All in onetm mirna qrt pcr kit

Total RNA was extracted from the cells using TRIzol reagent (Invitrogen, MA, USA). MiRNA was extracted from cell lines using the miRcute adsorption column method (Tiangen Biotech Co., Ltd, Beijing, China). CircRNA was extracted using the RNeasy Mini Kit kit (Qiagen, Hilden, Germany). Expression of miR-940 and U6 was performed using the All-in-OneTM miRNA qRT-PCR kit (GeneCopoeia, Inc., USA) in a 7500 system (Applied Biosystems, Thermo Fisher Scientific, USA) for reverse transcription and RT-qPCR reactions. CircMAN1A2 and GAPDH mRNA were detected using the TB Green Premix EX Taq^TM kit (Takara Bio Inc., Tokyo, Japan). ERBB2 mRNA was detected using FastKing RT Kit (With gDNase) reverse transcribed from cDNA and then evaluated using the Super Real Pre Mix Plus (SYBR Green) kit (Tiangen). MiR-940 primer (HmiRQ0845) was ordered from FulenGen (Guangzhou, China), and the U6 primer (CD201-0145) was ordered from Tiangen. Normalization was performed using U6 and GAPDH and quantified by the 2^−ΔΔCq method [34 (link)]. The primers are listed in Table 2.

According to the manufacturer’s protocol, total RNA was extracted from serum (volume, 200 µl) using a miRcute miRNA extraction isolation kit (DP501, Tiangen, China). These RNAs were then reverse-transcribed into cDNA using the instructions of All-in-OneTM miRNA qRT-PCR kit (GeneCopoeia, the US).

Total RNA was extracted using the PureLink^TM RNA Mini Kit (Ambion) according to the manufacturer's instructions, and reverse transcription was used to generate cDNAs using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). SncRNA was extracted using the mirVana^TM miRNA Isolation Kit (Ambion) according to the manufacturer's instructions, and detected using All-in-One^TM miRNA qRT-PCR Kit (Genecopoeia) according to the manufacturer's instructions. Real time PCR was performed using SYBRPremix Ex Taq^TM (TaKaRa) and the 7500 Real-Time PCR System (Applied Biosystems). The reaction parameters were 95°C for 30 s followed by 40 two-step cycles of 95°C for 5 s and 60°C for 34 s. All the primer pairs used to PCR amplification were shown in Supplementary Table 4. 18s rRNA and 5s rRNA were used as reference genes. Ct values were calculated using Sequence Detection System software (Applied Biosystems), and the amount of target sequence normalized to the reference sequence was calculated as 2^−ΔΔCt.