Mesa green 231qpcr master mix plus

Total RNA extraction from M. truncatula roots was performed using the innuPREP RNA kit (Analytik Jena AG, Germany) and quantified using a DeNovix Ds-11+Spectrophotometer (DeNovix Inc., United States). cDNA synthesis was performed using the reverse transcriptase SuperScriptII (Invitrogen by Thermo Fisher Scientific, Germany) as described before (Kuhn et al., 2010 (link)). Quantitative real-time expression analyses were carried out using the MESA Green 231qPCR Master Mix Plus (Eurogentec, Germany) in a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories GmbH, Germany). A total of 1 μl cDNA (1:5) was used per well as template with the following PCR protocol: 5 min 95°C, 15 s 95°C, 20 s 56°C, 30 s 72°C (40 cycles). Plant transcript levels and transcript levels of the translation elongation factor 1-alpha of R. irregularis (RiTEF1α, DQ282611) were normalized to the translation elongation factor 1-alpha of M. truncatula (MtTEF1α, Medtr6g021800) while fungal transcripts were normalized to RiTEF1α. Transcript levels of genes were determined in three technical replicates in each independent biological replicate. Numbers of biological replicates are indicated in the corresponding figure legends. All primers are listed in Supplementary Table S2.

Total RNA was extracted using the innuPREP RNA Kit (Analytik Jena AG). cDNA was synthesized as described in Kuhn et al. (2010) (link) with the reverse transcriptase SuperScript II (Invitrogen, United States). Control PCRs were carried out using the StActin gene (XM_006345899) to check for the absence of genomic DNA contamination in the cDNA samples. Real time expression analyses were carried out using an iCycler MyIQ (Bio-Rad, United States) and MESA Green 231qPCR Master Mix Plus (Eurogentec, Germany) with 3–5 independent biological replicates depending on the experiment. Expression of StActin gene was used for normalization of the expression of plant genes as well as of RiTEF (DQ282611). Fugal genes’ expression was normalized to RiTEF transcript levels. StPT4 (AY793559), StInvCD141 (Z22645), RiTEF, RiMST2 (HM143864), and StFatM (PGSC0003DMP400059797) were used as indicators of symbiosis status. The PCR program consisted in a 1 min incubation at 95°C, followed by 40 cycles of 30 s at 95°C, 30 s at 56°C and 30 s at 72°C, where the fluorescence signal was measured. The specificity of the PCR amplification procedure was checked with a heat-dissociation protocol (from 57 to 95°C) after the final cycle of the PCR. Oligonucleotides used can be found in Supplementary Table 1.