We isolated total RNA from samples of Eca109, EC9706, and TE-1 cells using TRIzol reagent (Invitrogen, Carlsbad, California, USA). We detected RNA purity and concentration using NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA was reversely transcribed into complementary deoxyribonucleic acid (cDNA) using a PrimeScript RT-PCR kit (TaKaRa, Dalian, China). We used an IQ5 RT-PCR system (Bio-Rad, Laboratories, Hercules, CA, USA) with SYBR Select Master Mix (Boster, Wuhan, China) to conduct qRT-PCR per manufacturers' instructions. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal reference for normalization. We evaluated the relative expression of target gene messenger ribonucleic acid (mRNA) via the 2-ΔΔCt method. The primers were shown in Table 1S .
Iq5 rt pcr system
Manufactured by Bio-Rad
Sourced in United States
The IQ5 RT-PCR system is a real-time PCR instrument designed for gene expression analysis and quantification. It provides thermal cycling, detection, and data analysis capabilities for real-time PCR experiments.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Primescript rt pcr kit, by Takara Bio (1 mentions)
Powertrack sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Goscript rt system, by Promega (1 mentions)
Transstart top green qpcr supermix, by Transgene (1 mentions)
Smart mmlv reverse transcriptase, by Takara Bio (1 mentions)
All in one mirna qrt pcr detection kit, by GeneCopoeia (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq 2 kit, by Takara Bio (1 mentions)
6 protocols using iq5 rt pcr system
1
RNA Extraction and qRT-PCR Analysis for Cell Lines
2
Quantitative PCR Analysis of Gene Expression
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Total RNAs were extracted from tissues and cells using TRIzol reagents (Invitrogen, USA) according to the manufacturer’s protocol and quantified by Nanodrop (Thermo Fisher Scientific, USA). Synthesis of cDNAs was executed using the GoScript RT system (Promega). Quantitative PCR was done as previously described [18]. The qRT-PCR analysis was performed using PowerTrack SYBR Green Master Mix (Applied Biosystems) on an IQ5 RT-PCR system (BioRad, USA). 18S or GAPDH was used as the internal control to normalize the data to determine the relative expression of the target genes by using the 2−ΔΔCt method. The primers for RT-qPCR were listed in Table S2 .
3
Quantitative Analysis of miRNA Expression
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Through real-time fluorescence quantitative analysis, it is verified that miR160b, miR396g, miR6300, ApUFGT, ApSUS, and ApUGP2. With SMART MMLV Reverse Transcriptase (Takara Bio Inc., China) Reverse transcription of RNA; Reverse transcription reaction of miRNA was performed with Green miRNA First-Strand cDNA Synthesis SupperMix (TransGene, China).According to the screened miRNA and target gene sequence, fluorescence quantitative PCR primers were designed (Table S2 and S3
4
Comprehensive Gene Expression Analysis
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Total RNAs were isolated using Trizol reagent (Invitrogen), assessed for the purity and concentration spectrophotometrically, and then reversely transcribed into cDNA with Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc, MA, USA). RT-qPCR analysis were carried out in a 20 μL reaction system consisting of 5 μL cDNA template, 1 μL forward and reverse primers, and 10 μL SYBR Green qPCR Mix (Dong-sheng Bio, Guangzhou, China) on a Bio-Rad iQ5 RT-PCR System (Bio-Rad Laboratories, CA, USA). The PCR assays were performed as follows: 95°C for 2min, followed by 40 cycles of 95 °C 15 s and 60 °C for 20s, and subsequently 72 °C for 20 s. β-actin was used as an endogenous control. Forward and reverse primers against FLG, LOR, IVL, and IL-13Rα1 for PCR amplification were designed based on their sequences in the GenBank, consistent with our preliminary study 12 (link).
Also, the reverse transcription of miRNA into cDNA and RT-qPCR analysis was performed using All-in-One miRNA qRT-PCR Detection Kit (GeneCopoeia, MD, USA). The PCR process was 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 20 s, and subsequently 72 °C for 20 s. A human U6 snRNAs was used as the endogenous control. Primers for the amplification of the cDNAs of hsa-miR-143a and hsa-U6 were purchased from GeneCopoeia.
Also, the reverse transcription of miRNA into cDNA and RT-qPCR analysis was performed using All-in-One miRNA qRT-PCR Detection Kit (GeneCopoeia, MD, USA). The PCR process was 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 20 s, and subsequently 72 °C for 20 s. A human U6 snRNAs was used as the endogenous control. Primers for the amplification of the cDNAs of hsa-miR-143a and hsa-U6 were purchased from GeneCopoeia.
5
ErbB1 and ErbB2 Gene Amplification Analysis
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ErbB1 and ErbB2 genes amplification for 7 test and one control samples was analyzed by RT-PCR using a BioRad IQ5 RT-PCR system. The PCR reaction mixture contained 3 µl of genomic DNA (100 ng, 5-fold diluted), 1 µl of 5 mmol/l solutions of each of the forward and reverse primers, and 12.5 µl of 2× SYBR green DNA PCR Master Mix (BioRad, USA) in a total volume of 25 µl. PCRs for each primer set were performed in duplicates, and the means were reported. All PCR reaction conditions were as follows: an initial denaturation at 95°C for 5 min and 35 cycles (15 s at 95°C, 30 s at specific annealing temperature as listed in Table 1 and 30 s at 72°C). The primer sequences for amplifications are summarized in Table 1 . The agarose gel (2.0% w/v) electrophoresis and melting curve analysis (Tm) were employed to confirm the specificity of the products. The efficiency of amplifications was measured by the slope of a standard curve, derived from five-fold serial dilutions of human normal genomic DNA. In all cases, the amplification efficiency was between 95 and 105%, respectively. Amplification of ErbB1 and ErbB2 were normalized to INFγ as the single copy reference gene and fold changes were calculated by using the formula of 2 -ΔΔCt.
6
Total RNA Extraction and RT-PCR Analysis
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Total RNA from cultured cells was extracted using Trizol reagent (Invitrogen, USA) according to the manufacturer’s protocol. Then, 1 μg of RNA was used to synthesize cDNA using the Superscript II reverse transcriptase (Invitrogen-Life Technologies, UK). Next, RT-PCR was performed using the SYBR Premix Ex Taq II kit (TaKaRa) in the Bio-Rad IQ5 RT-PCR system according to the manufacturer’s instructions. Relative fold changes in mRNA were analyzed by using the 2−ΔΔCT method. β-actin served as the internal control.
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