Sybr premix ex taq polymerase

qRT-PCR analysis was performed using a LightCycler 2.0 instrument system (Roche, Germany). The action 11 gene (AhACT11) was selected as the reference gene [41] (link). Three pairs of gene-specific primers (Table 1) were designed after analyses of the target genes’ sequences. qRT-PCR reactions were performed using the SYBR Premix Ex Taq polymerase (Takara) according to the manufacturer’s instructions. Each 20-µl reaction was comprised of 2 µl of template, 10 µl of 2× SYBR Premix, and 0.4 µl (200 nM) of each primer. The reactions were subjected to an initial denaturation step of 95°C/10 s, followed by 40 cycles of 95°C/5 s, 60°C/30 s and 72°C/10 s. A melting curve analysis was performed at the end of the PCR run over the 60–95°C range, increasing the temperature stepwise by 0.5°C every 10 s. The baseline and quantification cycle (CP) were automatically determined using the LightCycler Software. Zero template controls were included for each primer pair, and each PCR reaction was carried out in triplicate. The relative quantification method (delta-delta Cp) was used to evaluate quantitative variation.

Chicken liver, serum, and cell RNA was purified using TRIzol^® Reagent (Invitrogen). Amplification of mRNA for expression analysis was performed with SYBR Premix Ex Taq polymerase (Takara Bio, Inc., Shiga, Japan) using an ABI Q5 Real-time PCR System (Applied Biosystems, Foster City, CA, USA) with glyceraldehyde-3-phosphate dehydrogenase as internal references. The miRNA was reverse-transcribed into cDNA using the miScript II RT Kit (QIAGEN GmbH, Hilden, Germany) and amplified by RT-qPCR using a miScript SYBR Green PCR Kit (QIAGEN GmbH) with an ABI Q5 Real-time PCR System (Applied Biosystems). The miScript primers for selected miRNAs are the property of Qiagen. U6 was used as the internal control.

Relative transcript level expression profiles of OsOFPs were evaluated by real-time quantitative PCR (RT-qPCR) on Mx3005P (Stratagene, La Jolla, CA, USA) using the SYBR Premix ExTaq polymerase (TaKaRa, Bio Inc.). Each reaction contained 12.5 μl of the 2× SYBR Premix ExTaq, 50–100 ng of the cDNA template, 0.5 μl of 10 mM of each primer, and 10.5 μl of double-distilled H₂O for a final volume of 25 μl. The PCR reaction parameters were 95°C for 30 s (1 cycle), 95°C for 5 s, and 60°C for 20 s (40 cycles), which was followed by a melting curve analysis at 95°C for 60 s, 55°C for 30 s, and 95°C for 30 s. The relative fold differences were calculated based on the comparative 2^{-ΔΔ Ct} method. Approximately 100–200-bp PCR products, unique to each OsOFP, were amplified; the housekeeping gene ACTIN (X15863.1) was used to normalize the transcript level of each OsOFP in the samples. Expression profile analysis of OsOFP genes in each type tissue was displayed using relative percentage [15 (link)] in this study. The specific primer pairs used were listed in S3 Table.

RT-qPCR was performed using a DNA Engine (MJ Research, New Haven, CT, USA) and the SYBR Premix Ex Taq polymerase (TaKaRa, Toyoto, Japan). The PCR reaction volume was 25 μL containing 2 μL of diluted cDNA, 12.5 μL 2 × SYBR Premix and 0.2 μM of each primer. The RT-qPCR programs were as follows: a pre-denaturation of 95 °C/2 min, followed by 40 cycles of 94 °C/15 s, 60 °C/20 s, and 72 °C/15 s. The amplification efficiency was calculated using the following formula: efficiency = 10^(−1/slope) − 1. Moreover, a melting curve analysis was performed at the end of program to determine the validity of experimental results. All reactions were performed in triplicate for technical and biological replicates, and each biological repetition was performed by mixing the tissue sample from three individuals.

First-strand cDNA was synthesized using reverse transcriptase (TaKaRa Bio Inc, Dalian, China) with random hexamer primers. Real-time PCR was performed using SYBR Premix Ex Taq polymerase (TaKaRa Bio Inc, Dalian, China) with an ABI 7500 Fast Real-time PCR System (Applied Biosystems, Foster City, CA, USA). 16S rRNA was used to normalize RNA levels. Expression levels of galE, yywkA, araA, sinI, tasA and srfAA were measured at different timepoints during the plant-microbe interactions. Microarray results were validated by measuring the expression levels of some randomly chosen differentially expressed genes.

A measure of 1 µg total RNA was used to synthesize cDNA using the Prime Script™ RT Reagent Kit (Takara, Kyoto, Japan). The qRT-PCR was performed with the SYBR Premix Ex Taq polymerase (Takara) according to the manufacturer’s instructions. The relative expression of selected DEGs were detected by CFX Connect Real-Time System (Bio-Rad Laboratories, Hercules, CA, USA). Pear PbrGAPDH were used as the internal controls [50 (link)]. The 2^−ΔΔCT method was used to analyze the results [50 (link)]. Three biological replicates were performed for each sample. Primers used for RT-qPCR are listed in Supplementary Table S2.