Total tissue RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and sample concentration and purity verified through standard methods. Analysis of miR-155 expression was performed using a Hairpin-it qRT-PCR kit (GenePharma, Shanghai, China) according to the manufacturer’s instructions using U6 snRNA as internal control. TLR3 expression was assayed with a SYBR Green PCR kit (GenePharma) with GAPDH as internal control. Reactions containing neither reverse transcriptase nor template were used as negative controls. PCR reactions were carried out on an Applied Biosystems 7500 Real-Time PCR system. Each assay was repeated 3 times. Relative gene expression was quantified by the 2-ΔΔCT method. Convergent primers were used to detect mRNAs and stem-loop RT primers were used to detect miR-155. Primer sequences are shown in Supplementary Table 2 .
Hairpin it qrt pcr kit
Manufactured by GenePharma
Sourced in China
The Hairpin-it qRT-PCR kit is a laboratory equipment product designed for real-time quantitative reverse transcription PCR (qRT-PCR) analysis. It enables the detection and quantification of RNA transcripts.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr kit, by GenePharma (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Hairpin it mirna qpcr quantitation kit, by GenePharma (1 mentions)
Mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using hairpin it qrt pcr kit
1
Quantitative Analysis of miR-155 and TLR3 Expression
2
Quantification of Gene Expression in PBMNCs
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Total RNA of peripheral blood mononuclear cells (PBMNCs) was extracted using TRIzol (Invitrogen, CA, United States). The purity and concentration of total RNA samples were quantified using the NanoDrop ND-1000 (NanoDrop Technologies/Thermo Scientific, Wilmington, DE, United States). Total RNA (2 μg) from each sample was reverse-transcribed using the Hairpin-it qRT-PCR kit (GenePharma, Shanghai, China) according to the manufacturer’s instructions. 20 μl reaction volume contained 10 μl Real-Time PCR Master Mix (SYBR), 0.4 μl 10 μM Gene-Specific Primer Set (0.2 μM final), 0.4 μl ROX reference dye (50×), 0.2 μl Taq DNA polymerase, and 2 μl of template cDNA and DEPC-treated water. GAPDH was used as an internal control to quantify and normalize the results. The 2-△△CT value was used for comparative quantitation. All qRT-PCRs were performed in triplicate. The primers were obtained from GenePharma (Shanghai, China), and the sequences are shown in Supplementary Table S2 .
3
RNA Extraction, Quantification, and qRT-PCR Analysis
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We used TRIzol reagent (Invitrogen, USA) to isolate the total RNA from frozen tissue. Before further experiments, the purity and concentration of the extracted RNA samples were quantified using NanoDrop ND-1000 equipment. The steps described in the Hairpin-it qRT-PCR Kit (GenePharma Co., Shanghai, China) were followed to reverse-transcribe the total RNA (2 μg) of each sample. Then, the qRT-PCR ran according to the qRT-PCR Kit instructions, and the CT values were obtained after the end of the reaction. Finally, the relative changes in gene expression were calculated by the 2−ΔΔCT method. In this experiment, GAPDH was used as an internal control. The primer sequences were purchased from Invitrogen (Waltham, MA, USA) and are shown in Table S2 . All PCR runs were performed in triplicate.
4
Comprehensive RNA Extraction and Analysis
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Total RNA was extracted using TRIzol (Invitrogen, CA, USA). The purity and concentration of RNA samples were quantified using a NanoDrop ND-1000 device (NanoDrop Technologies/Thermo Scientific, Wilmington, DE, USA). Total RNA (2 μg) from each sample was reverse transcribed using a Hairpin-it qRT-PCR kit (GenePharma Co., Shanghai, China) according to the manufacturer’s instructions. LncRNAs and circRNAs levels were normalized to GAPDH mRNA. Isolation of miRNAs was performed using a miRNA Isolation kit (Thermo Fisher Scientific, Waltham, USA), and a Hairpin-it miRNA qPCR Quantitation kit (GenePharma Co.) was used to measure miRNA levels according to the manufacturer's instructions. Relative miRNA gene expression was normalized to U6. Reverse transcription reactions were conducted using convergent primers for mRNAs and lncRNAs, divergent primers for circRNAs, and stem-loop RT primers for miRNAs (GenePharma). Relative changes in gene expression were measured using the 2−ΔΔCT method. Primer sequences are shown in Supplementary Table 7 .
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