Cfx connect real time pcr detection system

Total RNA was extracted from fresh brain, spleen, liver, kidney, heart, lung, or cultured cells by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s guidelines. Then, cDNA was synthesized using TransScript First-Strand cDNA Synthesis Super Mix (TransGen Biotech, China). qPCR reactions were performed in triplicate with FastStart Universal SYBR Green Master Mix (Roche, Germany) on a CFX ConnectTM Real-Time PCR Detection System (Bio-Rad, USA). Relative gene expression was normalized to the housekeeping gene GAPDH and calculated using the 2^−ΔΔCt method. The primers used for amplification are shown in Additional file 1: Table S2.

RNA was isolated from micro-dissected dentate gyri using RNeasy Micro Kit (QIAGEN) and 500 ng were used for cDNA synthesis with Superscript^TM II Reverse Transcriptase (Thermo Fisher Scientific). Quantitative PCRs were performed using QuantiFAST SYBR Green PCR Kit (QIAGEN) and the CFX Connect^TM Real-Time PCR Detection System (Bio-Rad). Primer sequences were designed using the online tool Primer 3 version 0.4.0 and are listed in Supplementary Data 9. Results were analyzed using the delta Ct method. Housekeeping genes Actb (Fig. 1) or Oaz1 (aging experiments, Fig. 5) were used for the normalization of sample concentrations.

Total RNA was extracted from the peri-infarct tissues using RNAprep pure Tissue Kit (Tiangen, China, cat. No. DP431) and reverse transcribed into cDNA using FastQuant RT Kit (Tiangen, China, cat. No. KR106). RT-PCR was performed on a CFX Connect^TM Real-Time PCR Detection System (Bio-Rad) using SuperReal PreMix Plus (Tiangen, China, cat. No. FP205). The primer sequences were presented as follows (forward and reverse, respectively): VEGF, 5′-GCACGTTGGCTCACTTCCAG-3′ and 5′-TGGTCGGAACCAGAATCTTTATCTC-3′; Ang-1, 5′-ACCGTGAGGATGGAAGCCTAGA-3′ and 5′-AATGAA CTCGTTCCCAAGCCAATA-3′; Ang-2, 5′-CATGTCTAACGCCGTGCAGAG-3′ and 5′-GATCATCACAGCCGTCTGGTTC-3′; Netrin-1, 5′-TGCCAAAGGCTACCAGCAGA-3′ and 5′-GAAGCCTTGCAGTAGGAGTCACAG-3′; DCC, 5′- GATGGAGGTTATTGGCCAGTTGA-3′ and 5′-GTGACCACGGTTATGACAAGCAG-3′; Slit-2, 5′-GCCAAGGTTCGACCTCAGACA-3′ and 5′-CGCCCTCGATAGAGTTCCACA-3′; Robo-1, 5′-GCAACATGAGTGCTGCTGTAA-3′ and 5′-AGCTGTGCCTTGGACTGGA-3′; β-actin, 5′-GGAGATTACTGCCCTGGCTCCTA-3′ and 5′-GACTCATCGTACTCCTGCTTGCTG-3′. The quantitative data were presented as relative expression according to the formula of 2^{−Δ Δ Ct}. Actin was used as an internal standard (Chen J. et al., 2017 (link)).

BV2 cells (4 × 10⁵ cells/ml) were plated into 6-well plates and were pretreated with each sample for 2 h and then induced by LPS for 4 h. After that the total RNA was isolated using Trizol and converted to cDNA. CFX ConnectTM real-time PCR detection system (Bio-Rad, Hercules, CA, USA) was used for the experiment. And the primers used are listed in Table 3. The level of GAPDH gene was used for standardization^{9 (link)}.Table 3

Primer sequence used in Real-time PCR assay.

