Total RNA was isolated using TRIzol Reagent (Solarbio Co., Beijing, China), and cDNA synthesis was carried out using a cDNA Synthesis Kit (Solarbio Co., Beijing, China). According to the manufacturer’s instructions, PCR amplification was performed with SYBR Green PCR Mix (Solarbio Co., Beijing, China). The sequences of the primers for miR-184 and U6 were as follows: miR-184, forward: 5′-AGT GCA GGG TCC GAG GTA TT-3′, reverse: 5′-CGC GTG GAC GGA GAA CTG AT-3′, stem-loop: 5′-GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACA CCC TT; U6, forward: 5′-AGA GAA GAT TAG CAT GGC CCC TG-3′, reverse: 5′-AGT GCA GGG TCC GAG GTA TT-3′, stem-loop: 5′-GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACA AAA TA-3′. Relative gene expression was determined by the 2–ΔΔCt method. Gene expression levels were normalized to the level of U6.
Sybr green pcr mix
Manufactured by Solarbio
Sourced in China
SYBR Green PCR Mix is a ready-to-use solution for real-time quantitative PCR (qPCR) applications. It contains SYBR Green I dye, DNA polymerase, dNTPs, and necessary buffers and reagents.
Automatically generated - may contain errors
2 protocols using sybr green pcr mix
1
miRNA-184 Expression Analysis Protocol
2
Quantifying Gene Expression in Aortic Tissues
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Total RNAs were isolated from aortic tissues or RAW264.7 cells using a RNAsimple kit (DP419, Tiangen, China) and quantified with a spectrophotometer. The reverse-transcribed complementary DNA (cDNA) was used to gene amplification using a qPCR system (Exicycler 96, Bioneer, Korea) with SYBR Green PCR mix (SY1020, Solarbio, China). All specific primer sequences used in qPCR experiments are shown in Table 1 . Expression level of GAPDH mRNA was used as a housekeeping control. The method of 2−ΔΔCT was carried out to analyze the results.
Primer Sequences Used in This Study
| Gene | Primer Sequences (5ʹ-3ʹ) |
|---|---|
| Arg1 | F: GGAAGACAGCAGAGGAGGTG |
| R: TCAGTCCCTGGCTTATGGTT | |
| Mrc1 | F: AGTGATGGTTCTCCCGTTTC |
| R: TGGGCTCAGGTAGTAGTGTTTT | |
| Retnla | F: CGTGGAGAATAAGGTCAAGGA |
| R: ACACCCAGTAGCAGTCATCCC | |
| Chi3l3 | F: ATGGCCTCAACCTGGACTG |
| R: TCCTGCTCCTGTGGAAGTG | |
| CD86 | F: ATGGGCTCGTATGATTGT |
| R: CTTCTTAGGTTTCGGGTG | |
| iNOS | F: CACCACCCTCCTCGTTC |
| R: CAATCCACAACTCGCTCC | |
| MARCO | F: AGGCGAATCTTTCCAACG |
| R: CCCAGAGCCACCTCCATA | |
| GAPDH | F: TGTTCCTACCCCCAATGTGTCCGTC |
| R: CTGGTCCTCAGTGTAGCCCAAGATG |
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