Revertra ace α qpcr rt kit

TRIzol Reagent (Life Technologies) was used to extract total RNA according to the manufacturer's protocol. RNA was quantified and reverse transcribed into cDNA using the ReverTra Ace‐α qPCR RT Kit (Toyobo, Japan). RT‐PCR of the mature miRNAs was performed using miRcute miRNA First‐Strand cDNA Synthesis Kit (Tiangen, Beijing, China). According to the user guide of the SYBR^® Green Realtime PCR Master Mix (Toyobo, Japan), the qRT‐PCR amplification was done on ABI7500 Fast system. Melting curve analysis was used to monitor the specificity of the PCR products. GAPDH was used as a control. The HCP5 primers were as follows: forward, 5′‐GACTCTCCTACTGGTGCTTGGT‐3′; reverse, 5′‐CACTGCCTGGTGAGCCTGTT‐3′. The BIRC3 primers were as follows: forward, 5′‐CCAAGTGGTTTCCAAGGTGT‐3′; reverse, 5′‐TGGGCTGTCTGATGTGGATA‐3′. The GAPDH primers were as follows: forward, 5′‐CGGAGTCAACGGATTTGGTCG‐3′; reverse, 5′ ‐TCTCGCTCCTGGAAGATGGTGAT‐3′. The miR‐219a‐5p primers were as follows: 5′‐GCTGATTGTCCAAACGCAATTCT‐3′; U6 was used as a control and the primers were as follows: forward, 5′‐CTCGCTTCGGCAGCACA‐3′; reverse, 5′‐AACGCTTCACGAATTTGCGT‐3′. All of these primers were purchased from Sangon Biotech, Shanghai, China. All experiments were performed in triplicate. The qRT‐PCR results were analyzed and expressed as relative miRNA or mRNA levels of the CT (cycle threshold) value and then were converted to fold change.

Total RNA was extracted from tissues and cells using TRIzol reagent (Invitrogen, Carlsbad, CA) following manufacturer’s protocol. The RNA was reverse transcribed into first stranded cDNA using ReverTra Ace-α qPCR RT Kit (Toyobo, Osaka, Japan). The qPCR was performed using SYBR Green Master Mix (Roche, Basel, Switzerland) on a CFX96 system (Bio-Rad, Hercules, CA). GAPDH and U6 were used as internal controls for mRNA and miRNA respectively. The relative expression of gene was calculated using 2^−ΔΔCT [27 (link)]. The primer sequences were listed in Table 1.Table 1

Sequence of primers

Primer	Sequence
MIAT-F	5′-GCACCTTGAGTGAATGTCAAGGCAG-3′
MIAT-R	5′-TGGCAGCATCCAGCCGACACACAGG-3′
LASP1-F	5′-TGCGGCAAGATCGTGTATCC-3′
LASP1-R	5′-GCAGTAGGGCTTCTTCTCGTAG-3′
COLCA1-F	5′-CTTATGACAGGAAAGTGGAAG-3′
COLCA1-R	5′-TAGCATCAAGTTCCCATCCAC-3′
SHNG14-F	5′-TGCACAAAATAAGCCTGGCTGT-3′
SHNG14-R	5′-TCAATATTTAATACAGGCATGCA-3′
SHNG15-F	5′-TTCAGACAATGACTTCCTCCCTCCT-3′
SHNG15-R	5′-TAGCTCCTGGGGCACTCAGCTC-3′
RNU12-F	5′-TGCCTTAAACTTATGAGTAAGG-3′
RNU12-R	5′-GGGCCGGACTTATCTTTCTGAA-3′
LINC00667-F	5′-CTGAAATCACAGCAATGCCAGTTT-3′
LINC00667-R	5′-TATAGCTTTGATTTTCTTGCAGTGT-3′
GAPDH-F	5′-TTTGGTCGTATTGGGCGCCTGGTCA-3′
GAPDH-R	5′-TTGTGCTCTTGCTGGGGCTGGTGGT-3′
Stem-loop	5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCCAGCA-3′
miR-324-3p-F	5′-GCCGAGCCCACTGCCCCAGG-3′
miR-324-3p-R	5′-CTCAACTGGTGTCGTGGA-3′
U6-F	5′-CTCGCTTCGGCAGCACA-3′
U6-R	5′-AACGCTTCACGAATTTGCGT-3′