Gene	Forward primer	Reverse primer
IL-1β	5′-TGACGGACCCCAAAAGATGA-3′	5′-TCTCCACAGCCACAATGAGT-3′
IL-6	5′-TAGTCCTTCCTACCCCAATTTCC-3′	5′-TTGGTCCTTAGCCACTCCTTC-3′
GAPDH	5′-AGGTCGGTGTGAACGGATTTG-3′	5′-TGTAGACCATGTAGTTGAGGTCA-3′

Total RNA was extracted from ECFCs using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and plasma using TRIzol LS reagent (Invitrogen) following the manufacturer’s protocol. For miRNA detection, 150 ng of total RNA was used for RT using Universal cDNA Synthesis and SYBR^® Green Master Mix kits (EXIQON, Vedbaek, Denmark). Real-time PCR reactions were performed with miRCURY LNA^TM Universal RT microRNA PCR LNA^TM PCR primer set (EXIQON) using CFX connect^TM real-time PCR detection system (Bio-Rad, Hercules, CA, USA) in Bio-Rad CFX96 Real-Time PCR Detection System. The expression of miRNAs was normalized to U6 small nuclear RNA. For mRNA detection, 2 μg of total RNA was used for RT with Oligo(dT)_12-18 using SuperScript^III RT (Invitrogen). The expression of each gene was quantified by iQ^TM SYBR Green Supermix (BioRad) using CFX96 Real-Time PCR Detection System and normalized to GAPDH.

After 168 h of culture, the cells were rinsed with HBSS and homogenized by TriReagent^®, Sigma Aldrich, Poznań, Poland. Total RNA was isolated using phenol–chloroform method as previously described by Chomczynski & Sacchi [56 (link)]. The RNA was diluted in DEPC-treated water and analyzed using a nano-spectrometer (WPA Biowave II, Biochrom, Cambourne, Cambridge, UK). Genomic DNA digestion and cDNA synthesis were performed using PrimeScript kit (Takara, Clontech, Mountain View, CA, USA). For each reaction, 150 ng of total RNA was used. Both processes were performed in accordance with the manufacturers’ instructions using a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA).
The qRT-PCR reactions were performed using a CFX ConnectTM Real-Time PCR Detection System (BioRad, Hercules, CA, USA). The reaction mixture contained 2 μL of cDNA in a total volume of 20 μL using SensiFast SYBR & Fluorescein Kit (Bioline, Cincinnati, OH, USA). Primer concentration in each reaction equaled 500 nM; primer sequences used in individual reactions are listed in Table 1. The average fold change in the gene expression of experimental cultures was compared with control cultures and calculated by the 2−DDCt method in relation to the housekeeping gene GAPDH.

On the seventh day of the experiment, cells were homogenized using 1 ml TRI Reagent. Procedure of homogenization was performed directly in the culture dish. Total RNA was isolated using the phenol–chloroform method as previously described by Chomczynski & Sacchi 35. Obtained samples were diluted in DEPC‐treated water. Both, quality and quantity of isolated total RNA were determined using nano‐spectrometer (WPA Biowave II, Hercules, California, USA). Genomic DNA digestion and cDNA synthesis were performed with PrimeScript kit (Takara, Clontech, Kusatsu, Shiga, Japan). For each reaction, 150 ng of total RNA was used. Both processes were performed in accordance with the manufacturers’ instructions using a T100 Thermal Cycler (Bio‐Rad, Hercules, California, USA).
The qRT‐PCR reactions were performed with a CFX ConnectTM Real‐Time PCR Detection System (BioRad). Reaction mixture contained 2 μl of cDNA in a total volume of 20 μl using SensiFast SYBR & Fluorescein Kit (Bioline, Cincinnati, Ohio, USA). The concentration of primers in each reaction equalled to 500 nM; primer sequences used in individual reactions are listed in Table 1. Relative gene expression analysis (Qn) was calculated in relation to the GAPDH housekeeping gene.
Moreover, we evaluated the ratio between BCL‐2 and BAX expression in each group by dividing Qn of BCL‐2 by Qn of BAX.