Protocol: low cost fast and efficient generation of molecular tools for small RNA analysis

Background Small RNAs are sequence-dependent negative regulators of gene expression involved in many relevant plant processes such as development, genome stability, or stress response. Functional characterization of sRNAs in plants typically relies on the modification of the steady state levels of these molecules. State-of-the-art strategies to reduce plant sRNA levels include molecular tools such as Target Mimics (MIMs or TMs), Short Tandem Target Mimic (STTMs), or molecular SPONGES (SPs). Construction of these tools routinely involve many different molecular biology techniques, steps, and reagents rendering such processes expensive, time consuming, and difficult to implement, particularly high-throughput approaches. Results We have developed a vector and a cloning strategy that significantly reduces the number of steps required for the generation of MIMs against any given small RNA (sRNA). Our pGREEN-based binary expression vector (pGREEN-DLM100) contains the IPS1 gene from A. thaliana bisected by a ccdB cassette that is itself flanked by restriction sites for a type IIS endonuclease. Using a single digestion plus a sticky-end ligation step, the ccdB cassette that functions as a negative (counter) selection system is replaced by a pair of 28 nt self-annealing primers that provide specificity against the selected target miRNA/siRNA. The method considerably reduces the number of steps and the time required to generate the construct, minimizes the errors derived from long-range PCRs, bypasses bottlenecks derived from subcloning steps, and eliminates the need for any additional cloning technics and reagents, overall saving time and reagents. Conclusions Our streamlined system guarantees a low cost, fast and efficient cloning process that it can be easily implemented into high-throughput strategies, since the same digested plasmid can be used for any given sRNA. We believe this method represents a significant technical improvement on state-of-the-art methods to facilitate the characterization of functional aspects of sRNA biology.

the BsmBI sites. On the other hand, the IPSA and IPSB fragments were amplified from the Arabidopsis thaliana genome using IPSA1/IPSA2 and IPSB1/IPSB2 primers, respectively, and fused to the Cm R -ccdB cassette by overlapping PCR, giving rise to the IPSA-Cm R /ccdB-IPSB fragment. Then, the 2x35S promoter and Nos terminator were PCR-amplified from a pBINX' plasmid [3], using 35S-F/35S-R and NosT-F/NosT-R primers, respectively, and fused to IPSA-Cm R /ccdB-IPSB fragment via overlapping PCR to produce the final DNA fragment containing 2x35S-IPSA-Cm R /ccdB-IPSB-NosT. Afterwards, this PCR product was ligated into the EcoRV site of the pGreen0029 derivative lacking the BsmBI site generated previously, and transformants were selected on LB plates supplemented with Cm. Finally, an inverted PCR was performed on the recombinant plasmid obtained to remove a BsmBI site present in the Cm R -ccdB cassette, using primers BsmBI-Ccdb-removeA/BsmBI-Ccdb-removeB and the NZYMutagenesis Kit. All primers used in this study are detailed in Supplementary Table S1. Unless otherwise stated, PCRs were performed using Q5 High-Fidelity DNA Polymerase (NEB, USA) following the manufacturer's instructions. A graphic overview of the process is included in Fig. S1.

RNA extraction and Northern blot
Total RNA was extracted as previously described [4] or using TRISURE (Bioline, UK) for RT-qPCRs. Northern blot analyses were carried out as previously described in [5], with some modifications. In brief, for low molecular weight Northern blot analyses used to detect miR319, 10 µg of total RNA was suspended into 2x RNA loading buffer (95% formamide, 18 mM EDTA pH 8.0, 0.025% sodium dodecyl sulfate (SDS), 0.01% bromophenol blue, 0.01% xylene cyanol) and denatured at 90ºC for 5 min. Afterwards, samples were analysed in a 7M urea, 0.5X TBE, 17% polyacrylamide gel run at 180 V until bromophenol blue runs out of the gel. Then, RNA was electro-blotted onto an Amersham Hybond-N + nylon membrane (GE Healthcare Life Sciences, USA) at 80 V for 1 hour in cold 0.5x TBE. After transfer, RNA was fixed onto the membrane using UV light (0.120 J) and dried for 1 h at 80ºC. After this step, the membrane was pre-hybridize in Church buffer (1% BSA, 1 mM EDTA, 0.5 M phosphate buffer, 7% SDS) for 1 h at 40ºC. Hybridization was carried out using the same buffer with the added probe and incubated overnight at 40ºC. The next day, the membrane was washed 4 times with a 2x SSC, 0.1% SDS solution at 40ºC (10 min per wash) and detection was carried out as previously described by [5]. For detection of the IPS1 transcript, we used the same protocol described above with samples separated into a 7M urea, 0.5X TBE, 5% polyacrylamide gel.

cDNA synthesis, RT-qPCRs and semi-quantitative PCRs
For quantification of mature miRNAs, we used the stem-loop RT-qPCR method [6]. Pulsed-RT (1 step at 16ºC of 30 min, followed by 60 cycles at 30ºC for 30 s, 42ºC for 30 s and 50ºC for 1 min, and 1 cycle of 85ºC for 5 min) was performed using Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA) with an specific RT stem-loop primer for miR319, and oligo (dT) primers.
Stem-loop RT-qPCRs were performed in a CFX96 detection system (Bio-Rad, USA) following a previously described protocol [6], using 2 µl of a 1/2 dilution of the cDNA generated previously. Primers used to detect miR319 are listed in Supplementary Table S1. For each of the TCP2, TCP4 and actin RT-qPCRs, we added 2 µl of a 1 in 2 dilution of the cDNA generated previously, to a reaction containing 5 µl of SsoFast EvaGreen (Bio-Rad, USA), 0.5 µl of each of the corresponding Forward and Reverse primers (10 µM), and 2 µl of H2O. RT-qPCRs were performed using a CFX96 detection system (Bio-Rad, USA) with a first denaturing step at 95ºC 1 min, followed by 40 cycles of 95ºC 10 s and 60ºC 17 s. In all cases actin was used as internal control. Relative expression was calculated using the 2 -∆∆Ct method [7]. Primers used to detect TCP2, TCP4 and actin are listed in Supplementary Table S1.
For semi-quantitative PCRs, we used 2 µl of the cDNA generated previously, and a non-saturating number of cycles (22 cycles). PCRs were performed using GoTaq DNA Polymerase (PROMEGA, USA) following the manufacturer's instructions. Primers IPSA1 and MIM319-R (Supplementary Table S1) were used for MIM319 detection.