors = Gebert LF, Rebhan MA, Crivelli SE, Denzler R, Stoffel M, Hall J | title = Miravirsen (SPC3649) can inhibit the biogenesis of miR-122 | journal = Nucleic Acids Research | volume = 42 | issue = 1 | pages = 609–21 | date = January 2014 | pmid = 24068553 | pmc = 3874169 | doi = 10.1093/nar/gkt852 }}</ref> The miRNA target site of an mRNA transcript can also be blocked by a steric-blocking oligo.<ref name="pmid17761850">{{cite journal | vauthors = Choi WY, Giraldez AJ, Schier AF | title = Target protectors reveal dampening and balancing of Nodal agonist and antagonist by miR-430 | journal = Science | volume = 318 | issue = 5848 | pages = 271–64 | date = October 2007 | pmid = 17761850 | doi = 10.1126/science.1147535 | bibcode = 2007Sci...318..271C | s2cid = 30461594 | doi-access = free }}</ref> For the "in situ" detection of miRNA, LNA<ref name="pmid16670427">{{cite journal | vauthors = You Y, Moreira BG, Behlke MA, Owczarzy R | title = Design of LNA probes that improve mismatch discrimination | journal = Nucleic Acids Research | volume = 34 | issue = 8 | pages = e60 | date = May 2006 | pmid = 16670427 | pmc = 1456327 | doi = 10.1093/nar/gkl175 }}</ref> or Morpholino<ref>{{cite journal | vauthors = Lagendijk AK, Moulton JD, Bakkers J | title = Revealing details: whole mount microRNA in situ hybridization protocol for zebrafish embryos and adult tissues | journal = Biology Open | volume = 1 | issue = 6 | pages = 566–69 | date = June 2012 | pmid = 23213449 | pmc = 3509442 | doi = 10.1242/bio.2012810 }}</ref> probes can be used. The locked conformation of LNA results in enhanced hybridization properties and increases sensitivity and selectivity, making it ideal for detection of short miRNA.<ref name="pmid16752924">{{cite journal | vauthors = Kaur H, Arora A, Wengel J, Maiti S | title = Thermodynamic, counterion, and hydration effects for the incorporation of locked nucleic acid nucleotides into DNA duplexes | journal = Biochemistry | volume = 45 | issue = 23 | pages = 7347–55 | date = June 2006 | pmid = 16752924 | doi = 10.1021/bi060307w }}</ref>

High-throughput quantification of miRNAs is error prone, for the larger variance (compared to [[Messenger rna|mRNAs]]) that comes with methodological problems. [[Messenger rna|mRNA]]-expression is therefore often analyzed to check for miRNA-effects in their levels (e.g. in<ref name="pmid19689821">{{cite journal | vauthors = Nielsen JA, Lau P, Maric D, Barker JL, Hudson LD | title = Integrating microRNA and mRNA expression profiles of neuronal progenitors to identify regulatory networks underlying the onset of cortical neurogenesis | journal = BMC Neuroscience | volume = 10 | pages = 98 | date = August 2009 | pmid = 19689821 | pmc = 2736963 | doi = 10.1186/1471-2202-10-98 | doi-access = free }}</ref>). Databases can be used to pair [[Messenger rna|mRNA]]- and miRNA-data that predict miRNA-targets based on their base sequence.<ref name="pmid17612493">{{cite journal | vauthors = Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP | title = MicroRNA targeting specificity in mammals: determinants beyond seed pairing | journal = Molecular Cell | volume = 27 | issue = 1 | pages = 91–105 | date = July 2007 | pmid = 17612493 | pmc = 3800283 | doi = 10.1016/j.molcel.2007.06.017 }}</ref><ref name="pmid17991681">{{cite journal | vauthors = Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ | title = miRBase: tools for microRNA genomics | journal = Nucleic Acids Research | volume = 36 | issue = Database issue | pages = D154–58 | date = January 2008 | pmid = 17991681 | pmc = 2238936 | doi = 10.1093/nar/gkm952 }}</ref> While this is usually done after miRNAs of interest have been detected (e. g. because of high expression levels), ideas for analysis tools that integrate [[Messenger rna|mRNA]]- and miRNA-expression information have been proposed.<ref name="pmid19420067">{{cite journal | vauthors = Nam S, Li M, Choi K, Balch C, Kim S, Nephew KP | title = MicroRNA and mRNA integrated analysis (MMIA): a web tool for examining biological functions of microRNA expression | journal = Nucleic Acids Research | volume = 37 | issue = Web Server issue | pages = W356–62 | date = July 2009 | pmid = 19420067 | pmc = 2703907 | doi = 10.1093/nar/gkp294 }}</ref><ref name="pmid22723856">{{cite journal | vauthors = Artmann S, Jung K, Bleckmann A, Beissbarth T | title = Detection of simultaneous group effects in microRNA expression and related target gene sets | journal = PLOS ONE | volume = 7 | issue = 6 | pages = e38365 | year = 2012 | pmid = 22723856 | pmc = 3378551 | doi = 10.1371/journal.pone.0038365 | veditors = Provero P | bibcode = 2012PLoSO...738365A | doi-access = free }}</ref>