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• W2093799458 abstract "Dear Editor, microRNAs (miRNAs) are short non-coding RNAs that play crucial roles in plant development and responses to biotic and abiotic stresses. miRNA biogenesis starts with the transcription of a hairpin-forming pri-miRNA that is sequentially cleaved yielding a duplex RNA molecule consisting of the mature miRNA (typically 21–22 nt in length) and miRNA*. Typically, mature miRNAs are the active forms and are incorporated into different RNA-induced silencing complexes that regulate gene expression by inhibition of translation or cleavage of complementary mRNAs (reviewed in Axtell et al., 2011Axtell M.J. Westholm J.O. Lai E.C Vive la difference: biogenesis and evolution of microRNAs in plants and animals.Genome Biol. 2011; 12: 221Crossref PubMed Scopus (338) Google Scholar). Reliable quantification of mature miRNA levels is crucial to understand their function. Large-scale methods such as high throughput sequencing and miRNA microarrays are used primarily for miRNA discovery and global expression analyses, respectively, while Northern hybridization is the gold standard for assaying individual miRNAs. Northern hybridization requires large quantities of RNA, the use of radioactivity, and is generally less sensitive (Supplemental Table 1). qPCR assays are more sensitive, but mature miRNAs are too short to serve as templates in a classical RT–qPCR assay. Therefore, several modified methods for cDNA synthesis from mature miRNAs have been developed (reviewed in Benes and Castoldi, 2010Benes V. Castoldi M Expression profiling of microRNA using real-time quantitative PCR, how to use it and what is available.Methods. 2010; 50: 244-249Crossref PubMed Scopus (293) Google Scholar). Two most commonly used methods are (1) ‘polyA-tailing’, which involves in vitro polyadenylation followed by oligo dT-mediated reverse transcription (Supplemental Figure 1; Shi and Chiang, 2005Shi R. Chiang V.L Facile means for quantifying microRNA expression by real-time PCR.Biotechniques. 2005; 39: 519-525Crossref PubMed Scopus (619) Google Scholar), and (2) ‘hairpin priming’ (or ‘stem-loop RT’), which involves reverse transcription using a hairpin primer that is complementary to the 3’ end of miRNA (Supplemental Figure 1; Chen et al., 2005Chen C. Ridzon D.A. Broomer A.J. Zhou Z. Lee D.H. Nguyen J.T. Barbisin M. Xu N.L. Mahuvakar V.R. Andersen M.R. et al.Real-time quantification of microRNAs by stem-loop RT–PCR.Nucleic Acids Res. 2005; 33: e179Crossref PubMed Scopus (4049) Google Scholar; Varkonyi-Gasic et al., 2007Varkonyi-Gasic E. Wu R. Wood M. Walton E.F. Hellens R.P Protocol: a highly sensitive RT–PCR method for detection and quantification of microRNAs.Plant Methods. 2007; 3: 12Crossref PubMed Scopus (875) Google Scholar). cDNA generated by either method can be used in SYBR® green or Taqman® qPCR assays (Supplemental Table 1). Both these methods are used to assay the levels of plant miRNAs. However, plant miRNAs possess a 2’-O-methyl modification on the ribose of their 3’ termini catalyzed by HUA ENHANCER 1 (HEN1), a small RNA methyltransferase (Yu et al., 2005Yu B. Yang Z. Li J. Minakhina S. Yang M. Padgett R.W. Steward R. Chen X Methylation as a crucial step in plant microRNA biogenesis.Science. 2005; 307: 932-935Crossref PubMed Scopus (824) Google Scholar), and this modification protects against the addition of nucleotides to the 3’ end both in vivo and in vitro (Li et al., 2005Li J. Yang Z. Yu B. Liu J. Chen X Methylation protects miRNAs and siRNAs from a 3’-end uridylation activity in Arabidopsis.Curr. Biol. 2005; 15: 1501-1507Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar; Ren et al., 2012Ren G. Chen X. Yu B Uridylation of miRNAs by hen1 suppressor1 in Arabidopsis.Curr. Biol. 2012; 22: 695-700Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar; Zhao et al., 2012Zhao Y. Yu Y. Zhai J. Ramachandran V. Dinh T.T. Meyers B.C. Mo B. Chen X The Arabidopsis nucleotidyl transferase HESO1 uridylates unmethylated small RNAs to trigger their degradation.Curr. Biol. 2012; 22: 