7900ht fast real time pcr system

RNA was extracted using RNeasy Plus Mini kit or QIAzol protocol (Qiagen, Milan, Italy) and converted to cDNA using the High Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, Monza, Italy). qRT-PCR was performed using the 7900 HT Fast Real Time PCR system (SDS version 2.3 software) or CFX384 Touch Real-Time PCR Detection System (Bio-Rad, Segrate, Italy) using commercially available primers (TaqMan Gene Expression Assays; Thermo Fisher Scientific, Monza, Italy) listed in Text S1. Relative gene expression was calculated as described [46 (link)].

For quantitative miRNA analysis, Bulge-LoopTM miRNA qPCR Primer Sets (RiboBio, Guangdong, China) were used to detect selected miRNAs expressions by quantitative reverse transcription polymerase chain reactions (qRT-PCRs) with iTaqTM Universal SYBR Green Supermix (BIO-RAD, Guangdong, China) in the 7900HT Fast Real-Time PCR System as previously reported. All qRT-PCR reactions were performed in triplicate, and the signal was collected at the end of every cycle. As there is no consensus on endogenous stable miRNAs in the circulation to act as house-keepers, the expression level of miRNAs in serum was normalized using spike-in cel-miR-39, which lacks sequence homology to human miRNAs.

Quantitative-RT-PCR (qRT-PCR) experiments were performed on tissue collected after control, F. oxysporum (see ‘Pathogen assays’) or MeJA treatment (see ‘Microarray analysis’). Three biological replicates were taken for all experiments comprising tissue pooled from 5–30 plants. RNA extraction, cDNA synthesis and Q-RT-PCR were conducted as described by McGrath et al. (2005) (link) using an Applied Biosystems 7900HT Fast Real-Time PCR System (Foster City, CA) or by Thatcher et al. (2015) using a CFX384 (Bio-Rad) system. Absolute gene expression levels relative to the previously validated reference genes β-actin 2, β-actin 7 and β-actin 8 (At1g49240, At3g18780 and At5g09810, respectively) were used for each cDNA sample using the equation: relative ratio gene of interest/actin=(Egene^{-Ct gene})/(Eactin^{-Ct actin}) where Ct is the cycle threshold value. The gene specific primer sequences are listed in Supplementary Table S3.

Pelleted cells were lysed in RIPA buffer (50 mM Tris pH 7.4; 150 mM NaCl; 1 mM EDTA; 1% NP-40; 0.1% SDS; 0.1% sodium deoxycholate; 1 mM PMSF; PhosSTOP and cOmplete PIC (Roche)) sonicated, and quantified by BCA assay. Equal sample amounts were then immunoblotted using Bolt gels and buffers (Thermo Fisher). Blots were blocked in 5% non-fat dry milk in TBSt (0.05% tween), washed in TBSt and incubated overnight at 4 °C with the following antibodies: anti-TDP-43 (ProteinTech; 12,892–1-AP; 10,782–2-AP); anti-Actin (Millipore; MAB1501); anti-α-synuclein (BD 610787); anti-ATXN2 (BD Biosciences; 611,378); anti-VCP (Thermo.; MA3–004); anti-AHR (Thermo.; MA1–514); anti-α-tubulin (Sigma-Aldrich; T5168). After washing, HRP-conjugated secondary antibodies (Jackson) were incubated with the blots the following day. Blots were activated with Pierce ECL chemiluminescent substrates (Thermo Fisher) and imaged using a ChemiDoc XRS+ Imager (BioRad). Band densitometries were assessed using Image Lab Software (BioRad).
RNA was collected from cultured cells by RNeasy minikit (Qiagen). cDNA was generated using High-Capacity cDNA Reverse Transcriptase (ABI). qPCR was performed using iQ SYBR green Supermix (Bio-Rad) on a 7900HT Fast Real-Time PCR system and the data was analyzed on SDS software. qPCR primer sequences are available in Additional file 1: Table S1.