disadvantages of cdna synthesis by hairpin loop method
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Explanation:
We recently reported that hairpin (or stem-loop) priming is better-suited than polyA tailing to generate cDNA for plant microRNA qPCR. One major limitation of this method is the need to perform individual cDNA synthesis reactions for the reference gene and test miRNAs. Here, we report a novel fusion primer that allows multiplexed hairpin cDNA synthesis of the most-commonly used reference gene, nucleolar small RNA U6, together with test miRNAs. We also propose the use of miR1515 as a house keeping control for tropical legumes. We show that multiplexed cDNA synthesis does not result in loss of sensitivity and reduces the amount of RNA required for miRNA gene expression assays.
Keywords: hairpin cDNA qPCR, U6, miR1515, multiplexing, miRNA
microRNAs (miRNAs) are short non-coding RNAs that regulate gene expression at the post-transcriptional level in both plants and animals (reviewed in refs. 1–4). In plants, their biogenesis starts with pri-miRNA transcripts which are capped and generally poly-adenylated and then cleaved by DICER-LIKE 1 to give rise to short duplex RNA molecules consisting of the mature miRNA (typically 21–22 nt in length) and miRNA* molecules. Both miRNA and miRNA* are then 2′ O-methylated at the 3′ end. Mature miRNAs are incorporated into RNA-induced silencing complexes and direct inhibition of translation or cleavage of complementary mRNAs (reviewed in refs. 5–7). Reliable quantification of individual mature miRNA levels is necessary to understand their biological roles. Hairpin/stem-loop priming8 and PolyA tailing9 are the two most commonly used cDNA synthesis methods for plant miRNA qPCR. We recently showed that hairpin priming method is better suited than polyA tailing method to synthesize cDNA for plant miRNA qPCR.10 One disadvantage of hairpin priming method is the use of specific RT primers for the cDNA synthesis and therefore individual cDNA synthesis reactions for each miRNA. This not only requires more input RNA, but also results in possible differences in cDNA synthesis efficiencies between test miRNAs and reference genes. It is crucial that the reference genes and test miRNAs undergo identical reaction conditions during cDNA synthesis and qPCR. This ensures equal efficiency of the reaction between the reference gene and the test miRNA(s) leading to accurate and reliable measurements.
In general, other small endogenous noncoding RNAs such as U6, U24 or U26 are used to normalize the expression of both plant and animal miRNAs.11,12 In Northern hybridization and polyA technique, the small nucleolar RNA U6 has been successfully used as a normalization control for a wide range of experiments (e.g., see refs. 13 and 14). However, no suitable method exists for U6 cDNA synthesis coupled with hairpin priming. To resolve this issue, we designed cDNA synthesis and qPCR primers for U6 from a highly conserved region of the gene (Fig. S1A) For U6 cDNA synthesis primer design, we added the “universal reverse” primer used in hairpin cDNA qPCR to the 5′ end of U6-specific reverse primer sequence (Fig. S1B). When this modified U6 reverse primer is used for cDNA synthesis, U6 expression can thereafter be assayed using hairpin cDNA qPCR technique, using U6-specific forward primer and universal reverse primer. “Hairpin-primed” cDNAs were generated using U6-specific cDNA synthesis primer from RNA samples obtained from different plant species. RNA preparations were obtained from rice (leaf and seed), wheat (leaf), Arabidopsis (seedlings), medicago (leaf), corn (leaf meristematic tissue), grape (root), soybean (root) and prairie cord grass (rhizomes). All cDNA synthesis reactions were performed on an ABI thermocycler (GeneAmp 9700). All qPCR assays were performed using a MX3000P thermocycler (Stratagene/Agilent Technologies) and SYBR Advantage qPCR premix (Product# 639676, Clontech). Three different dilutions (1/50, 1/500, 1/5,000) of cDNA from each plant species were examined for U6 expression by qPCR (Fig. 1). Suitability of these primers to synthesize cDNA and assay U6 expression were determined by examining the linearity of amplification, quality of the amplification curves and dissociation curves. We tested a range of monocotyledonous and dicotyledonous (both legumes and non-legumes) species. The primers we designed were able to successfully detect U6 expression with reliable amplification efficiencies and linearity in different plant species (Fig. 1).
figure psb-8-e24918-g1
Figure 1. Validation of the novel U6 fusion primer for cDNA synthesis on multiple plant species; (A) Arabidopsis, (B) soybean, (C) medicago, (D) grape root, (E) corn, (F) wheat, (G) rice leaf, (H) rice seed and (I) prairie