Pcr master mix kit

Total RNA was extracted from frozen surgical specimens and UC cell lines using Trizol reagent (Invitrogen, Carlsbad, USA). it was reverse transcripted into cDNA by random primers and M-MLV reverse transcriptase (Tiangen, Beijing, China). RNA quality was evaluated by electrophoresis on a 1% agarose gel. PCR Master Mix kit (Tiangen, Beijing, China) was used for cDNA amplification, and quantitative real-time PCR was performed by a Mastercycler real-time PCR machine (Eppendorf, Hamburg, Germany) using SYBR Green kit (Applied Biosystems, CA, USA) according to the manufacturer’s instruction. The relative mRNA expression levels were analyzed using the 2^–ΔΔCt method. All sequences (Table 2) were achieved by Primer Premier 5.0 software and GAPDH acted as an internal control.Table 2.

Sequences of primers and siRNA oligonucleotides

Primers	Forward primer (5'-3')	Reverse primer (5'-3')	Length of PCR products (bp)
PPARγ	TAGTCGAGGCACCTAGAGA	CTTGTGAATGGAATGTCTTCG	122
RXRα	TGACGTGCGACGTCGACAA	ACCTTGAGGACGCCATTGAG	110
GAPDH	GAAGGTGAAGGTCGGAGTC	GAAGATGGTGATGGGATTTC	226
siRNA	Sense oligonucleotide (5'-3')	Antisense oligonucleotide (5'-3')	Target gene sequence (5'-3')
LEF1	AAGAGAAAGAGAAGUUUGCC	GCAAACUUCUCUUUCUCUUCC	TGGCAAACTTCTTTCTCTTCT
Negative control	GUACCGCACGUCAUUCGUAUC	UACGAAUGACGUGCGGUACGU

The total RNA was extracted from transgenic rice leaves (for OsPDS, OsROC5, OsLAZY1, and OsActin gene detection) or endosperm (for OsGBSS gene detection) at the same development time by TRIzol method, and reverse transcription was carried out by RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, United States). RT-PCR was performed with PCR master mix kit (Tiangen, China) through the manufacturer’s instructions. OsActin (OsActin-F: 5′-CCTT GATTATGAGCAGGAGCTG-3′, OsActin-R: 5′-AAGTGATCTCCTTGCTCATAC-3′) fragment was used as an internal control to detect differences in expression levels of target gene in wild type and transgenic plants. Three biological replicates (three random-selected independent transgenic lines for each transgenic plants with different vectors) were examined to ensure reproducibility and each experiment was performed three times independently.