Icycler iq5 system

Total RNA was isolated using an RNAprep Pure Plant Kit (Tiangen, Beijing, China). First-strand complementary DNA (cDNA) was synthesized using the RevertAid First-Strand cDNA Synthesis Kit (Fermentas, St. Leon-Roth, Germany). Three replicates were prepared for each sample. Quantitative real-time polymerase chain reaction (qPCR) analyses were conducted using 50 ng/μL cDNA as the template, SYBR Green PCR Master Mix (TransGen Biotech, Beijing, China), and the iCycler iQ5 system (Bio-Rad, Hercules, CA, USA). Details regarding the qPCR primers are provided in Supplementary Table S1. MdActin was used as the internal control.

Following manufacturer’s protocol (Invitrogen), Trizol reagent was used to extract total RNA from shoot and root samples of FM. To remove any possible DNA contamination, total RNA (4–5 g) was digested by DNase 1 (Takara Biomedicals, Kyoto, Japan). Reverse transcription of RNA samples into cDNA was carried out using M-MLV reverse transcriptase (Invitrogen). Quantitative real-time PCR (qRT-PCR) in a Bio-Rad iCycler iQ5 system (Bio-Rad, Hercules, CA, United States) was used to determine gene expression levels using the SYBR^® Premix Ex TaqTM (Takara) along with specific primers constructed according to the genes of interest having elongation factor 1-α (EF-1α) as internal control or reference gene (Table 1). The primers were designed using Primer Premier 5.0. The program was as follows: pre-incubation for 10 min at 95°C, 40 cycles of determination at 95°C for 15 s, annealing at 60°C for 30 s, and finally, extension at 72°C for 30 s. The standard comparative ΔΔC(t) method (Livak and Schmittgen, 2001 (link)) was used to calculate relative gene expression levels. The treatments used for the analysis had four biological replicates and three technical replicates of every biological replicate. The IDs and names of genes examined were given in Table 1.

RNA was isolated from pericarp of fruits at Br+ 5 d stage by using the InviTrap Spin Plant RNA kit (Stratec) and cDNA was synthesized by the iScript cDNA synthesis kit (Bio-Rad). Two replicates were analysed per plant containing one fruit each from two plants per line. Primers used for qRT-PCR are listed in Supplementary Table 1. iQ SYBR Green Supermix (Bio-Rad) and iCycler iQ5 system (Bio-Rad) were used for quantitative RT PCR. Actin was used as a reference and relative expression changes of AP2a were calculated according to 2^−ΔΔCt method as described^{50 (link)}. Student’s t-test was performed to detect significant differences.

Real time RT-PCR was used to quantify any differences in the expression of GFAP, ALDH1L1, IL-6 or STAT3 between the brain and spinal cord developmentally or in adulthood. Whole brain or spinal cords were harvested from three individual mice on postnatal day P0, 7, 21 or 45 (adulthood) and RNA extracted with STAT-60 (Tel-Test, Friendswood, TX). The level of RNA encoding GFAP, ALDH1L1, IL-6 or STAT3 was determined in 0.10 μg of RNA in triplicate using an iCycler iQ5 system (BioRad) with primers described in Table 1 [35 (link), 36 (link)]. The relative amount of RNA in each case was normalized to the constitutively expressed gene Rn18S. All PCR cycle conditions followed those recommended by BioRad using the iTaq Universal SYBR Green One-Step kit (#172–5151) for conventional primers, and the iTaq Universal Probes One-Step Kit (#172–5141) for hybridization probes.

Brain tissues (about 20 mg) were mixed with 1 mL Trizol and homogenized on ice followed by total RNA extraction. Real-time PCR was conducted in the iCycler iQ5 system (Bio-Rad Laboratories, Hercules, CA, USA) to measure the mRNA levels of target genes, and the protocols were the same as described in our previously published papers [21 (link),25 (link)]. The expression levels of the genes for tyrosine hydroxylase (TH), GPX1, GPX4, and Selenoprotein P (Sepp1) were analyzed. Then, an 18S ribosomal RNA (18S rRNA) gene or GAPDH gene was used as the endogenous reference for normalization. Each sample was detected in duplicate. Levels of mRNA were shown as a quantitative ratio of the target gene to 18S rRNA or GAPDH gene and relative to the group mean of the Normal group. The sequences of the primers were shown in Supplementary Table S1.

Total RNA was isolated from different tissues of the apple using the CTAB method. The iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) was used to reverse-transcribed cDNA from total RNA. Primer premier 5.0 software was used to design all the specific quantitative primers for the MdCIPK genes in apple were listed in the supplemental materials (Table A1). Following the manufacturer’s instruction, qRT-PCR was performed on an Icycler iQ5 system (Bio-Rad) using the SYBR Green Supermix Kit (Bio-Rad) and the actin gene was used as controls (Table A1). Compared to untreated plants the relative expression level of each MdCIPK gene was calculated as 2^−ΔCT values, and each reaction was replicated three times [46 (link)].