Total RNA was extracted using the Trizol reagent (MRC, TR1187) and cDNA was converted from 1μg total RNA using the ReverTraAce Kit (TOYOBO, 34520B1). The qPCR reactions were done on Roche LightCycler® 96 PCR system with the SYBR Premix Ex Taq™ Kit (TAKARA, RR420A). Gene expression levels were normalized to GAPDH and compared to gene expression levels in hESCs. Three or more biological replicates were performed for each assay and data bars represent mean ± SD. Primers used in this study are listed in Table S1.
Lightcycler 96 pcr system
Manufactured by Takara Bio
The LightCycler® 96 PCR system is a real-time PCR instrument designed for quantitative and qualitative analysis of nucleic acid samples. It is capable of performing high-throughput, high-precision gene expression analysis and genetic variation detection.
Automatically generated - may contain errors
2 protocols using lightcycler 96 pcr system
1
Quantifying Gene Expression in hESCs
2
Quantification of Intestinal Tight Junction Genes
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Total RNA was extracted from the ileum using TRIzol Reagent, and reverse transcription was performed using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Dalian, China). Quantitative real-time PCR was performed using TB Green Premix Ex Taq II (Takara) by a Light Cycler 96 PCR System to examine the mRNA levels. Relative RT-PCR was performed to measure gene expression of Claudin-2, Occludin, and Zo-1mRNAs. The primers used were shown in Table 2 . Relative quantification of gene expression was determined by 2−ΔΔCt.Table 2
Primer sequences of target and reference genes.
| Gene name | Primer sequence (5′–3′) |
|---|---|
| Claudin-2 | F: CATACTCCTGGGTCTGGTTGGT |
| R: GACAGCCATCCGCATCTTCT | |
| Occludin | F: ACGGCACCTACCTCAA |
| R: GGGCGAAGAAGCAGATGAG | |
| Zo-1 | F: CAACGTAGTTCTGGCATTATTCG |
| R: GGAGGATGCTGTTGTCTCGG | |
| β-actin | F: AGTGTCTTTTTGTATCTTCCGCC |
| R: CCACATACTGGCACTTTACTCCTA |
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