0.2 to 1 million cells grown in 6 well dishes were trypsinized, pelleted, resuspended in Buffer RLT (Qiagen), and homogenized with QIAshredder (Qiagen). RNA was further extracted using RNeasy Mini Kit (Qiagen). 2 μg of extracted RNA from each sample was converted to cDNA using TaqMan reverse transcription reagents (Life Technologies). qPCR primers were designed to detect all splicing variants using Primer Express 2.0 (Applied Biosystems). The designed primers were confirmed to amplify only the designated gene transcripts using in-silico PCR (http://genome.ucsc.edu/cgi-bin/hgPcr ). For qPCR reactions, primers, cDNA and Power SYBR Green PCR Master Mix (Applied Biosystems) was mixed, and quantitation was performed using a StepOnePlus real-rime PCR system (Applied Biosystems). Experiments were done in biological triplicates, and the mean and the standard error of a representative result was shown.
Steponeplus real rime pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The StepOnePlus real-time PCR system is a compact, easy-to-use instrument designed for real-time PCR analysis. It provides a reliable platform for gene expression studies, SNP genotyping, and other real-time PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (2 mentions)
Primer express 2, by Thermo Fisher Scientific (2 mentions)
Rlt buffer, by Qiagen (2 mentions)
Taqman reverse transcription reagent, by Thermo Fisher Scientific (2 mentions)
Qiashredder, by Qiagen (2 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Gdna eraser, by Takara Bio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
3 protocols using steponeplus real rime pcr system
1
Quantifying Gene Expression Profiles by qPCR
2
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was isolated using TRIzol reagent (Invitrogen, Grand Island, NY, USA). Six microlitres of total RNA was subjected to reverse transcription by using a PrimeScript™ RT reagent kit with gDNA Eraser (Takara, Shiga, Japan). QRT–PCR was performed using the StepOnePlus™ real-rime PCR system (Applied Biosystems, Foster City, CA, USA). The sequences of the PCR primers were as follows: NEAT1: F-CAGTTAGTTTATCAGTTCTCCCATCCA, R-GTTGTTGTCGTCACCTTTCAACTCT; miR-101: F-UACAGUACUGUGAUAACUGAA, R-CAGUUAUCACAGUACUGUAUU; U6: F-CTCGCTTCGGCAGCACA, R-AACGCTTCACGAATTTGCGT; VEGF-C: F-AGAAGGAGGAGGGCAGAAT, R-GTCTCGATTGGATGGCAGTAG; and GAPDH: F-GTGGTCTCCTCTGACTTCAAC, R-CCTGTTGCTGTAGCCAAATTC. The data were recalculated by the 2–ΔΔCt method after normalization against the corresponding endogenous controls (GAPDH and U6).
3
Quantifying Gene Expression Profiles by qPCR
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