To verify the high-throughput sequencing results from the RNA-Seq data, the upregulated or downregulated genes were detected by RT-qPCR. The primers (Table 1
Real time fluorescence quantitative pcr system
Manufactured by Bio-Rad
Sourced in United States, China
The Real-time fluorescence quantitative PCR system is a laboratory instrument designed for the amplification and detection of nucleic acid sequences in real-time. It utilizes fluorescent dyes to monitor the progress of the PCR reaction, allowing for the quantification of target DNA or RNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
Reverse transcriptase kit, by Accurate Biology (1 mentions)
Trizol reagent, by Accurate Biology (1 mentions)
Quantscript rt kit, by Tiangen Biotech (1 mentions)
Taq pro universal sybr qpcr master mix, by Vazyme (1 mentions)
Cell total rna isolation kit, by Vazyme (1 mentions)
Hiscript 3 all in one rt supermix, by Vazyme (1 mentions)
Reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Sybr green premix pro taq hs qpcr kit ag11701, by Accurate Biology (1 mentions)
Nanodrop nd 2000, by Thermo Fisher Scientific (1 mentions)
Novoscript plus all in one 1st strand cdna synthesis supermix, by Novoprotein (1 mentions)
Sybr qpcr mix, by Novoprotein (1 mentions)
7 protocols using real time fluorescence quantitative pcr system
1
Validating RNA-Seq Data via RT-qPCR
2
Quantifying YBX1 mRNA Expression
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Total RNA was extracted using TRIzol reagent (Accurate Biotechnology, China) and 1 µg of total RNA was used for synthesis of first-strand cDNA with a reverse transcriptase kit (Accurate Biotechnology, China). The mRNA level was measured by qPCR with Kit AG11701 (Accurate Biotechnology, China) on a Real-time fluorescence quantitative PCR system (Bio-Rad, USA). The primers used in the study were as follows: YBX1 Forward: AAGGAGAAAAGGGTGCGGAG; YBX1 Reverse: CCTACGACGTGGATAGCGTC; GAPDH Forward: CGAGATCCCTCCAAAATCAA; GAPDH Reverse: TTCACACCCATGACGAACAT. GAPDH was used as control. Relative expression was determined with GAPDH control through the 2−∆∆Ct method.
3
Real-time qPCR Validation of Transcriptome
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Real-time fluorescence quantitative PCR (RT-qPCR) was used to verify the transcriptome data using a real-time fluorescence quantitative PCR system (Bio-Rad, Hercules, CA, USA). Ten genes were selected and Primer Premier 5 was used to design primers (Supplementary Table 1 ). One microgram of RNA in each sample was reverse-transcribed into cDNA using the QuantScript RT Kit (Tiangen, China), and then amplified by RT-qPCR. Three PCR were performed for each sample. Evaluation of relative gene expression by 2–ΔΔCT method (Livak and Schmittgen, 2001 (link)).
4
Goldfish Immune and Apoptosis Gene Expression
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To analyze the expression of immune-related genes and apoptosis-related genes, as well as to validate sequence quantification, quantitative real-time PCR (qRT-PCR) was performed targeting some selected DEGs associated with immunity (IL-1β, IL-10, IL-8) and apoptosis (Caspase 3, Bad, Bax). As an internal control, the β-actin gene of goldfish was used to normalize the expression level of genes. qRT-PCR was performed in a total volume of 10 μL containing 5 μL of TB Green™ Premix Ex Taq™ II, 0.2 μL of Rox, 1 μL of cDNA, 0.8 μL of each primer (specific primers outlined in Table 1
5
Ti3C2-PVP Nanosheet-Induced Osteogenesis
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The BMSCs were cultured in 12-well plates and exposed to 12.5 μg mL−1 Ti3C2–PVP nanosheets and NIR light. Osteogenesis induction medium was prepared by supplementing the complete medium with 10 nM dexamethasone, 10 mM sodium β-glycerol phosphate disodium, and 50 μg mL−1 ascorbic acid. After osteogenesis induction for 14 days, total RNA was extracted using Cell Total RNA Isolation Kit (Vazyme, China). Reverse transcription was performed to generate cDNA using HiScript III All-in-one RT SuperMix (Vazyme, China). Then, a 20 μL quantitative real-time PCR system was established using the Taq Pro Universal SYBR qPCR Master Mix (Vazyme, China). Real-time fluorescence quantitative PCR analysis was performed using a real-time fluorescence quantitative PCR system (Bio-Rad, USA). Relative gene expression levels were determined by the ΔΔCt method and normalized by GAPDH. Primer sequences used in the analysis are listed below:
GAPDH: F-5′-AGGTCGGTGTGAACGGATTTG-3′, R-5′-TGTAGACCATGTAGTTGAGGTCA-3′;
ALP: F-5′-TCCGTGGGCATTGTGACTAC-3′, R-5′-TGGTGGCATCTCGTTATCCG-3′;
OCN: F-5′-GGTAGTGAACAGACTCCGGC-3′, R-5′-GGCGGTCTTCAAGCCATACT-3′;
OPN: F-5′-ATCTCACCATTCGGATGAGTCT-3′, R-5′-TGTAGGGACGATTGGAGTGAAA-3′;
RUNX2: F-5′-GACTGTGGTTACCGTCATGGC-3′, R-5′-ACTTGGTTTTTCATAACAGCGGA-3′;
COL1: F-5′-GCTCCTCTTAGGGGCCACT-3′, R-5′-ATTGGGGACCCTTAGGCCAT-3′.
