Tb green qrt pcr

Total RNA was isolated from the liquid nitrogen tissue samples of 16 BCa patients using the KZ-II grinder TRIzol reagent (Cat. abs9331-100ml, Absin, China) in an RNase-free atmosphere. The quality of total RNA was evaluated using a NanoDrop (Cat. #N60, Implen, Germany). Next, in a 20 ul total reaction volume using a PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Japan), the cDNA of mRNAs and lncRNAs was synthesized from the < 1000 ng of total extracted RNA, which was then amplified in a 25 ul total reaction volume with TB Green^® Premix Ex Taq™ II (Tli RNaseH Plus, Takara) using a CFX96 Real-Time PCR Detection System. Additionally, miRNA cDNA was synthesized from 250 ng < total RNA < 800ng in a 10 ul total reaction volume, and amplified in a 25 ul total reaction volume using Mir-X™ miRNA First- Strand Synthesis and TB Green™ qRT-PCR (Takara), according to the manufacturers protocol. The housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was used as an internal control to calculate the comparative quantification of gene expression levels via the 2-ΔΔCt method. All primers, the sequences of which are listed below, were synthesized by Sangon Biotech (Shanghai).

Total RNA was extracted from tissues or cells using Trizol (Takara, Japan) and cDNA prepared and subsequently quantitated using Mir-X™ miRNA First-Strand Synthesis and TB Green^® qRT-PCR (Takara, Japan) performed on ABI 7500 qRT-PCR System U6 was used as the internal control for quantitative PCR of miRNA, while the beta-actin was used as internal control used for quantitative PCR of CCND2. 2-delta delta CT was used to calculate the gene level in each sample. The primers used in this study were listed below (Table 1).Table 1

Primer sequences used for qPCR

Primer name	Sequences (5′–3′)
miR-93-5p	F: GCCGCCAAAGTGCTGTTC
	R: CAGAGC AGGGTCCGAGGTA
U6	F: CTCGCTTCGGCAGCACA
	R: AACGCTTCACGAATTTGCGT
CCND2	F: TTGTGATGCCCTGACTGAGC
	R: CACGTTGGTCCTGACGGTACT
ACTIN	F: AGCGAGCATCCCCCAAAGTT
	R: GGGCACGAAGGCTCATCATT