We extracted total RNAs from ccRCC cell lines and si-IL4I1-transfected 786-O or 769-P, as well as the ccRCC and adjacent normal samples of 10 KIRC patients using EZ-press RNA Purification Kit (EZBioscience, USA) and PrimeScript RT reagent kit (EZBioscience, China). The level of the mRNA IL4I1 was further examined through qRT-PCR with SYBR Green PCR reagent (EZBioscience, China) on Applied Biosystems™ QuantStudio™ 5 Real-Time PCR System in triplicate. Each mRNA expression was calculated with the 2-ΔΔCt method. All specific primers used in quantitative real-time PCR (qRT-PCR) are shown in Additional file 4 : Table S3. Human participants in these studies were reviewed and approved by the Institutional Ethics Committee for Clinical Research and Animal Trials of the First Affiliated Hospital of Sun Yat-sen University [(2021)144] and conformed to the standards set by the Declaration of Helsinki.
Primescript rt reagent kit
Manufactured by EZBioscience
Sourced in China, United States
The PrimeScript RT reagent kit is a set of reagents used for the reverse transcription of RNA to cDNA. It includes a reverse transcriptase enzyme, reaction buffer, and other necessary components to facilitate the conversion of RNA to complementary DNA for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Sybr green pcr reagent, by EZBioscience (5 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
Sybr green qpcr master mix, by EZBioscience (4 mentions)
Ez press rna purification kit, by EZBioscience (1 mentions)
Quantstudio 5 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr green premix ex taq, by Takara Bio (1 mentions)
Lightcycler 480 system, by Roche (1 mentions)
11 protocols using primescript rt reagent kit
1
Quantifying IL4I1 mRNA in ccRCC Samples
2
Quantifying TREM1 Expression by qRT-PCR and Immunoassays
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After extracting total RNA by TRIzol, PrimeScript RT reagent kit (EZBioscience, China) and SYBR Green PCR reagent (EZBioscience, China) were used for reverse transcription and further qRT-PCR according to the manufacturer’s instruction. ACTB was used as an internal control. The primer sequences are presented in Table S2
3
Quantitative Real-Time PCR for Gene Expression
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The total cellular RNA was extracted using TRIzol (Invitrogen, USA) reagent based on the protocol provided by the manufacturer and used to synthesize cDNA with the PrimeScript RT reagent kit (EZBioscience, China). EZBioscience 2× SYBR Green qPCR Master Mix (EZBioscience) was used for the procedure. Primers for qPCR were as follows. CLIC1 forward (5′- ACCGCAGGTCGAATTGTTC-3′) and CLIC1 reverse (5′- ACGGTGGTAACATTGAAGGTG-3′); ACTB forward (5′- CATGTACGTTGCTATCCAGGC-3′) and ACTB reverse (5′-CTCCTTAATGTCACGCACGAT-3′). TSINGKE produced every primer (Beijing TSINGKE Biotech Co., Ltd., China). After real-time PCR was carried out using the QuantStudio 5 Real-Time PCR machine, data were examined using the QuantStudio Design and Analysis software. The 2–ΔΔCt technique was implemented to measure relative gene expression, and ACTB was employed to normalize the results.
4
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was isolated from cells using TRIzol (Thermo Fisher Scientific, Waltham, MA, USA), and RNA was reverse-transcribed into cDNA by the PrimeScript RT reagent kit (EZBioscience, Roseville, USA). SYBR Green Premix Ex Taq (Takara Bio, Tokyo, Japan) was used for subsequent qRT-PCR amplification on an LC480 system. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 were used as internal controls.
5
Gene Expression Analysis via qRT-PCR
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Total RNA was extracted using TRIzol Reagent (Invitrogen, United States) according to the manufacturer’s instructions. NanoDrop was used to detect RNA concentration by A260/A280 ratio. We performed cDNA synthesis and conducted qRT-PCR with PrimeScript RT reagent kit (EZBioscience, China), and SYBR Green PCR reagent (EZBioscience, China). The reaction was incubated at 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. ACTB was used as an internal control. The primer sequences were exhibited in Supplementary Table S4 . Data were analyzed using the 2−ΔΔCT relative quantification method.
6
Validating Signature Genes in PDAC
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To further validate the potential roles of signature genes in PDAC, six paired PDACs and matched normal adjacent tissue samples were collected from the SHDSYY. Ethical approval was confirmed by the ethical committee of the hospital. Tissue specimens were frozen in liquid nitrogen and stored at − 80 °C until used. Total RNA was extracted with a TRIzol Reagent (ThermoFisher: #15596018), and the concentration was calculated by the A260/A280 ratio. The PrimeScript RT reagent kit (EZBioscience: #A0010CGQ) and SYBR-Green PCR reagent (EZBioscience: #A0012-R2-L) were used to perform cDNA synthetization and further conduct RT-qPCR based on the LightCycler ® 480 System (Roche). The housekeeping gene GAPDH was used as an endogenous control. The 2−DDCT cycle threshold method was used to calculate the relative expression. Supplementary Table 3 lists the primers used in this study.
7
Quantifying lncRNA Expression by qRT-PCR
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The TRIzol (Invitrogen, USA) reagent was utilized to extract total cellular RNA based on the protocol. RNA was reverse transcribed to cDNA by the PrimeScript RT reagent kit (EZBioscience, China). EZBioscience 2 × SYBR Green qPCR Master Mix (EZBioscience, China) conducted the procedure. Primers for qRT-PCR were provided by TSINGKE (Beijing TSINGKE Biotech Co., Ltd., China) and shown in Additional file 2 : Table S2. ACTB was chosen for internal reference. Expression levels of lncRNAs were measured as 2-ΔΔCT.
8
RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted using TRIzol reagent (Invitrogen, USA). The extracted RNA was then reverse‐transcribed to cDNA using a PrimeScript RT Reagent Kit (EZ Bioscience, China) according to the manufacturer's protocol. qRT‐PCR was conducted using a SYBR Green Master MIX Kit (EZ Bioscience, China) according to the manufacturer's instructions. The qRT‐PCR assays were performed in triplicate, and the relative gene expression was determined using the 2−ΔΔCt method. The primers used are listed in Table S1 (Supporting Information).
9
Quantification of gene expression
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The TRIzol Reagent (Invitrogen, USA) was applied to extract total cellular RNA according to the protocol. RNA was reverse transcribed to cDNA by the PrimeScript RT reagent kit (EZBioscience, China). EZBioscience 2 × SYBR Green qPCR Master Mix (EZBioscience, China) conducted the procedure. Primers for mRNAs were provided by TSINGKE (Beijing TSINGKE Biotech Co., Ltd., China) and shown in Additional file 5:Table S4
10
Quantifying lncRNA Expression via qRT-PCR
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The TRIzol (Invitrogen, United States) reagent was utilize to extract total cellular RNA based on the protocol. RNA was reverse transcribe to cDNA by the PrimeScript RT reagent kit (EZBioscience, China). EZBioscience 2 × SYBR Green qPCR Master Mix (EZBioscience, China) conducted the procedure. Primers for qRT-PCR were provided by TSINGKE (Beijing TSINGKE Biotech Co., Ltd., China) and shown in Supplementary Table S1 . ACTB was chosen for the internal reference. Expression levels of lncRNAs were measured as 2−ΔΔCT.
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