GAPDH: F-5′-AGGTCGGTGTGAACGGATTTG-3′, R-5′-TGTAGACCATGTAGTTGAGGTCA-3′;
ALP: F-5′-TCCGTGGGCATTGTGACTAC-3′, R-5′-TGGTGGCATCTCGTTATCCG-3′;
OCN: F-5′-GGTAGTGAACAGACTCCGGC-3′, R-5′-GGCGGTCTTCAAGCCATACT-3′;
OPN: F-5′-ATCTCACCATTCGGATGAGTCT-3′, R-5′-TGTAGGGACGATTGGAGTGAAA-3′;
RUNX2: F-5′-GACTGTGGTTACCGTCATGGC-3′, R-5′-ACTTGGTTTTTCATAACAGCGGA-3′;
COL1: F-5′-GCTCCTCTTAGGGGCCACT-3′, R-5′-ATTGGGGACCCTTAGGCCAT-3′.
6
Quantifying mRNA Expression via qPCR
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Isolation of total RNA from cells entailed the use of Trizol reagent, followed by reverse transcription of 1 μg of total RNA into first-strand cDNA utilizing a reverse transcriptase kit (Thermo Fisher, America). To quantify mRNA levels, qPCR was executed employing the SYBR® Green Premix Pro Taq HS qPCR Kit AG11701 (Accurate Biotechnology, China) on a Real-time fluorescence quantitative PCR system (Bio-Rad, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was enlisted as the internal control, with the 2-∆∆Ct method serving as the calculation methodology. Comprehensive insight into primer sequences employed for qRT-PCR, along with those pivotal for RIP, was collated within Table Supplementary 2 for qRT-PCR and Table Supplementary 3 for RIP.
7
Quantitative Real-Time PCR Analysis of Inflammatory Markers
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An RNA-Quick Purification kit (ESscience, China) was used to extract total RNA from lung tissue. RNA was quantified spectrophotometrically (Nanodrop ND-2000; Thermo Scientific, Waltham, MA) and reverse transcribed into cDNA using a NovoScript Plus All-In-One 1st Strand cDNA Synthesis SuperMix (NovoProtein, Shanghai, China). qRT-PCR employed a SYBR qPCR Mix (NovoProtein) and real-time fluorescence quantitative PCR system (Bio-Rad, Hercules, CA). Specific primers were synthesized by ThingKe Biotechnology (Guangzhou, China) and are listed in Table 1 . Relative expression levels were calculated by normalizing expression of the GAPDH housekeeping gene using the 2−ΔΔCt method and are presented as the fold increase compared with the control.
Quantitative real-time polymerase chain reaction primers
| Gene name | Forward primer (5′-3′) | Reverse primer (5′-3′) |
|---|---|---|
| Interleukin 1beta (IL-1β) | GTCGCTCAGGGTCACAAGAA | GTGCTGCCTAATGTCCCCTT |
| Interleukin 6 (IL-6) | TCTTCAACCAAGAGATAAGCTGGA | CGCACTAGGTTTGCCGAGTA |
| Interleukin 8 (IL-8) | TGTTCACAGGTGACTGCTCC | AGCCCATAGTGGAGTGGGAT |
| Transforming growth factor beta (TGF-β) | CTGCTGACCCCCACTGATAC | GGGCTGATCCCGTTGATTTC |
| Macrophage Inflammatory Protein 1 Alpha (MIP-1α) | TGCCAAGTAGCCACATCGAG | GAGATGGGGGTTGAGGAACG |
| GAPDH | CAGTGGCAAAGTGGAGATTGTTG | TCGCTCCTGGAAGATGGTGAT |
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