We first analyzed the relationship between hub TFs and various clinicopathological characteristics, and explored their expression characteristics. Then, 10 pairs of gastric cancer clinical surgical specimens were collected. All procedures were approved by the patient’s informed consent and the ethics committee of the Second Affiliated Hospital of Nanchang University. After the sample was homogenized, the total RNA was extracted with Trizol (Thermo Fisher, USA), and the RNA obtained was reverse transcribed using the reverse transcription kit RR047A (Takara, Japan). ACTB was used as the internal reference gene, and the mRNA expression of hub TFs was analyzed by rt-PCR using the RR820 kit (Takara, Japan) on the 7900-HT system (Thermo Fisher, USA). The primers were all synthesized by Shanghai Shenggong, see the attached table for details. In addition, the HPA database was used to analyze the protein expression of hub TFs [14 (link)]. Finally, the prognosis of hub TFs in GSE51105 was verified on Kaplan–Meier Plotter.
Rr820 kit
Manufactured by Takara Bio
Sourced in Japan
The RR820 kit is a laboratory equipment product from Takara Bio. It is designed for the amplification and detection of target DNA or RNA sequences. The kit includes all the necessary components to perform the reverse transcription and real-time PCR reactions.
Automatically generated - may contain errors
3 protocols using rr820 kit
1
Transcription Factors in Gastric Cancer
2
Validating Hub RBPs Expression and Prognosis
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In order to verify the prognosis of hub RBPs, we used Kaplan-Meier Plotter (http://kmplot.com/ ) to verify it in the GSE29272 data set 24 (link). For the verification of hub RBPs expression, we first use the GEPIA network tool for verification, which contains data from the TCGA and GTEx databases 25 (link). In addition, we also collected surgical samples from 10 pairs of gastric cancer patients. The process was approved by the patient's informed consent and the ethics committee of the Second Affiliated Hospital of Nanchang University. After homogenizing the clinical samples, the Trizol (Thermo Fisher, USA) method was used to extract total RNA. The obtained RNA was reverse transcribed using reverse transcription kit RR047A (Takara, Japan). ACTB was used as the internal reference gene, and the mRNA expression of hub RBPs was analyzed by fluorescence quantitative PCR using the RR820 kit (Takara, Japan) on the 7900-HT system (Thermo Fisher, USA). The primers used are all synthesized by Shanghai Shenggong Company, and the sequences of all primers are in Supplementary Table 2 .
3
Validation of Hub Genes Expression in Colorectal Cancer
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The expression of six hub genes was validated following MR in HCT116, HT29 and SW480. Total RNA was extracted from 80–90% confluent cells using TRIzol® Reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. After the concentration and purity of the RNA were qualified, an RR047a reverse transcription (RT) kit (Takara Bio, Inc.) was used for RT, according to the manufacturer's instructions. β-actin was used as the internal reference gene, and calculate the 2−ΔΔCq value for each hub gene. The calculation formula was as follows: ΔΔCq=ΔCqexperimentalgroup-ΔCqcontrolgroup, ΔCq=Cqtargetgene-Cqinternalreference (12 (link)). The expression level of the target gene was analyzed using an RR820 kit (Takara Bio, Inc.) in the ABI7900-HT system (Thermo Fisher Scientific, Inc.). The qPCR conditions were: Denaturation, 95°C, 30 sec; annealing, 95°C, 3 sec; extension, 60°C, 30 sec; 40 cycles. All primers used were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and the primer sequences are shown in Table I . The above experiments were repeated three times.
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