The Total RNA Small Amount Extraction kit (Axygen; Corning, Inc.) was used to lyse T98G or U87 cells and extract their total RNA. Prime script RT Master mix (Takara Biotechnology Co., Ltd.) used to reverse transcribe the extracted RNA into cDNA according to the manufacturer's protocol. HIF1α and GAPDH were amplified using primers purchased from Sangon Biotech Co., Ltd. SYBR Green Master Mix (Tiangen Biotech Co., Ltd.) was used for RT-qPCR following the manufacturer's instructions. The thermocycling conditions used for PCR were: Initial denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 60 sec. The 2−ΔΔCq method (36 (link)) was used to calculate the relative mRNA expression levels. GAPDH was used as the endogenous control. The primer sequences were as follows: HIF1α forward (F) 5′-GTGGTGGTTACTCAGCACTTT-3′ and reverse (R), 5′- ATCTCCGTCCCTCAACCTCT-3′; and GAPDH F, 5′-AGGTCGGTGTGAACGGATTTG-3′ and R, 5′-GGGGTCGTTGATGGCAACA-3′.
Total rna small amount extraction kit
Manufactured by Corning
Sourced in United States
The Total RNA Small Amount Extraction kit is a laboratory product designed to extract small amounts of total RNA from various sample types. It provides a streamlined process for RNA isolation, suitable for applications where limited sample material is available.
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Lab products found in correlation
2 protocols using total rna small amount extraction kit
1
Quantifying HIF1α Expression in Glioma Cells
2
Investigating TMED3's Immunomodulatory Role
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The correlation between TMED3 and immune in ltration degree and immune checkpoint of CD4 T lymphocytes, CD8 T lymphocytes, B cells, macrophages, and dendritic cells was carried out in the sangerbox (http://vip.sangerbox.com/home.html).
RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
The Total RNA Small Amount Extraction Kit (Axygen, Corning, USA) was used to extract total RNA from cell lines according to the manufacturer's instructions. Sangon Biotech developed primers for the ampli cation of TMED3 and the endogenous control actin (Shanghai, China). The SYBR Green master mix kit (Tiangen Biotech, Beijing, China) was used for qRT-PCR according to the manufacturer's instructions. The 2-ΔΔCt method was used to calculate fold changes in target gene expression.
Western blot and antibodies SDS-PAGE (Solar Bio, Beijing, China) was used to separate the protein, which was then transferred to a polyvinylidene uoride membrane (Millipore, Billerica, MA, USA) and mixed with TMED3(1: 2000), GAPDH (1: 10000; Proteintech, USA) was incubated at 4℃for 12 hours. The HRP-bound anti-mouse or rabbit IgG antibody (1:10000, a nity) was incubated for 2 hours at 25°C. ECL chemiluminescence reagent(UE,Suzhou,China) was used to detect the target protein band. The strip density was measured and compared to the internal control using a gel imaging technique.
RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
The Total RNA Small Amount Extraction Kit (Axygen, Corning, USA) was used to extract total RNA from cell lines according to the manufacturer's instructions. Sangon Biotech developed primers for the ampli cation of TMED3 and the endogenous control actin (Shanghai, China). The SYBR Green master mix kit (Tiangen Biotech, Beijing, China) was used for qRT-PCR according to the manufacturer's instructions. The 2-ΔΔCt method was used to calculate fold changes in target gene expression.
Western blot and antibodies SDS-PAGE (Solar Bio, Beijing, China) was used to separate the protein, which was then transferred to a polyvinylidene uoride membrane (Millipore, Billerica, MA, USA) and mixed with TMED3(1: 2000), GAPDH (1: 10000; Proteintech, USA) was incubated at 4℃for 12 hours. The HRP-bound anti-mouse or rabbit IgG antibody (1:10000, a nity) was incubated for 2 hours at 25°C. ECL chemiluminescence reagent(UE,Suzhou,China) was used to detect the target protein band. The strip density was measured and compared to the internal control using a gel imaging technique.
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