Sybr green real time pcr 2 premix kit

For absolute qPCR, the pMD-18T plasmid (mycoplasmal sequence inserted) was diluted in a 1:10 serial dilution five times and utilized as the standard template The absolute copy number of mycoplasmas in the mycoplasma-containing cell culture supernatant was determined by comparison with the standard curve, and the multiplicity of infection (MOI) was determined accordingly.
For relative qPCR, the intracellular mycoplasma copy numbers were normalized to host genome copy numbers. The host cells were suspended in 0.01 M PBS and washed five times to remove extracellular planktonic mycoplasma. The genomic DNA was then extracted from total cell extracts and purified as previously described (20 (link)).
qPCR was performed using the LightCycler^® 480 Real-Time PCR Detection System (Roche, Basel, Switzerland) and an SYBR Green Real-Time PCR 2× premix kit (Takara). The reaction conditions were as follows: 15 sec at 95ºC followed by 45 cycles of denaturation for 20 sec at 95ºC and annealing and extension for 20 sec at 60ºC. The melting curve of the products was determined and found to be specific. Primers used for the M. pulmonis 16s–23s gene spacer region were as follows: forward, 5′-GGAGCTGGTAATGCC CAAAGT-3′ and reverse, 5′-ACGTTCTCGTAGGGATAC CTTG-3′. Primers for host cell genome (β-hemoglobin) were as follows: forward, 5′-GAAGAGCCAAGGACAGGTAC-3′ and reverse, 5′-CCAACTTCATCCACGTT CAC-3′.

Total RNA was extracted from HeLa cells using TRIzol (Invitrogen, Carlsbad, CA, USA), and first strand cDNA reverse transcription was performed using a reverse-transcription RT-PCR kit (Takara) in the presence of Oligo (dT) 15 primers. qPCR was conducted using a LightCycler® 480 Real-Time PCR Detection System (Roche) and SYBR Green Real-Time PCR 2× premix kit (Takara). The reactions were performed under the following conditions: 15 sec at 95ºC followed by 40 cycles of denaturation for 15 sec at 95ºC and annealing and extension for 15 sec at 60ºC. The melting curves for Rab7, LC3 and p62 were determined and found to be specific. A two standard curve method was used to quantify the expression levels of Rab7, LC3 and p62, and the mRNA levels were normalized to that of β-actin. Primers used for Rab7, LC3, p62 and β-actin were as follows: Rab7 forward 5′-CATCCTGGGAGATTCTGGAGTC-3′ and reverse, 5′-TGT GTCCCATATCTGCATTGTG-3′; LC3 forward, 5′-GAGCAG CATCCAACCAAAATC-3′ and reverse, 5′-GCCTGATTA GCATTGAGCTGTAAG-3′; p62 forward, 5′-GGACTTGGT TGCCTTTTCCAGTG-3′ and reverse, 5′-GCAGCCGTC GCAGATCACAT-3′; β-actin forward, 5′-GCACCCAGC ACAATGAAGATC-3′ and reverse, 5′-CTCGTCATACTC CTGCTTGCTG-3′.

DNA was extracted from AGS or BGC823 cells after M. hyorhinis infection by DNA lysis buffer (50 mM Tris pH 8.5, 1 mM EDTA, 0.5% Tween-20, and 200 mg/L proteinase K) according to the standard protocol. qPCR was performed with 30 ng DNA and SYBR Green Real-time PCR 2×premix kit (Takara, Otsu, Japan) using Step One system from ABI (Foster City, CA, USA). The reaction programs and p37-specific primers (forward: 5’-TATCTCATTGACCTTGACTAAC-3’ reverse: 5’-ATTTTCGCCAATAGCATTTG-3’) were reported previously [22 (link)]. To compare M. hyorhinis DNA levels in cells, GAPDH (forward: 5’-TGAAGGTCGGAGTCAACGG-3’, reverse: 5’-CCTGGAAGATGGTGATGGG-3’) was amplified as control and the data were analyzed using the 2^-ΔΔCt method. Primers were synthesized by Sangon (Shanghai, China).