Primescriptvr rt reagent kit

Total RNA was extracted from the HOEC or OSCC cell lines and tissues with RNAiso Plus (TaKaRa, Japan) or the mirVana Paris kit (Life Technologies, USA), according to the manufacturer’s guidelines. For mRNA quantification, the PrimeScriptVR RT reagent kit (TaKaRa, Japan) was utilized to reverse transcribe the RNA extracted with RNAiso Plus to cDNA. After that, the RNA was subjected to specific primer-based reverse transcription-quantitative PCR (RT-qPCR) using SYBR Premix Ex Taq (TaKaRa, Japan). For miRNA quantification, the total RNA extracted with the mirVana Paris kit was reverse transcribed and quantified with the All-in-One miRNA RT-qPCR detection kit (GeneCopoeia, China). After that, RT-qPCR was performed with the 7900HT Fast real-time PCR system (Thermo Fisher Scientific, USA). Subsequently, the threshold cycle (2^−ΔΔCT) method was utilized to calculate relative expression of RNA, with GAPDH and U6 as the mRNA internal control and the miRNA internal control, respectively. Table 2 indicates the primer sequences used in this study.

Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized for the detection of NF-κB1, c-Rel, and ELK1 expression using the PrimeScriptVR ^RT Reagent Kit (Takara, Dalian, China). A qRT-PCR assay was performed to evaluate the mRNA expression using the SYBR® Select Master Mix (Applied Biosystems Life Technologies, Beijing, China) on the ABI PRISM 7300 Sequence Detection System (Applied Biosystems Life Technologies). GAPDH mRNA was employed as an endogenous control and relative expression levels in each sample were measured by the 2^−ΔΔCT method. The primers for the analysis of NF-κB1, c-Rel, ELK1, and TAB1 expression are shown in Supplementary Table 2. For qRT-PCR analysis of miR-134 levels, cDNA was synthesized from 10 ng of total RNA using TaqMan^TM miRNA hsa-miR-134 specific primers (Applied Biosystems Life Technologies) and a TaqMan^TM microRNA Reverse Transcription kit (Applied Biosystems Life Technologies). All reactions were performed in triplicate.

All the RNAs were extracted from AML or normal bone marrow tissues and cells with the TRIzol reagent (Invitrogen, USA). After quantifying and assessing the RNAs with NanoDrop 2000 (Thermo Fisher Scientific, USA), the All-in-One miRNA qRT-PCR Detection Kit (GeneCopoeia, China) was utilized to examine miR-4458 expression. As for the measurement of LINC00665 and DOCK1 mRNA expression, the PrimeScriptVR RT reagent Kit (Takara, Japan) was applied to reverse-transcribe PCR and generate cDNA. Then, the SYBR Premix Ex Taq (Takara, Japan) was used qRT-and subjected to qRT-PCR was conducted through ABI 7900 system (Thermo Fisher Scientific, USA). The relative expression of LINC00665 and DOCK1 were subsequently normalized by GAPDH, while that of miR-4458 was normalized by U6. The 2^−ΔΔCT method was used to calculate their expression levels. The primer sequences are illustrated in Table 2.Table 2

The primer sequences for RT-qPCR.

GENE	Primer sequences (5′–3′)
miR-4458	Forward: AGAGGTAGGTGTGGAAGAA
	Reverse: GCGAGCACAGAATTAATACGAC
U6	Forward: CTCGCTTCGGCAGCACA
	Reverse: AACGCTTCACGAATTTGCGT
LINC00665	Forward: GGTGCAAAGTGGGAAGTGTG
	Reverse: CGGTGGACGGATGAGAAACG
DOCK1	Forward: CCGCCGCAAACTTTTTCCTC
	Reverse: AGATGTGCACAGTGTCTCCG
GAPDH	Forward: AGCCACATCGCTCAGACAC
	Reverse: GCCCAATACGACCAAATCC