MicroRNAs (miRNAs) are 20–24 nucleotide RNAs that are predicted to play regulatory roles in animals and plants. Here we report a simple and sensitive real-time PCR method for quantifying the expression of plant miRNAs. Total RNA, including miRNAs, was polyadenylated and reverse-transcribed with a poly(T) adapter into cDNAs for real-time PCR using the miRNA-specific forward primer and the sequence complementary to the poly(T) adapter as the reverse primer. Several Arabidopsis miRNA sequences were tested using SYBR® Green reagent, demonstrating that this method, using as little as 100 pg total RNA, could readily discriminate the expression of miRNAs having as few as one nucleotide sequence difference. This method also revealed miRNA tissue-specific expression patterns that cannot be resolved by Northern blot analysis and may therefore be widely useful for characterizing miRNA expression in plants as well as in animals.
MicroRNAs (miRNAs) are released from long hairpin-containing miRNA precursors (pre-miRNAs) as 20–24 nucleotide single-stranded mature miRNAs and enter and guide the RNA-induced silencing complex (RISC) to identify target messages for silencing through either direct mRNA cleavage or translational repression (for recent reviews, see References 1 and 2). Such miRNA-mediated gene silencing has been predicted to regulate various developmental, metabolic, and cellular processes (1,2). Thus, the spatiotemporal expression patterns of miRNAs are important to the verification of their predicted functions. Northern blot analysis, the principal technique for detecting RNA transcripts, is however often insensitive for miRNAs (3,4). As miRNA identification efforts have been shifted from cloning to computation (5,6,7,8), a more sensitive and high-throughput method for quantifying the expression of a large number of in silico miRNAs is needed for verifying their authenticity and functions.
In addition to Northern blot analysis, many miRNA detection systems have recently been developed, such as mirVana™ miRNA Detection (Ambion, Austin, TX, USA), the invader assay-based detection (9), mirMASA™ miRNA profiling (Genaco Biomedical Products, Huntsville, AL, USA) (10), and modified microarrays (11,12,13,14,15,16). All these hybridization-based methods require large quantities of RNA. A more sensitive real-time PCR method has been developed for quantifying the expression of pre-miRNAs (17). However, this method cannot detect mature miRNAs. Here we report the establishment and validation of a simple, high-throughput real-time PCR method for quantifying plant miRNAs based on techniques and materials readily available to the general scientific community.Materials and Methods RNA Isolation, Polyadenylation, and Reverse Transcription
Total RNA was isolated from leaf and stem tissues of Arabidopsis thaliana (Columbia accession) using TRIZOL® reagent (Invitrogen, Carlsbad, CA, USA) and treated with RNase-free DNaseI (Promega, Madison, WI, USA). The treated total RNA (1 µg) was polyadenylated with ATP by poly(A) polymerase (PAP) at 37°C for 1 h in a 20-µL reaction mixture following the manufacturer's directions for the Poly(A) Tailing Kit (Ambion). After phenol-chloroform extraction and ethanol precipitation, the RNAs were dissolved in diethyl-pyrocarbonate (DEPC)-treated water and reverse-transcribed with 200 U SuperScript™ II Reverse Transcriptase (Invitrogen) and 0.5 µg poly(T) adapter [3′ rapid amplification of complementary DNA ends (RACE) adapter in the FirstChoice® RLM-RACE kit; Ambion] according to the manufacturer's protocols (Invitrogen).Real-Time PCR Primer Design and Melting Temperature Evaluation
Seven miRNAs were selected, covering a variety of miRNA sequence features. AthmiR159a and AthmiR161 each represent the single member in the corresponding miRNA families (3,18). AthmiR165a and AthmiR166a differ by 1 nucleotide (3), and AthmiR167a (3) and its two paralogues, AthmiR167c and AthmiR167d, have 1 to 2 nucleotide differences (5,18). AthmiR159a, AthmiR165a, and AthmiR166a are each located at the 3′ arm of their cognate pre-miRNAs, whereas all the other tested miRNAs are on the 5′ arm of the corresponding pre-miRNAs. Arabidopsis 5.8S ribosomal RNA (rRNA) was selected as the internal reference gene for PCR quantitation. The reverse primer for these miRNAs and 5.8S rRNA was a 3′ adapter primer (3′ RACE outer primer in the FirstChoice RLM-RACE kit), and the forward primer was designed based on the entire tested miRNA sequence. However, for those forward primers containing more than three G/C within the five 3′-end nucleotides, one or two As were added to the 3′ end of these primers to ensure their binding to the target site encompassing the miRNA sequence and Ts in the poly(T) adapter ((Figure 1)). For 5.8S rRNA, the forward primer contained sequences complementary to those located at the 3′ end. These primer designs were expected to result in 63–65 bp products. All primers used in this study were synthesized by MWG Biotech (Highpoint, NC, USA) and are listed in (Table 1). The true melting temperature (Tm) values of primers were experimentally determined from the thermal dissociation curves generated from the target primers and their corresponding antisense sequences using an ABI PRISM® 7000HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Briefly, 100 pmol each of the primer and its antisense oligonucleotide were mixed with 12.5 µL SYBR® Green PCR Master Mix (Applied Biosystems) in a total volume of 25 µL for DNA melting analysis with a programmed temperature ramp from 45° to 95°C in 5 min to produce a dissociation curve, from which the Tm was calculated.