Mesa blue qpcr mastermix plus

RNA was extracted with Quick-DNA/RNA Miniprep Kit (Zymo Research) and DNAse treated with the TURBO DNA-free Kit (Ambion). cDNA was then prepared with SuperScript III Reverse Transcriptase (Invitrogen). Target sequences (primer list in Additional file 6) were amplified with MESA BLUE qPCR MasterMix Plus (Eurogentec) or KAPA SYBR FAST Roche LightCycler 480 qPCR Master Mix (Kapa Biosystems, KK4611) and quantified on a Light-Cycler 480 (Roche). Prior to RNA-seq library construction, 10–100 ng of total RNA was treated with the NEBNext rRNA Depletion Kit. Library construction was performed with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina, according to the manufacturer’s protocol.

Cells were plated in triplicate at 312 000 cells per well in 12 well plates, cultured for 48 h and then switched to differentiation medium (treated with biochanin A or following PGC1α siRNA-mediated knock-down) for 3 days before cells were harvested. RNA was isolated using miRNeasy kit and reverse-transcribed using the Reverse Transcription Kit with genomic DNA wipeout (Qiagen, Manchester, UK), RT-qPCR was performed on a Viia7 qPCR system (Life Technologies) with MESA Blue qPCR MasterMix Plus and ROX reference dye (Eurogentec Ltd, Hampshire, UK) using TBP expression as a control.
The primers used were as follows:
ESRRA forward: 5′-AAGACAGCAGCCCCAGTGAA-3′
ESRRA reverse: 5′-ACACCCAGCACCAGCACCT-3′
PPARGC1A forward: 5′-GTGAAATTGAGGAGTGCACAGTAAA-3′
PPARGC1A reverse: 5′-TCACAGGTATAACGGTAGGTAATGAAA-3′
TBP forward: 5′-CGGCTGTTTAACTTCGCTTC-3′
TBP reverse: 5′-CACACGCCAAGAAACAGTGA-3′

PCR products for sequencing were amplified with S7 Fusion Polymerase^™ (Mobidiag, Finland) according to the manufacturer’s instructions and using the oligonucleotides provided in Supplementary Information, Table S1. A homemade Taq polymerase was used with standard protocols for co-amplification PCRs of splicing patterns. All PCR products were separated on 2% or 3% agarose gels, stained with ethidium bromide, and visualized under UV light. Quantitative PCR for relative quantification of cDNAs transcripts was performed using the MESA BLUE qPCR MasterMix Plus (Eurogentec) according to manual and the CFX384 real-time PCR system (Bio-Rad). GFP was used as reference transcript for the samples from infiltrated N. benthamiana leaves.