Superscript 3 preamplification system

The TRIzol reagent (Ambion, Cartsbad, CA, USA) was used to extract the cells and isolate the total RNA. The superscript III preamplification system (Invitrogen, Carlsbad, CA, USA) was used to synthesize the cDNA. TaqMan^Tm gene expression master mix and PCR FAM dye-labeled probes for human MT2A (Hs01591333_g1). HO-1 (Hs00157965_m1), β-actin (Hs01060665_g1), and 18S (Hs03003631_g1) were from Applied Biosystems (Foster City, CA, USA). The CFX Connect Real-Time PCR system (Bio-Rad Laboratories, Foster city, CA, USA) was used to perform the quantitative real-time PCR as described previously [23 (link)]. The mean cycle threshold (Ct) values for the 18S or β-actin control probe were used in the normalization of target gene expression. All the reactions were carried out in triplicate, and each experiment was performed at least 3 times independently.

The total RNA was isolated with a Trizol reagent, and cDNA was synthesized using the superscript III preamplification system (Invitrogen). The real-time polymerase chain reaction (PCR) was performed using a CFX Connect Real-Time PCR system (Bio-Rad Laboratories, Foster city, CA, USA). The FAM dye-labeled TaqMan MGB probes as well as PCR primers for human maspin (Hs00985283_m1), p21 (Hs00355782_m1), p27 (Hs01597588-m1), MMP2 (Hs01548728_m1), MMP9 (Hs00234579_m1), vimentin (Hs00185584_m1), Cyclin D1 (Hs00277039_m1), PTEN (Hs02621230_sl), p53 (Hs01034249_m1), 18S (Hs03003631_g1), and β-actin (Hs01060665_g1) were purchased from Applied Biosystems (Foster City, CA, USA). Either 18S or β-actin was used as an internal positive control. The mean cycle threshold (C_t) values for target genes were normalized against the 18S or β-actin control probe to calculate the ΔC_t values by using the StepOne software program v2.0 (Applied Biosystems). All the reactions were conducted in triplicate, and each experiment was conducted on at least three independent occasions.

The cDNAs were synthesized using the superscript III pre-amplification system (Invitrogen) after the total RNA was extracted from the cells using a TRIzol reagent (ambion, Life technologies, Carlsbad, CA, USA). The ARv7 primers (5′-GGAAATGTTATGAAGCAGGGATG-3′ and 5′-GGTCATTTGAGATGCTTGCA), ARFL primers (5′-TGCAGCCTATTGCGAGAGA-3′ and 5′-TGATCTCTGCCATCATTTCC), and 18S (5′-ACCGCAGCTAGGAATAATGGA-3′ and 5′-GCCTCAGTTCCGAAAACCA-3′) were used according to the previous report [50 (link)]. The qPCR reactions were performed in a 12 μL reaction volume that consisted of 6 μL of 2 x iQ^TM SYBR Green supermix (Bio-Rad Laboratories, Foster City, CA, USA), 1 μL of the cDNA sample, 1 μL of gene-specific primers (5 μM), and 4 μL of H₂O. For the MALT1 and NDRG1 assays, TaqMan^TM gene expression master mix and FAM dye-labeled TaqMan MGB probes for human MALT1 (Hs01120052_m1), NDRG1 (Hs00608387_m1), and β-actin (Hs01060665_g1) from Thermo Fisher Scientific Inc. (Vilnius, Lithuania) were used as described previously [35 (link)]. The qPCR analysis was performed using a CFX Connect Real-PCR system (Bio-Rad Laboratories, Foster City, CA, USA). The cycle threshold (Ct) values for the target genes were normalized against the 18S or β-actin control probe to calculate the mean cycle threshold (ΔCt) values using the Bio-Rad CFX manager 3.1 (Bio-Rad Laboratories, Foster City, CA, USA).

Total RNA from tissues or cells was isolated using Trizol reagent (Ambion, Life Technologies, Carlsbad, CA, USA). The cDNA was synthesized using Superscript III pre-amplification system (Invitrogen), as described previously [45 (link)]. The PCR probes for human p53 (Hs01034249_m1), PTEN (Hs02621230_Sl), TAGLN (Hs01038777_g1), β-actin (Hs01060665_g1), and 18S (Hs03003631_g1) were purchased from Applied Biosystems (Foster City, CA, USA). Real-time polymerase chain reactions (qPCR) were performed using an CFX Connect Real-Time PCR system (Bio-Rad Laboratories, Foster city, CA, USA) and the mean cycle threshold (C_t) values were calculated for internal control and target genes, as described previously [44 (link)].

The total RNA was extracted from cells with Trizol reagent and cDNA was synthesized by using the superscript III preamplification system (Invitrogen). FAM dye-labeled TaqMan MGB probes as well as PCR primers, which are used for human GDF15 (Hs00171132_m1), NDRG1 (Hs00608387_m1), Maspin (Hs00985283_m1), GFRAL (Hs01087628_m1), and β-actin (Hs01060665_g1), and were purchased from Applied Biosystems. The real-time PCR (qPCR) was performed using a CFX Connect Real-Time PCR system (Bio-Rad Laboratories, Foster City, CA, USA) as described previously [6 (link)]. Mean cycle threshold (C_t) values for target genes were normalized against the β-actin control probe to calculate Δ C_t values. All reactions were conducted on at least three independent occasions.