Hairpin it mirna qpcr kit

Total RNA was extracted from tumor tissues and cells with TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. cDNA was generated with a reverse transcription kit from Takara (Dalian, China). Amplification reactions included 2 µl of Mix, 2 µl of RNA and 6 µl of ddH₂O. The thermal cycling protocol was as follows: 37 °C for 15 min, 85 °C for 5 s and 4 °C for 15 min. The expression of SNHG16 and SLC2A4 was detected by using a SYBR Green Real-Time PCR Kit (Qiagen, Germany). miR-302b-3p expression was analyzed with a Hairpin-it^™ miRNA qPCR kit (GenePharma, China). Amplification of the transcripts involved an initial denaturation step at 95 °C for 2 min followed by 40 cycles at 95 °C for 5 s, 55 °C for 30 s, and 72 °C for 30 s. The mRNA and miRNA expression data are presented as fold changes in the mRNA/miRNA abundance normalized to β-actin or U6 snRNA expression. The primer sequences used in this study are presented in Table 1.
Table 1

Primer sequences used for qRT-PCR

Gene	Primer sequences
SNHG16
F	5ʹ-CAGAATGCCATGGTTTCCCC-3ʹ′
R	5ʹ-TGGCAAGAGACTTCCTGAGG-3ʹ
miR-302b-3p
Loop	5′-GTCGTATCCAGTCCAGGGACCGAGGACTGGATACGACCTACTA-3′
F	5′-GCGTAAGTGCTTCCATGTT-3′
R	5′-TCCAGGGACCGAGGA-3′
SLC2A4
F	5′-TGGCTGGGTTCTCCAACTG-3′
R	5′-CTGGAAACCCATGCCAATG-3′
β-actin
F	5′-ACCGAGCGCGGCTACAG-3′
R	5′-CTTAATGTCACGCACGATTTCC-3′
U6 snRNA
F	5′ -CTCGCTTCGGCAGCACA-3′
R	5′-AACGCTTCACGAATTTGCGT-3′