Total RNA from WT and adjacent normal tissues was extracted using Trizol reagent and the concentration was measured using a UV spectrophotometer. Reverse transcription to cDNA was performed using the PrimeScriptTM RT kit (Qiagen) according to the manufacturer’s instructions. RT-qPCR (SYBR Premix Ex TaqTM, TAKARA) was performed using primers with the following sequences: XIST, F: 5ʹ-AACCACCACACGTCAAGCTCTTC-3ʹ, R: 5ÁGTGCCAGGCATGTTGATCTTCAG-3ʹ; miR-194-5p, F: 5ʹ-cgTGTAACAGCAACTCCATGTGGA-3ʹ; YAP: F: 5ʹ-TAGCCCTGCGTAGCCAGTTA-3ʹ, R: 5ʹ-TCATGCTTAGTCCACTGTCTGT-3ʹ. The following RT-qPCR reaction conditions were used: Cycle: 95°C for 30 s, 95°C for 5 s, and 60°C for 45 s, repeated for 40 cycles. Quantification of lncRNA and miRNA in tissues was calculated using the 2−ΔΔCT method. Three experimental replicates of each sample were obtained and the average was calculated.
Primescript rt kit
Manufactured by Qiagen
Sourced in Germany
The PrimeScript RT kit is a reverse transcription reagent system used for the conversion of RNA to cDNA. It contains the necessary components for efficient first-strand cDNA synthesis, including the reverse transcriptase enzyme, reaction buffer, and random primers.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Sybr premix ex taqtm, by Takara Bio (1 mentions)
Sybr green pcr kit, by Qiagen (1 mentions)
Q6 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Miscript pcr kit, by Qiagen (1 mentions)
Qiazol reagent, by Qiagen (1 mentions)
Paris kit, by Thermo Fisher Scientific (1 mentions)
Abi prism 7900 sequence detection system, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Bulge loop mirna qrt pcr starter kit, by RiboBio (1 mentions)
Cfx96 touch sequence detection system, by Bio-Rad (1 mentions)
Sybr green premix ex taq 2, by Takara Bio (1 mentions)
Sybr premix ex taq, by Qiagen (1 mentions)
Mirnease mini kit, by Qiagen (1 mentions)
Lightcycler 480 sybr green 1 master, by Roche (1 mentions)
7 protocols using primescript rt kit
1
Quantification of lncRNA and miRNA Expression
2
Quantitative RNA Expression Analysis
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Total RNA was extracted with a Trizol reagent. A NanoDrop 2000 spectrophotometer (Thermo Scientific, United States) was used to determine the concentration and OD260/OD280 of extracted RNA. To determine the integrity of RNA, the 5S rRNA, 18S rRNA, and 28S rRNA bands of total RNA were detected by agarose gel electrophoresis. Immediately thereafter, total RNA was reverse transcribed into complementary DNA (cDNA) using a PrimeScriptTM RT Kit (Qiagen, Germany). Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was conducted using an SYBR Green PCR kit (Qiagen, Germany). The PCR procedure was: 95°C for 5 min; 95°C for 10 s, and 60°C for 30 s, for 40 cycles. At the end of the cycle, the specificity of the product was identified by melting curve, the temperature was gradually elevated from 60°C to 97°C, and the fluorescence signal was collected 5 times per degree. The RT-qPCR amplification for each experiment was carried out in triplicate, and the 2-ΔΔCt method [20] (link) was used to calculate the expression of RNA.
3
Real-Time Quantification of Gene and MicroRNA Expression
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Total RNA was isolated from tissue samples or cultured cells using Qiazol reagent (Qiagen, Gaithersburg, MD, USA) or TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA was reverse‐transcribed into miRNA cDNA or total cDNA using the Prime Script miRNA cDNA synthesis kit (Qiagen) or PrimeScript RT kit (Qiagen), and then subjected to quantitative RT‐PCR (qRT‐PCR). mRNA or miRNA expression was determined by qPCR on a Q6 real‐time PCR System (Applied Biosystems, Carlsbad, CA, USA) using SYBR Green Master Mix (4309155, Applied Biosystems) or the miScript PCR Kit (Qiagen), using GAPDH or U6, respectively, as the internal loading control. Reverse transcription and PCR were performed as described in our previous report.55 Specific primers for human MMP9 (Forward: 5′‐TTC CAA ACC TTT GAG GGC GA‐3′ and Reverse: 5′‐CTG TAC ACG CGA GTG AAG GT‐3′), human SMURF1 (Forward: 5′‐AAC TGA AAC CCA ATG GCA GAA ATGT‐3′ and Reverse: 5′‐TTG CCA GAA CCA CCG CAC GAT G‐3′), and human GAPDH (Forward: 5′‐GAC TCA TGA CCA CAG TCC ATG C‐3′, Reverse: 5′‐CAG GTC AGG TCC ACC ACC ACT GA‐3′) were used for PCR amplification. The primer for miR‐516a (5′‐TGC TTC CTT TCA GAG GGT‐3′) was synthesized by Sunny Biotechnology (Shanghai, China), and U6 was used as the internal loading control through the primer provided in the miScript PCR Kit. The data were analyzed as previously described.55
4
Quantification of lncRNA, mRNA, and miRNA
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Total RNA was extracted with the RNeasy Mini Kit (Qiagen, New York, USA). lncRNAs and mRNAs were reversely transcribed with the PrimeScript RT Kit (Qiagen, New York, USA). miRNA was reversely transcribed with the Bulge-Loop miRNA qRT-PCR Starter Kit (RiboBio, Guangzhou, China). Then, qRT-PCR analysis was done on ABI PRISM 7900 Sequence Detection System (Applied Biosystems, USA) according to the manual of SYBR Green Master Mix (Applied Biosystems, USA). Both lncRNA and mRNA took GAPDH as internal reference, whereas miRNA took U6 as internal reference. Relative expressions of VPS9D1-AS1, miRNA-30a-5p, and KIF11 were obtained by 2−ΔΔCT method. Primer sequences were shown in Table 1 .
