Power sybrs green pcr master mix

Total RNA of OSCC cells was extracted by applying a Trizol reagent following the instructions. Utilizing a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, UK), cDNA was synthesized. Then qPCR was executed on a ABI7300 real-time PCR machine (Helsinki, Finland) utilizing Power SYBRs Green PCR Master Mix (Applied Biosystems) by employing GAPDH as an endogenous control. The relative expression level was calculated utilizing the 2^-ΔΔCt method. Table 1 illustrated the primes utilized in qRT-PCR.
Table 1

The primers utilized in qRT-PCR

Name	Sequences
E2F7 forward	5′-TCTGAACCCGACTGTCCCTCTT-3’
E2F7 reverse	5′-TTTGGCAGCCACATCCAGAGTG-3’
GAPDH forward	5′-TGTGTCCGTCGTGGATCTGA-3’
GAPDH reverse	5′-CCTGCTTCACCACCTTCTTGA-3’

Total RNAs were extracted using TRIzol reagent (Invitrogen). cDNAs were synthesized using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCRs were conducted using the Power SYBRs Green PCR Master Mix (Applied Biosystems) on the StepOne Plus™ Real-Time PCR System. The PCR profile was 94°C for 10 min, and 42 cycles of 94°C for 10 s and 58°C for 15 s. PCR products were verified by melting curve analysis as well as by 2.0% agarose gel electrophoresis. Data analysis was conducted using the 2^−ΔΔCt method.

TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used for total RNA extraction, following the manufacturer's instructions. Then, 2 μg RNA was used for the reverse transcription of RNA to cDNA using M-MLV reverse transcriptase (Promega Corporation). qPCR was conducted using Power SYBRs Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) to detect the mRNA expression of LINC00630 and FGF7. miR-199a expression was detected using the TaqMan™ MicroRNA Reverse Transcription kit and TaqMan Universal Master Mix II kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). GAPDH and U6 were used as the endogenous reference for mRNA and miRNA, respectively. All forward and reverse primers were obtained from Biomics. The primer sequences used were as follows: LINC00630, 5'-TACCGTTATTATTTCCC-3' forward and 5'-TCCTAAGTATTGACCCT-3' reverse; FGF7, 5'-AATGACATGAGTCCGGAGCAA-3' forward and 5'-CCATAGGAAGAAAATGGGCTG-3' reverse; GAPDH, 5'-AGGTGAAGGTCGGAGTCAACG-3' forward and 5'-AGGGGTCATTGATGGCAACA-3' reverse; miR-199a, 5'-CGCGCCCAGTGTTCAGACTAC-3' forward and 5'-AGTGCAGGGTCCGAGGTATT-3' reverse; U6, 5'-CTCGCTTCGGCAGCACA-3' forward and 5'-AACGCTTCACGAATTTGCGT-3' reverse. The expression of miRNA and mRNA was expressed as a fold change using the 2^-∆∆Cq method 9 (link).

Trizol reagent (Invitrogen) was used to extract total RNA following kit specifications. Meanwhile, 2 μg RNA was collected to prepare cDNA through reverse transcription via M-MLV (Promega, Madison, WI, USA). Thereafter, qRT-PCR assays via Power SYBRs Green PCR Master Mix (Applied Biosystems, CA, USA) were performed for detecting the expression levels of MALAT1, β-catenin, caspase-3, TCF4, and p300. The miR-1297 expression with Taqman Universal Master Mix II kit and Taqman MicroRNA Reverse Transcription Kit (Applied Biosystems) was tested. U6 and GAPDH served as endogenous references of miRNA and mRNA. The sense/anti-sense chain primers based on Biomics were acquired. Table 1 shows the primer sequences. miR-1297 and mRNAs gene expression was denoted as fold changes using 2^−ΔΔCT method [27 (link)].Table 1.

Primer sequences for different genes

Genes	Primers	Sequences (5’-3’)
MALAT1	Forward	TGGGGGAGTTTCGTACTGAG
	Reverse	TCTCCAGGACTTGGCAGTCT
β-catenin	Forward	AAAGCGGCTGTTAGTCACTGG
	Reverse	CGAGTCATTGCATACTGTCCAT
TCF4	Forward	CCTGGCTATGCAGGAATGTT
	Reverse	CAGGAGGCGTACAGGAAGAG
p300	Forward	CTTCCCCACTGTCGCACAAT
	Reverse	TTTGTCGAGAAGATGCACAGTGT
Caspase-3	Forward	TTTGTTTGTGTGCTTCTGAGCC
	Reverse	ATTCTGTTGCCACCTTTCGG
GAPDH	Forward	CATGTTCCAATATGATTCCACC
	Reverse	GATGGGATTTCCATTGATGAC
miR-1297	Forward	TCGGCAGGTTCAAGTAATT
	Reverse	CTCAACTGGTGTCGTGGA
U6	Forward	ATTGGAACGATACAGAGAAGATT
	Reverse	GGAACGCTTCACGAATTTG

Trizol reagent (Invitrogen) was used for extraction of total RNA from C28/I2 cells under the guideline of the kit instructions. Specifically, RNA (2 μg) was reverse transcribed to cDNA using M-MLV (Promega, Madison, WI, USA). Quantitative PCR assays were conducted on Power SYBRs Green PCR Master Mix (Applied Biosystems, CA, USA) to detect the expression of HOTAIR, BBC3, and caspase-3. The expression of miR-221 was detected using the Taqman MicroRNA Reverse Transcription Kit and Taqman Universal Master Mix II kit (Applied Biosystems). GAPDH and U6 were used as the endogenous reference for mRNA and miRNA, respectively. All the primers for the sense and anti-sense chains were obtained from Biomics. The primer sequences are listed as follows: HOTAIR forward: 5’-TAGGCAAATGTCAGAGGGTT-3’, reverse: 5’-ACACAAGTAGCAGGGAAAGG-3’; BBC3 forward: 5’-TTGTGCTGGTGCCCGTTCCA-3’, reverse: 5’-AGGCTAGTGGTCACGTTTGGCT-3’; caspase-3 forward: 5’-TTTGTTTGTGTGCTTCTGAGCC-3’, reverse: 5’-ATTCTGTTGCCACCTTTCGG-3’; GAPDH forward: 5’-AGGTGAAGGTCGGAGTCAACG-3’, reverse: 5’-AGGGGTCATTGATGGCAACA-3’; miR-221 forward: 5’-GGGAAGCTACATTGTCTGC-3’, reverse: 5’-CAGTGCGTGTCGTGGAGT-3’; U6 forward: 5’-CTCGCTTCGGCAGCACA-3’, reverse: 5’-AACGCTTCACGAATTTGCGT-3’. The gene expression of miRNA and mRNA was indicated as fold changes by employing 2^−ΔΔCT method [17 (link)].