Evagreen 2 qpcr mastermix

Among the annotated unigenes that were involved in the 16 immune system KEGG pathways, seven genes that were differentially expressed at different developmental stages were selected and confirmed by qRT-PCR. The primers of the seven immune-relevant genes were designed for qRT-PCR analysis according to the sequences from our transcriptome data, and they are listed in Table S1. qRT-PCR analysis was used to validate the reliability of the Illumina Sequencing. Three individuals from each group (two-week-old goslings and adult geese) were euthanatized for the collection of spleen samples. Total RNA was extracted as described for the cDNA library preparation and then reverse transcribed using EvaGreen 2× qPCR MasterMix (Abm, Milton, ON, Canada). The qRT-PCR analysis was performed by QuantiFast SYBR Green PCR Kit (QIAGEN, Dusseldorf, Germany) and the Bio-Rad CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA). The goose GAPDH was chosen as the internal control gene and the negative control used no cDNA template. The differential mRNA expression levels of the seven genes were verified in triplicate.

The total RNA in infected cells was extracted, using TRIzol reagent (TaKaRa Bio, Kyoto, Japan). Then, the RNA was reverse transcribed into cDNA. The level of DTMUV was determined by RT-qPCR using Eva green 2× qPCR master mix (Abm, Canada) in at 7300 real-time PCR system (Applied Biosystems, Foster City, CA, USA). Two pairs of specific primers were designed to amplify the DTMUV E gene and the cellular β- actin in the conserved region by utilizing DNAMan software. These were, respectively, (DTMUV-E-F: 5′-TGTCTTATGCAGGTACCGATG-3′, DTMUV-E-R: 5′-CGTATGGGTTGACTGTTATCA-3′; actin-F:5′-CTCCATCATGAAGTGCGACGT-3′, actin-R:GTGATCTCCTTCTGCATCCTGTC-3′). The data analysis is presented as the 2-ΔΔCT value from quadruplicate samples [20 (link)].

Total RNA was extracted from the frozen samples using a Total RNApure Kit (ZOMANBIO, http://zomanbio.com). First-strand cDNA was synthesized using a 5 × All-in-One RT Master Mix Reverse Transcription Kit (ABM, http://www.abmgood.com). RT-qPCR was performed on a LightCycler 480 II (Roche, Switzerland) with EvaGreen 2 × qPCR Master Mix (ABM, http://www.abmgood.com). The potato Ef1α gene was used as a control gene to normalize the expression data (Nicot et al., 2005 (link)). Gene expression levels were calculated via the 2^−ΔCq method described by Bio-Rad (Hercules, CA, USA; http://www.bio-rad.com/zh-cn/applications-technologies/real-time-pcr experimental-design). All primer sequences for RT-qPCR analysis are described in (Supplemental Table S2).

To evaluate the mRNA transcript levels of chondrocytes specific genes (SOX-9, COL-II and ACAN), chondrocytes and rBMSCs were cultured on the scaffolds for 7 and 14 days and processed for total RNA extraction by using a trizol kit (Ambion, CA) according to the manufacturer’s instructions, and the concentration of RNA was determined at 260 nm with a multifunction microplate reader (Synergy 2; BioTek, USA). The cDNA was reverse-transcribed using All-In-One RT MasterMix (ABM, CA), and quantitative real-time polymerase chain reaction (qRT-PCR) was performed using EvaGreen 2 × qPCR MasterMix (ABM, CA) with a Light Cycler 480 II (Roche, CHE). To study the underlying mechanism of IGF-1 promoting chondrogenesis, the expression level of HIF-1α was examined. Chondrocytes and rBMSCs were cultured on the scaffolds for 24 h, and then qRT-PCR analysis was performed as described above. GAPDH was used as the housekeeping gene for normalization. Primer sequences are listed in Table 1.

To assess the transcription levels of multiple genes, real-time quantitative PCR was performed on a 7500 Real-time PCR System (Applied Biosystems) using EvaGreen 2× qPCR Master Mix (Abm) with a 10 μL reaction volume containing 5 μL of 2× qPCR MasterMix, 1 μL of template (200 ng/μL), 0.15 μL of each forward and reverse primer (10 μM), and 3.7 μL of H₂O. The cycling conditions for all samples were: 95 °C for 30 s, then 40 cycles at 95 °C for 1 s and 58 °C for 10 s. Melt curve analysis was performed after the amplification cycle to confirm the accuracy of each amplicon. The expression of the target gene was calculated using the 2^−ΔΔCt method after normalized relative to the relative expression of ef-1α, which has been tested to be stable under the current experimental conditions. Table 2 lists the primer information used in this study.

Total RNA from RAOECs, PMCs, and BMSCs was isolated using a FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme, NanJing). The isolated RNA was then reverse-transcribed into cDNA using an All-In-One 5× RT MasterMix (Abm, Canada). Real-time quantitative polymerase chain reaction (RT-qPCR) was performed using an EvaGreen 2× qPCR MasterMix (Abm). The PCR automatic serialization analyzer (ABI QuantStudio 3, USA) was set to predenaturation at 95 °C for 10 min and 40 cycles of denaturation at 95 °C for 15 s, and annealing and extension at 60 °C for 60 s. GAPDH was used as an internal reference gene. The Ct values of each target gene were obtained, and semiquantitative calculation and analysis were carried out according to the 2^−∆∆Ct method. The primer sequences are shown in Table.

mRNA was isolated from the cells and embryonic fragments of epiblast with RNAzol (Mrcgene, Cat# RN190) according to the protocol of manufacturer. The concentration of mRNA was measured by NanoDrop 2000 (Thermo Fisher Scientific). For each sample, 500 ng mRNA was transcribed into cDNA with PrimeScript RT Reagent Kit (TaKaRa, Cat# RR037A). Quantitative PCR (qPCR) was performed with EvaGreen 2× qPCR MasterMix (ABM, MasterMix-S) on LightCycler 480 II (Roche). All results were repeated with at least three independent samples. Gadph or β-actin was used to normalize the gene expression. The 2^−ΔCt method was used for data analysis.^{71 (link)} All primer sequences for qPCR were listed in Supplementary information, Table S6.