689-694Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). We reasoned that this might warrant a systematic comparison of the two methods to assay plant miRNAs. We used polyA and hairpin cDNA qPCR to assay (1) synthetic RNA molecules with and without 2’-O-methylation, (2) selected miRNAs in the small RNA methylation-deficient Arabidopsis mutant, hen1, (3) selected miRNAs in respective Arabidopsis overexpression lines, and (4) miR164 in soybean roots during nodulation. We synthesized cDNAs using the polyA-tailing method (‘polyA cDNA’) or hairpin priming method (‘hairpin cDNA’) from different quantities of synthetic RNA molecules with and without 2’-O methylation at the 3’ end nucleotide (subsequently referred to as ‘miR393–2’OMe’ and ‘miR393–2’OH’, respectively). The efficiency of cDNA synthesis was measured using SYBR green-based qPCR assays (Figure 1A–1D). For each cDNA synthesis method, we made pairwise comparison of Ct values between reactions containing cDNA synthesized from identical amounts of miR393–2’OH or miR393–2’OMe RNA template (Supplemental Table 2). In hairpin cDNA qPCR, there was virtually no difference in Ct values (P = 0.87; Student’s t-test) between reactions using miR393–2’OH or miR393–2’OMe (compare Figure 1A with 1C; Supplemental Table 2). In contrast, polyA cDNA qPCR assays using miR393–2’OMe had ∼8 Ct values (P < 0.0001; Student’s t-test) higher than those using miR393–2’OH (compare Figure 1B with 1D; Supplemental Table 2). We obtained similar results when assaying mixtures of different ratios of miR393–2’OH and miR393–2’OMe RNA molecules (Supplemental Figure 2). Together, these results suggest that hairpin cDNA qPCR was equally efficient in assaying both 2’-OH and 2’-OMe RNA molecules, while polyA cDNA qPCR was less efficient in assaying 2’-OMe RNA molecules. This is consistent with the observation that 2’-OMe inhibits in vitro polyadenylation of miRNAs (Li et al., 2005Li J. Yang Z. Yu B. Liu J. Chen X Methylation protects miRNAs and siRNAs from a 3’-end uridylation activity in Arabidopsis.Curr. Biol. 2005; 15: 1501-1507Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar). However, it should be noted that polyA cDNA qPCR appeared to be more sensitive (2’-OH RNA molecules) than or as sensitive (2’-OMe RNA molecules) as the hairpin cDNA method based on raw Ct values (Figure 1 and Supplemental Table 2). We examined the efficiency of these methods in assaying selected miRNAs (miR160, 164, 166, and 393) in wild-type and the 2’O-methylation-deficient hen1 (hen1–1; Yu et al., 2005Yu B. Yang Z. Li J. Minakhina S. Yang M. Padgett R.W. Steward R. Chen X Methylation as a crucial step in plant microRNA biogenesis.Science. 2005; 307: 932-935Crossref PubMed Scopus (824) Google Scholar) Arabidopsis seedlings. The hen1 mutant accumulates very low levels of mature miRNAs and those that accumulate have additional nucleotides of varying length at the 3’ end (Li et al., 2005Li J. Yang Z. Yu B. Liu J. Chen X Methylation protects miRNAs and siRNAs from a 3’-end uridylation activity in Arabidopsis.Curr. Biol. 2005; 15: 1501-1507Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar). Hairpin cDNA qPCR assays indicated that the levels of all four miRNAs were lower in hen1 seedlings compared to wild-type (Figure 1E) as previously reported. However, polyA cDNA qPCR assays indicated that miR160, 166, and 393 accumulated at higher levels in hen1 compared to Col (Figure 1E). The polyA-tailing method perhaps did not distinguish canonical miRNA molecules from those with additional nucleotides in the 3’ end, resulting in artificially higher abundance in hen1. Indeed, dissociation curves from these qPCR assays showed broader peaks (Supplemental Figure 3), indicating the presence of amplicons of different lengths and/or nucleotide composition. On the other hand, since hairpin priming depends on the last 6 nts at the 3’ end of mature miRNAs, this method could distinguish canonical miRNA molecules from the modified molecules in hen1 RNA preparations. We also examined miRNA expression in three Arabidopsis transgenic lines overexpressing miR160, 164, or 393 using polyA