Kapa sybr fast universal qrt pcr kit

To evaluate the transcript profiles of floral scent-related genes, the spadices at stage S2 were harvested for RNA extraction. Total RNA was extracted from the above samples using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. First-strand cDNAs were synthesized from 1.0 μg total RNA using the HiScript II RT-PCR system (Vazyme, Nanjing, China), according to the manufacturer’s instructions. Q-PCR reactions (20 microliter (μL) volume containing 1.0 μL cDNA as the template) were performed using the CFX connect Real-Time PCR System (Bio-Rad, Hercules, CA, USA) in standard mode with the KAPA SYBR FAST Universal qRT-PCR Kit (Kapa Biosystems, Wilmington, MA, USA). Anthurium Actin was used as the internal reference gene to quantify the relative expression level of target genes [40 (link)]. Primer sequences for qPCR were designed using NCBI Primer-BLAST, and are listed in Supplementary Table S1. The relative expression levels of target genes were calculated by the 2^−ΔΔCT method. Three biological replicates were performed per experiment.

The total RNA was isolated from soybean different tissues of root, stem, leaf, flower, seed, and hypocotyl using Trizol agent (Trans). cDNAs were synthesized from 800 ng total RNA using the M-MLV reverse transcriptase and oligodT primer according to the manufacturer’s instructions (Promega). qRT-PCR reactions (20 μl volume containing 2 μl cDNA as the template) were performed using the StepOne real-time PCR system (Applied Biosystems 7500) in standard mode with the KAPA SYBR FAST Universal qRT- PCR kit (KAPA Biosystems). Each tissue has three samples, and each sample of one tissue was performed in triplicate. The soybean Actin gene was used as the internal control. The primers for GmPRP2 were 5′- GCTCCTTAGTGCTGCTCCTT-3′ and 5′-TCAGTGGGAGGCTTGTACA-3′. The primers for Actin were 5′-CGTTTCATGAATTCCAGTAGC-3′ and 5′- GAGCTATGAATTGCCTGATGG-3′.

Total RNA samples were extracted from chrysanthemum apical buds, leaves, stem, and roots using TRIzol reagent (Invitrogen) and treated with RNase-free DNase I (Promega). First-strand cDNAs were synthesized from 1 μg total RNA using an oligo d(T) primer and the SuperScript III RT-PCR system, according to the manufacturer’s instructions (Invitrogen). qRT-PCR reactions (20 μl volume containing 1 μl cDNA as the template) were run using the StepOne Real-Time PCR System (Applied Biosystems) in standard mode with the KAPA SYBR FAST Universal qRT-PCR Kit (Kapa Biosystems). The chrysanthemum UBIQUITIN gene (GenBank accession NM_112764) was used as an internal control.
Stem-loop reverse transcription^{54 (link)} was performed to evaluate the expression of cmo-miR156. cDNAs were synthesized using a miR156 stem-loop primer and the SuperScript III RT-PCR system as described above. qRT-PCR reactions (20 μl volume containing 1 μl cDNA and the cmo-miR156F/stem-loop universal-R primer set) were run using the StepOne Real-Time PCR System (Applied Biosystems) as described above. The U6 gene was used as an internal control^{55 (link)} for stem-loop qRT-PCR.
Each reaction was performed using three biological replicates and verified by melting curve analysis. All reactions were performed with at least three biological replicates. PCR primers are listed in Supplementary Table 1.