Total RNA was extracted from the cells using an RNA-Quick purification Kit (ES Science, China), and the RNA was reverse transcribed into complementary DNA (cDNA) using a qPCR RT kit (TOYOBO, Japan). The cDNA was used for RT-PCR assays (SYBR Green Master Mix; YESEN, Shanghai, China) according to the manufacturer’s instructions. Relevant data were analyzed with the 2−ΔΔCt method normalized to β-actin. The primers for HGDF were 5ʹ-CTCTTCCCTTACGAGGAATCCA-3ʹ (forward) and 5ʹ-CCTTGACAGTAGGGTTGTTCTC-3ʹ (reverse).
Sybr green master mix
Manufactured by Yesen
Sourced in China
SYBR Green Master Mix is a ready-to-use solution containing all the necessary components for performing real-time PCR reactions, including SYBR Green I dye, DNA polymerase, dNTPs, and buffer. It is designed to detect and quantify DNA targets in real-time.
Automatically generated - may contain errors
Lab products found in correlation
Qpcr rt kit, by Toyobo (1 mentions)
M mlv rnase h, by Takara Bio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Icycler iq, by Bio-Rad (1 mentions)
Qrt pcr, by Bio-Rad (1 mentions)
Abi 7900ht system, by Thermo Fisher Scientific (1 mentions)
Trieasy total rna extraction reagent, by Yesen (1 mentions)
4 protocols using sybr green master mix
1
RT-PCR Analysis of HGDF Expression
2
Transcriptional Analysis of HK-2 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was acquired from HK-2 cells using TRIzol reagent (Invitrogen, USA). The absorbances at 260 and 280 nm were measured to confirm the quantity and purity of total RNA. cDNA was synthesized from total RNA using reverse transcriptase M-MLV (RNase H-) (Takara, Japan). SYBR Green Master Mix (YESEN, China) was used to measure the relative expression levels of mRNA. All the primers were designed and synthesized by Sangon, Shanghai, China, and the sequences of primers are listed in Supplementary Table S3 . The relative expression levels of targeting genes were normalized to β-actin gene and calculated using the 2ΔΔCT method.
3
Quantitative RT-PCR Analysis of H9C2 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA from H9C2 cells was extracted using the Trizol technique (Sigma-Aldrich, USA). The YESEN kit reverse transcribed cDNA. The SYBR Green Master Mix (YESEN, China) and the iCyclerIQ equipment were used to perform qRT-PCR (BioRad, USA). qRT-PCR was performed in an initial denaturation step at 95°C for an initial denaturation step of 2 min, and 40 cycles in 95°C/10 s and 60°C/30 s. The amount of endogenous control (β-actin) was used to standardize the expression of the mRNA. The primers targeting the genes were as follows: BST-1: forward 5'-GTACCACGCCTCACCTCCAGAG-3' , reverse 5'-CCTTGTCCAGCACCACCTTGAAG-3'; ANP: for-ward 5'-GAGAGTGAGCCGAGACAGCAAAC-3' , reverse 5'-GAAGAAGCCCTTGGTGATGGAGAAG-3'; BNP: forward 5'-CCAGTCTCCAGAACAATCCACGATG-3' , reverse 5'-GCCTTGGTCCTTTGAGAGCTGTC-3'; Myh7: forward 5'-CACCAGCCTCATCAACCAGAAGAAG-3' , reverse 5'-TCCTCTGCGTTCCTACACTCCTG-3'; β-actin: forward 5'-TGTCACCAACTGGGACGATA-3' , reverse 5'-GGGGTGTTGAAGGTCTCAAA-3'
4
Quantification of Tissue-Specific Genetic Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total mRNA from the lung, liver, kidney and heart were isolated using a total RNA extraction reagent (YESEN, Shanghai, China) following the manufacturer's protocol. The TRIeasyTM Total RNA Extraction Reagent (YESEN, Shanghai, China) and the Hifair 1 st Strand cDNA Synthesis SuperMix (YESEN, Shanghai, China) with qPCR. SYBR Green Master Mix (YESEN, Shanghai, China) were used on an ABI-7900HT system (Applied Biosystems, USA) for real-time quantitative PCR (RTqPCR). mRNA levels were calculated using the comparative Ct method (ΔΔCt) after normalization to GAPDH. The primers used for RTqPCR in this study were as the follows: HIF-2α (forward primer:5′-TCACTCATCCTTGCGACCAC-3′, reverse primer:5′-CAGGTGGCCGACTTAAGGTT-3′); EGLN1(forward primer:5′-AGGGCTAACGCTAATCACCT-3′, reverse primer:5′-TTGTTGTCTTGAGACGCAGC-3′); eNOS (forward primer:5′-CCCAGGAGAGATCCACCTCA-3′, reverse primer:5′-CGGAAGGGTGCAATACCAGT-3′); ApoA5(forward primer:5′-CACTCCCGTGGCTTCTAGTG-3′, reverse primer:5′-GGACTGGCGAGCCTTAGTTT-3′); VEGF (forward primer:5′-GCAGCGACAAGGCAGACTAT-3′, reverse primer:5′-GAGGGAGTGAAGGAGCAACC-3′); BNP (forward primer:5′-AGTCTCCAGAACAATCCACGATGC-3′, reverse primer:5′-CCGGAAGGCGCTGTCTTGAG-3′); TGF-βR (forward primer:5′-GCGATCTAACCTGTTGCCTGTG-3′, reverse primer: 5′-GGGCCATGTATCTCGCTGTTC-3′); GAPDH (forward primer:5′-AATGGTGAAGGTCGGTGTGAAC-3′, reverse primer:5′-AGGTCAATGAAGGGGTCGTTG-3′).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!