Sybr premix ex taq 2 pcr kit

qRT-PCR was performed as previously described
[27] (link). An RNeasy Plus Mini kit (Bio Teke, Beijing, China) was used to collect RNA from hPDLSCs. A NanoDropR spectrophotometer (Thermo Fisher Scientific, Waltham, USA) was utilized to measure the concentration of each group. A cDNA synthesis kit (K1622; Thermo Fisher Scientific) was used to obtain first-strand cDNA. A SYBR Premix ExTaq II PCR kit (TaKaRa, Shiga, Japan) was used for RT-qPCR on the iCycler (Bio-Rad, Hercules, USA) as previously described
[28] (link). The sequences of the primers are provided in
Supplementary Table S1. All genes were analyzed in triplicate. The relative expressions of genes were determined with the 2
^–ΔΔCt method, based on normalization to the
GAPDH gene.

TRIzol reagent (Invitrogen; Thermo Fisher Scientific) was used to extract total RNA and RNeasy Maxi kit (Qiagen GmbH) was applied according to kit protocols. Then, cDNAs were synthesized according to protocols of Prime-Script RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China). PCR was conducted on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.) using a SYBR Premix EX Taq II PCR kit (Takara Biotechnology Co., Ltd.) with a miRNA-specific 5ʹ primer (has-miR-503-5P; CTG CAG AAC TCT TCC CGC TGC) and the mRO 3ʹ primer supplied with the kit for all miRNAs. The sequences for MALAT1 primers were as follows: forward, 5ʹ-AAAGCAAGGTCTCCCCACAA-3ʹ, and reverse, 5ʹ-GGTCTGTGCTAGATCAAAAGGCA-3ʹ. GAPDH was used as an internal control for mRNAs with primers as follows: forward, 5ʹ-GAAGGTGAAGGTCGGAGTC-3ʹ and reverse, 5ʹ-GAAGATGGTGATGGGATTTC-3ʹ. U6 was used as an internal control for microRNAs whose primers were designed and synthesized by Sangon Biotech (Shanghai, China). Analysis of gene expressions was measured using the ΔΔC_q method.20 (link)

Total RNA extraction, and RT-qPCR were performed as previously described (29 (link)). Total RNA was isolated from the tissue samples or cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. First-strand cDNA was synthesized using PrimeScript reverse transcriptase (Takara Biotechnology Co., Ltd., Dalian, China) and oligo (dT). Following RT, qPCR was performed according to the manufacturer's instructions of the SYBR Premix Ex Taq™ II PCR kit (Takara Bio, Inc., Otsu, Japan). The thermocycling conditions were as follows: 94°C for 3 min, followed by 40 amplification cycles of 30 sec at 94°C, 30 sec at 58°C and 30 sec at 72°C. The relative expression levels of cPLA2α mRNA were calculated using SYBR green on the ABI Prism 7300 Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.) using β-actin as the endogenous reference gene amplified from the samples. The relative amounts of mRNA were calculated using the 2^−ΔΔCq method (30 (link)). The primers were as follows: cPLA2α forward, 5′-GGTGGGAGAGAAGAAAGAAGTC-3′ and reverse, 5′-AGGGATTTTGGATTGTGCGACC-3′; β-actin forward, 5′-GCTATGCTCTCCCTCACGCCAT-3′ and reverse, 5′-TCACGCACGATTTCCCTCTCAG-3′.

Total RNA was extracted from blood using the miRNeasy Mini Kit (Qiagen GmbH), and from NSCLC cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) per the manufacturers' instructions. Then, the total RNA was reverse transcribed into cDNA using the miScript II RT Kit (Qiagen GmbH) or PrimeScript™ RT Master Mix (Takara Bio, Inc.) according to the manufacturers' instructions. qPCR was performed using the SYBR Premix Ex Taq II PCR kit (Takara Bio, Inc.) according to the manufacturer's protocol. The thermocycling conditions were as follows: 95˚C for 5 min, followed by 35 cycles of 95˚C for 20 sec, 60˚C for 30 sec, and 72˚C for 20 sec, with a final extension at 72˚C for 5 min. The relative levels of miR-107 and HMGB1 expression were calculated using the 2^-ΔΔCq method (21 (link)), and U6 and GAPDH served as the internal controls for miRNA and mRNA expression, respectively. The qPCR primer sequences were as follows: miR-107 forward, 5'-GCCGAGAGCAGCAUUGUACA-3', and reverse, 5'-CTCAACTGGTGTCGTGGA-3'; HMGB1 forward, 5'-CCA GGGGCTTTTCTACCAGG-3' and reverse, 5'-AACCCCAAC AAACTGCTGGA-3'; U6 forward, 5'-GTGCTCGCTTCGGCA GCACATATAC-3' and reverse, 5'-AAAAATATGGAACGC TCACGAATTTG-3'; and GAPDH forward, 5'-ACCCAGAAG ACTGTGGATGG-3' and reverse, 5'-CACATTGGGGGTAG GAACAC-3'.

RT-qPCR was performed as described in our previous study (11 (link)). Briefly, total RNA was extracted from cells using the TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. qPCR was performed using the SYBR Premix Ex Taq II PCR kit (Takara Bio, Inc.).
A total of 4 mg of DNase-treated (Ambion; Thermo Fisher Scientific, Inc.) RNA was reverse transcribed into cDNA using oligo(dT) primers and reverse transcriptase (Promega Corporation) under standard conditions. β-actin was used as a control. qPCR was performed using the StepOne and StepOnePlus Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). Cycle thresholds for each test mRNA were recorded and normalized to the control. The qPCR conditions were as follows: Initial denaturation at 95°C for 2 min, then 40 cycles of 95°C for 15 sec and elongation at 60°C for 1 min. The 2^−ΔΔCq method (33 (link)) was used to measure the relative expression of miR-16-1 or HSP70 using β-actin as internal control. The levels of miR-16-1 or HSP70 were expressed as the fold change from the mean expression levels in normal or NC group. The sequences of the primers are listed in Table I.