After stimulation with TNF-α (10 ng/ml), RNA was extracted from treated RAW 264.7 cells by using Trizol reagent (Invitrogen). cDNA synthesis involved use of the PrimeScript RT reagent Kit with gDNA Eraser (TakaRa Biotechnology, Dalian, China). The mRNA expression of VEGF-A, ANGPT-1 and ANGPT-2 was quantified by qRT-PCR with SYBR Green qPCR Master Mix reagent (Takara Biotechnology). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The sequences of forward and reverse primers for VEGF-A, ANGPT-1, ANGPT-2 and GAPDH are in Table 2 . The 2−ΔΔCT method was used to analyze relative expression between treatments [40 (link)]. The results were normalized against GAPDH expression.
Sybr green qpcr master mix reagent
Manufactured by Takara Bio
Sourced in Japan
SYBR Green qPCR master mix reagent is a ready-to-use solution for quantitative real-time PCR (qPCR) experiments. It contains SYBR Green I dye, which binds to double-stranded DNA and emits fluorescence upon excitation, enabling the detection and quantification of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using sybr green qpcr master mix reagent
1
Quantification of Angiogenic Factors in RAW 264.7 Cells
2
Quantification of miRNA targeting CYP27A1
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Total RNA (2 ug) treated with RNase-free DNase I (Promega, USA) were polyadenylated by poly(A) polymerase using a poly(A) tailing kit (AM1350; Applied Biosystems), according to the manufacturer's instructions. Polyadenylated RNA was then dissolved and reverse transcribed using a poly(T) adapter. Realtime PCR was performed with SYBR Green qPCR master mix reagent (Takara) in triplicate using a microRNA (miRNA)-specific forward primer and a universal reverse primer complementary to part of the poly(T) adapter sequence.U6 small-nuclear RNA (U6 snRNA) was used as a reference gene to normalise the expression of miRNA [38 ]. The sequences of all porcine miRNA were acquired from miRBase (http://www.mirbase.org/ ). miRNA targeting CYP27A1were predicted with an online miRNA prediction tool [39 (link)]. Among all the predicted miRNA, eleven targeting CYP27A1were quantified by real-time PCR. The primer sequences used for miRNA analysis are listed in Table 3 .
3
Quantitative RT-PCR for Angiogenic Factors
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Total RNA was extracted from tissue samples using Trizol reagent (Invitrogen), and the IQ5 real‐time PCR thermocycler (Bio‐Rad) was used to perform the qRT‐PCR. mRNA levels of VEGF‐A, FGF2 and PDGF‐BB were quantified with SYBR Green qPCR master mix reagent (Takara Biotechnology). The results were normalized against glyceraldehyde 3‐phosphate dehydrogenase expression and calculated using the 2−ΔΔCT method. Primers are listed in Table S2 .
4
Quantification of miRNAs Targeting PC and PCK1
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Two micrograms of total RNA treated with RNase-free DNase I (Promega) was polyadenylated by poly (A) polymerase using Poly (A) Tailing Kit (AM1350, Applied Biosystems, USA) according to the manufacturer's instruction. Polyadenylated RNA was then dissolved and reverse transcribed using poly (T) adapter. Real-time PCR was performed with SYBR green qPCR master mix reagent (TaKaRa, Japan) in triplicates with a miRNA specific forward primer and a universal reverse primer complementary to part of the poly (T) adapter sequence. U6 small nuclear RNA (U6 snRNA) was used as a reference gene to normalize the expression of miRNAs.
Because the 3'UTR sequence of porcine PC gene has not been reported, we aligned the 3′ flanking sequence of this gene with the 3'UTR sequence of human PC gene to obtain the consensus sequence for miRNA prediction using an online miRNAs prediction tool, PITA algorithm, with the threshold of score set at -10 [26] (link). Twenty miRNAs were predicted to target PC and 15 miRNAs predicted to target PCK1. Among all these predicted miRNAs, 7 miRNAs for each gene were reliably and repeatedly quantified in real-time PCR. The primer sequences used for miRNAs analysis are listed inTable S6
Because the 3'UTR sequence of porcine PC gene has not been reported, we aligned the 3′ flanking sequence of this gene with the 3'UTR sequence of human PC gene to obtain the consensus sequence for miRNA prediction using an online miRNAs prediction tool, PITA algorithm, with the threshold of score set at -10 [26] (link). Twenty miRNAs were predicted to target PC and 15 miRNAs predicted to target PCK1. Among all these predicted miRNAs, 7 miRNAs for each gene were reliably and repeatedly quantified in real-time PCR. The primer sequences used for miRNAs analysis are listed in
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