Computational protocol: Silencing SlMED18, tomato Mediator subunit 18 gene, restricts internode elongation and leaf expansion

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Protocol publication

[…] The cDNA and protein sequence date of tomato mediator of RNA polymerase II transcription subunit 18 was found in NCBI (http://www.ncbi.nlm.nih.gov/) and Sol Genomics Network (SGN, http://solgenomics.net/). MED18 in Solanum lycopersicum (Sl), Arabidopsis thaliana (At), Oryza sativa (Os), Solanum tuberosum (St), Zea mays (Zm), Saccharomyces. cerevisiae (Sc) and Human (Hs) were obtained from the NCBI databases. GenBank accession numbers for multiple sequence alignment and phylogenetic analysis are as follows: SlMED18 (XP_010321804), AtMED18 (NP_565534), OsMED18 (XP_015625319), StMED18 (XP_006364546), ScMED18 (NP_011618) and HsMED18 (NP_001120822). Multiple sequence alignment of MED18 in tomato with other species was conducted by DNAMAN 6.0 programs. Alignments of amino acid sequences of MED18 subunits proteins were made using Clustal X2.1. A phylogenetic tree of Mediator of RNA polymerase II transcription subunit 18 protein sequence in 7 different species was constructed using MEGA7 (http://megasoftware.net/) and the phylogenic analysis was inferred using the Neighbor-Joining method. [...] Using RNAiso Plus (Takara) in accordance with the instructions, we extracted Total RNA from wild type and MED18-RNAi plant tissues. cDNA was synthesized with oligo(dT)20 as primer by RNA that reverse-transcribed using M-MLV reverse transcriptase (Promega). In addition, the synthesized cDNA need to fold dilute three times with nuclease-free water for quantitative real-time PCR analysis. Additionally, the qRT-PCR analysis was conducted with the GoTaq qPCR Master Mix (Promega), 1.0 μL mixture primers, 1.0 μL cDNA, 3.0 μL ddH2O by CFX96™ Real-Time System (Bio-Rad, USA). We performed the NRT (no reverse transcription control) and NTC (no template control) to eliminate the effect by genomic DNA and the environment factor. The organ-specific expression analysis was detected using the SlCAC gene (accession number: SGN-U314153) which showed stable expression across diverse tissues, as internal standard. The 2−ΔΔCT method was used to performed relative gene expression levels analysis. In addition, each sample was repeated three times and standard curves were run at the same time. All primers we used were shown in Supplementary Table  that designed by Primer premier 6.24 software (http://www.premierbiosoft.com/crm/jsp/com/pbi/crm/clientside/ProductList.jsp). […]

Pipeline specifications

Software tools DNAMAN, Clustal W, MEGA, Primer Premier
Databases SGN
Applications Phylogenetics, Non-coding RNA analysis
Organisms Solanum lycopersicum