Touch real time pcr detection system

ChIP was performed as described previously with minor modification (Zhang et al., 2017 (link)). 10 g of maize stem was homogenized in liquid nitrogen and re-suspended by TBS. The nuclei were purified in sucrose gradient and digested by MNase (Sigma). The digested nucleosome samples were incubated with 4% protein A Sepharose (GE Healthcare Bio-Sciences) for 3 h and centrifuged. Pan anti-Khib (PTM Biolabs, PTM-801) and pan anti-H4ac (Millipore, 06-598) was incubated with supernatant. The sample was incubated with 25% protein A agarose gel for 2.5 h. After centrifugation, the precipitate is eluted with an elution buffer to wash the eluted immune complex. The immunoprecipitated DNA was extracted by phenol/chloroform extraction and ethanol precipitation. Quantitative real-time PCR analysis of ChIPed DNA was performed on the Bio-Rad touch real-time PCR detection system to determine the relative enrichment factor of the modified histone-related sequences in the binding part. UBQ1 gene was used as an internal reference. Primers were designed and listed in Supplementary Table S3.

The mRNA expression of CD8 in liver allografts was measured at POD 7 using a PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Shiga, Japan, no. RR047A) and TB Green Premix Ex Taq II (TaKaRa, Shiga, Japan, no. RR820A) in accordance with the manufacturer’s instructions (Applied Biosystems). The primer sequences were as follows: β-actin: forward, 5′-CCGCGAGTACAACCTTCTTGC, and reverse, ATACCCACCATCACACCCTG-3′; CD8: forward, 5′-GCTTTTTGCCTTCGAGCTATCG, and reverse, GACTTCGTAGCGTACCTCTGG-3′. Reverse transcription was performed at 37°C for 15 min and 85°C for 5 s. The products were amplified at 95°C for 30 s followed by 40 cycles of 95°C for 5 s and 60°C for 30 s on a Touch Real-Time PCR Detection System (Bio-Rad, CA, USA, CFX96). The data were analyzed using Bio-Rad CFX96 Manager. The expression levels were normalized to the expression level of the housekeeping gene β-actin. We used the comparative Ct (ΔΔCt) method to calculate the relative mRNA expression.

Total RNA extraction was performed using the TRIzol reagent, following the manufacturer’s protocol. In summary, the samples were treated, to remove genomic DNA fragments with the kit (Promega™). Reverse transcription of RNA into cDNA was performed using Superscript III reverse transcriptase (Invitrogen™) and random hexamers (Invitrogen™), according to the manufacturer’s protocol. cDNA (assayed by NanoDrop™) was added to supermix SYBR Green (Bio-Rad) as well as some gene-specific primers sought in a 96-well plate. The following PCR procedure was then performed in the Touch Real-Time PCR detection system (Bio-Rad™), following the manufacturer’s protocol. The cycles being: 1. 95.0 o C for 3:00 2. 95.0 o C for 0:15 3. TM o C for 0:15 4. 72.0 o C for 0:30 5. Go to 2, 39 times more 6. Melt Curve 65.0 at 95.0°C, increase of 0.5°C by 0:05 seconds the results were analyzed using the ΔΔ method using the group control values for relative comparison. Data were submitted to GraphPad Prism 8™ for statistical analysis using ANOVA by Kruskal-Wallis`s test and uncorrected Dunn`s test for multiple comparisons.

Total RNA from EPCs was extracted using TRIzol reagent (15596-018, Invitrogen, Madison, MI, U.S.A.) following the protocols of the manufacturer and preserved in a 4°C or −80°C refrigerator. Concentration of total RNA was detected and quantified using a biological spectrometer (Nano Drop 2000, Thermo Fisher, Waltham, MA, U.S.A.). The cDNA was synthesized from 1 μg of total RNA with a First-Strand cDNA Synthesis Kit (E6300L; New England Biolabs, Beijing, China) according to the manufacturer’s instructions. QRT-PCR was conducted with SYBR Premix ExTaq II kit (RR820L, TaKaRa, Shiga, Japan) in Touch Real-Time PCR Detection system (CFX96, Bio-Rad, U.S.A.) under the following conditions: 95°C for 3 min, followed by 55 cycles at 53°C for 1 min, 72°C for 30 s. Primer sequences for this experiment are shown in Table 1. GAPDH (for FGF1) and U6 (for miR-361-5p) were used as internal reference. Relative expressions were quantified by 2^−ΔΔC_T calculation method [17 (link)].

Human neural astrocytes (No. CP-H122) and brain glioma cells (T98G (No. CL-0583) and HS 683 (CL-0362)) were purchased from Prosperity Life Sciences Ltd (Wuhan, China). All were cultured in the corresponding complete medium (CM-H122, CM-0583, CM–0362) and 95%; CO2, 5% environment. Assays for the extraction of total RNA and reverse transcription of first strand cDNA were performed using TRIzol reagent (No. 10296010, Invitrogen, Carlsbad, CA, USA). PrimeScript RT reagent Kits were used to generate first strand cDNA using Takara Bio RR047A. qPCR was performed using TB Green Premix Ex Taq II (No. RR820A, Takara Bio, Shiga, Japan) on a Touch Real-Time PCR Detection System (No. CFX96, Bio-rad, Hercules, CA, USA). The miRNA expression levels were normalised using U6 as an internal reference gene. Specific primer design and synthesis were performed by Ribo Biotech (Shanghai, China). Relative expression was calculated using the 2^(-ΔΔCt) method. All experiments were repeated three times and differences between groups were calculated using one-way ANOVA.