The cytoplasm and nucleus of A549 cells were isolated with the PARIS Kit (Thermo Fisher Scientific, USA). Then, RNA was separated from the two parts, with GAPDH as cytoplasmic control and U6 as nuclear control. Finally, qRT-PCR was used for analysis.
The cytoplasm and nucleus of A549 cells were isolated with the PARIS Kit (Thermo Fisher Scientific, USA). Then, RNA was separated from the two parts, with GAPDH as cytoplasmic control and U6 as nuclear control. Finally, qRT-PCR was used for analysis.
5
Quantitative Real-Time PCR Analysis of SFRP2 and β-Catenin
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Total RNA was isolated from EMS tissues and EEECs utilizing the TRIzol reagent (Invitrogen, Shanghai, China), and all cRNA transcripts were generated using a primeScript™ RT kit (Qiagen, Hilden, Journal of Molecular Histology1 Germany). All primers (Sangon Biotechnology, China) were listed as fellows: SFRP2, 5′-TGGGGGAAACGGTCGCACTC-3′, and 5′-GGCCACGAGACCATGAAGGAGG-3′. β-catenin, 5′-AAAGCGGCTGTTAGTCACTGG-3′ and 5′-CGAGTCATTGCATACTGTCCAT-3′. The qPCR was performed in triplicate to determine the relative levels of the target mRNA using SYBR premix Ex Taq™ Green II (Takara) in the CFX96 Touch sequence detection system (Bio-Rad, Hercules, CA, USA). Quantitative real-time PCR was conducted ABI 7500 Real-Time PCR System(Applied Biosystems/Life Tech).
6
Quantitative Real-Time PCR Analysis of Gene Expression
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Total RNA was extracted from cells using TRIzol® (Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. RNA was reverse transcribed into cDNA using the PrimeScript RT kit (Qiagen GmbH). RT-qPCR was performed with SYBR Premix Ex Taq (Qiagen GmbH) to quantify the RNA expression. The following thermocycling conditions were used for the qPCR: Initial denaturation at 95°C for 6 min; followed by 40 cycles of initiation at 94°C for 30 sec, annellation at 60°C for 30 sec and elongation at 73°C for 90 sec. The 2−ΔΔCq (25 (link)) method was employed to assay relative expression levels. The primers for SCNN1A were: Forward 5′-AGGACTCTAGCCCTCCACAG-3′, reverse 5′-GTGGATGGTGGTGTTGTTGC-3′. The primers for HOXD9 were: Forward 5′-ACGTCCCCAAGCCAGGCTC-3′, reverse 5′-CTATGTAGCTCAGGAATAA-3′. The primers for GAPDH were: Forward 5′-CACCATTGGCAATGAGCGGTTC-3′, reverse 5′-AGGTCTTTGCGGATGTCCACGT-3′.
7
Quantitative RT-PCR for Gene Expression
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In the light of the manufacturer's instructions, total RNA was extracted from the serum using the miRNease Mini kit (Qiagen, Hilden, Germany), and 500 ng of total RNA was implemented to synthesize complementary DNA according to the PrimeScript RT kit (Qiagen). Based on these gene sequences published in GenBank, Primer 5.0 software was applied to design primers (Table 1), and checked by NCBI Blast and oligo 7. Then, LightCycler 480 SYBR Green I Master (Roche Diagnostics, Penzberg, Germany) was utilized for PCR reaction. Each SYBR Green reaction took 0.25 µL of primers and 2 µL of cDNA as templates, with a total volume of 20 µL. U6 and glyceraldehyde phosphate dehydrogenase were selected as internal references. 2 -△△Ct method was implemented to calculate the relative expression level.
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