and hairpin cDNA qPCR assays. Results from hairpin cDNA qPCR indicated that the respective miRNAs were expressed at significantly higher levels in overexpression (OX) lines compared to Col (Supplemental Figure 4). However, polyA cDNA qPCR indicated significantly lower levels of miR393 in the OX line compared to Col (Supplemental Figure 4) while being able to detect overexpression of miR160 (levels comparable to hairpin cDNA qPCR) and 164 (levels lower than hairpin cDNA qPCR) in the respective OX lines. It is tempting to speculate that the miRNA methylation machinery might have kept up with miR393 in the miR393OX line which had the least overexpression (three to fivefold), but not with miR160 and 164 in their respective OX lines that had ∼25–60-fold overexpression. The presence of sufficient amounts of unmethylated miR160 and 164 molecules might have enabled polyA cDNA qPCR to detect them in the respective overexpression lines. Finally, we compared the accuracy of hairpin and polyA cDNA qPCR assays to Northern hybridization. We used all three methods to assay the expression of miR164 along a time course of Bradyrhizobium japonicum inoculation in soybean roots (Figure 1F and 1G). We compared each qPCR assay to Northern hybridization by calculating correlation coefficients between expression levels. The time course of miR164 expression assayed by hairpin cDNA qPCR was largely in agreement with Northern hybridization in both mock (R2 = 0.91) and B. japonicum-inoculated (R2 = 0.68) samples. However, expression pattern assayed by polyA cDNA qPCR was not in agreement with that of Northern hybridization (R2 values of –0.53 and –0.29 for mock and B. japonicum-inoculated samples, respectively). The disagreement is not due to the difference in ability of these methods to distinguish miRNA family members with different sequences, as all known miR164 precursors produce identical mature miRNAs in soybean (Turner et al., 2012Turner M. Yu O. Subramanian S Genome organization and characteristics of soybean microRNAs.BMC Genomics. 2012; 13: 169Crossref PubMed Scopus (63) Google Scholar). Therefore, hairpin cDNA qPCR can detect miRNA expression as reliably as Northern hybridization using much lower amounts of total RNA consistent with previous reports (e.g. Varkonyi-Gasic et al., 2007Varkonyi-Gasic E. Wu R. Wood M. Walton E.F. Hellens R.P Protocol: a highly sensitive RT–PCR method for detection and quantification of microRNAs.Plant Methods. 2007; 3: 12Crossref PubMed Scopus (875) Google Scholar). Together, these results suggested that hairpin cDNA qPCR is better suited to quantifying canonical plant miRNAs and that the use of polyA cDNA qPCR might lead to erroneous abundance measurements, perhaps depending on the methylation status of mature miRNAs. It is emerging that miRNA methylation might be a crucial mechanism that regulates miRNA stability/activity in plants (Ren et al., 2012Ren G. Chen X. Yu B Uridylation of miRNAs by hen1 suppressor1 in Arabidopsis.Curr. Biol. 2012; 22: 695-700Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar; Zhao et al., 2012Zhao Y. Yu Y. Zhai J. Ramachandran V. Dinh T.T. Meyers B.C. Mo B. Chen X The Arabidopsis nucleotidyl transferase HESO1 uridylates unmethylated small RNAs to trigger their degradation.Curr. Biol. 2012; 22: 689-694Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). This makes it necessary to specifically assay methylated miRNA molecules, as they are the active forms. We conclude that hairpin cDNA qPCR is better suited to assaying plant miRNAs compared with polyA cDNA qPCR, despite the latter’s apparent higher sensitivity. We developed an Excel spreadsheet that designs the hairpin primer and qPCR forward primer when the mature miRNA sequence is provided (supplemental file: miRNAqP-primerdesigntool.xlsx). Supplementary Data are available at Molecular Plant Online." @default.
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• W2093799458 title "Hairpin Priming Is Better Suited than In Vitro Polyadenylation to Generate cDNA for Plant miRNA qPCR" @default.
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