Faststart universal sybr green master rox

Total RNA was extracted using Invitrogen AmbionTRIzol® LS reagent
(Invitrogen Life Technologies) and reverse transcribed using Thermo Scientific
Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA,
U.S.A.) according to the manufacturer's instructions. qRT-PCR was carried
out with FastStart Universal SYBR Green Master (Rox) (Roche FastStart Universal
SYBR Green Master (Rox) (Roche Applied Science, Mannheim, Germany) on an ABI
Prism 7900 Fast instrument (Applied Biosystems, Foster City, CA, U.S.A.).
Reactions were performed in triplicate.
Primers for qRT-PCR reactions were as follows: human IL-6R, forward
5′-TTCTACAGACTACGGTTTGAG-3′ and reverse
5′-GGATGACACAGTGATGCT-3′; human IL-6ST, forward
5′-ACTGTTGATTATTCTACTGTGTAT-3′ and reverse
5′-AATTATGTGGCGGATTGG-3′; human BCL2, forward
5′-GATGACTGAGTACCTGAACC-3′ and reverse
5′-AGTTCCACAAAGGCATCC-3′; human CCND1, forward
5′-CGGAGGAGAACAAACAGA-3′ and reverse
5′-GCGGATTGGAAATGAACTT-3′; human MCL1, forward
5′-CAGGATTGTGACTCTCATT-3′ and reverse
5′-CCTCTACATGGAAGAACTC-3′; human MMP2, forward
5′-ACAAGAACCAGATCACATACAG-3′ and reverse
5′-TCACATCGCTCCAGACTT-3′; human GAPDH, forward
5′-GTCAAGGATTTGGTCGTATT-3′ and reverse
5′-AGTCTTCTGGGTGGCAGTGAT-3′. Then, the mRNA level of
IL-6R, IL-6ST, BCL2, CCND1, MCL1, and MMP2genes was normalized to GAPDH mRNA and expressed by
2^−ΔΔC_T(ΔΔC_t, the relative cyclic value)
[21 (link)].

RNA was reverse transcribed to cDNA using the High Capacity RNA to cDNA kit (Applied Biosystems, Foster City, CA). cDNA was then analyzed by quantitative RT-PCR. Real-Time PCR on microbial DNA was performed using primers found in Supplementary Table 1. Real-time PCR was performed using FastStart Universal SYBR Green Master (Rox) (Roche) and analyzed with ViiA™7 Real-Time PCR System (Applied Biosystems).
Real-time PCR was performed using FastStart Universal SYBR Green Master (Rox) (Roche) and analyzed with ViiA™7 Real-Time PCR System (Applied biosystems).

Total RNA was extracted using the Total RNA kit (OMEGA, USA) according to the manufacturer’s protocol. For this protocol, 1 μg of RNA was reverse transcribed into first strand cDNA using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). Real-time PCR was performed using a System 7500 instrument (Applied Biosystems) with 2× FastStart Universal SYBR Green Master (Rox) (Roche Applied Science, USA) using the relative quantity (2^–ΔΔCt) method. All reactions were performed in triplicate with internal duplicate determinations. The primer sequences were as follows: E-cadherin forward: 5’-GCCTCCTGAAAAGAGAGTGGAAG-3′, E-cadherin reverse: 5’-TGGCAGTGTCTCTCCAAATCCG-3′; Vimentin forward: 5′ -AGGCAAGCAGGAGTCCACTGA-3′, Vimentin reverse: 5′ -ATCTGGCGTTCCAGGGACTCAT -3′; MTA1 forward: 5′- CCAGGACCAAACCGCAGTAACA -3′, MTA1 reverse: 5′ -GTCAGCTTCGTCGTGTGCAGAT -3′; GAPDH forward: 5′- GTCTCCTCTGACTTCAACAGCG -3′, GAPDH reverse: 5′ -ACCACCCTGTTGCTGTAGCCAA -3′.