Total RNA was extracted from tissues and cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The purity and quantity of RNA was determined by measuring the absorbance at 260/280 nm using a SmartSpec Plus Spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Reverse transcription was carried out using the ReverTra Ace qPCR RT Kit (TOYOBO Life Science, Shanghai, China), according to the manufacturer’s instructions. qRT-PCR was performed using the Bestar SYBR Green qPCR Master Mix (TOYOBO) on a Bio-Rad S1000 system. For quantitative RT-PCR, GAPDH was used as an endogenous control, using the 2−ΔΔCT method. All primer sequences are listed in Supplementary Table 1
S1000 system
Manufactured by Bio-Rad
Sourced in China, Japan
The S1000 system is a versatile laboratory instrument designed for a range of applications. It features a compact and durable construction, with a user-friendly interface for easy operation. The system's core function is to provide precise and reliable results for researchers and scientists in various fields.
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Lab products found in correlation
3 protocols using s1000 system
1
Quantitative Gene Expression Analysis
2
Quantifying Gene Expression via RT-qPCR
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Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the control gene for assessing the impact of EIF3D knockdown. Standard procedures were followed for cDNA synthesis, and RT-qPCR was executed using the Bio-Rad S1000 system with HieffTMqPCR SYBR® Green Master Mix (Low Rox Plus; Yeasen, Shanghai, China). Supplementary Table 1 contains the information of the primers used. To standardize the concentration of each transcript, we normalised them to the GAPDH mRNA level using the 2−ΔΔCT method [24 (link)]. Statistical comparisons were conducted using the paired Student’s t-test, facilitated by GraphPad Prism software (Version 8.0, San Diego, CA, USA).
3
Quantitative RT-PCR Analysis of Lung Tissue
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TRIzol™ Reagent (Invitrogen, United States) was used to extract RNA from the lung tissues (n = 5 per group). Complementary-DNA was synthesized from 1 μg of total RNA (Toyobo, Osaka, Japan) on an S1000 system (Bio-Rad Laboratories). Quantitative RT-PCR was performed using AceQ Universal SYBR qPCR Master Mix (Q511-02/03, Vazyme, Nanjing, China). The messenger (m) RNA level was standardized to that of endogenous glyceraldehyde 3-phosphate dehydrogenase. Expression of target genes was analyzed by the 2−△△ct method. The primer sequences for rat samples were:
Mapk14-F:5′- GATGCCAAGCCATGAGGCAA.
Mapk14-R:5′- GGGTCGTGGTACTGAGCAAA.
Vegfa-F:5′- ACTCATCAGCCAGGGAGTCT.
Vegfa-R:5′- GGGAGTGAAGGAGCAACCTC.
Tgfbr1-F:5′- ACTCCCAACTACAGAAAAGCA.
Tgfbr1-R:5′- AAGGGCGATCTAGTGAGGGA.
Dusp1-F:5′- ATATCGTGCCGAACACCGAA.
Dusp1-R:5′- ACGCTTCATATCCTCCTTGG.
Gapdh-F:5′- GTCGGTGTGAACGGATTT.
Gapdh-R:5′- ACTCCACGACGTACTCAGC.
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