Clone amv first strand cdna synthesis kit

MDMs were infected with H37Rv at a MOI of 1:2.5 (2.5 M. tb to one MDM), as outlined above, and treated with medium alone or recombinant D4GDI. In some experiments, CD4⁺CD25⁺ or CD4⁺CD25^‑ cells were added in Transwells. After 3 days, RNA was isolated from 10⁶ MDMs, and reverse transcribed, using the Clone AMV First-Strand cDNA synthesis kit (Life Technologies). For analysis of M. tb mRNA, host cells were lysed, bacteria were recovered by centrifugation, and then RNA was extracted by bead-beating in Trizol, followed by further purification.
Mycobacterial primer and probe sets were designed with primer express software (Applied Biosystems); probes were labeled with 5’-fluorescein phosphoramidite and 3’-TAMRA. The primers used to amplify human and M. tb cDNA are shown in S2 Table. Real-time PCR was performed using the Quantitect SYBR Green PCR kit (Qiagen) in a sealed 96-well microtiter plate (PE Applied Biosystems) on a spectrofluorometric thermal cycler (7700 PRISM, Applied Biosystems). PCR reactions were performed in triplicate as follows: 95°C for 10 min, and 45 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. Expression of human and M. tb genes were normalized to the amount of GAPDH and 16S rRNA transcripts in each sample, respectively.

Total RNA was extracted from lung leukocytes or lung tissue as described previously [39 ]. Total RNA was reverse transcribed using the Clone AMV First-Strand cDNA synthesis kit (Life Technologies). Real-time PCR was performed using the Quantitect SYBR Green PCR kit (Qiagen) in a sealed 96-well microtiter plate (Applied Biosystems) on a spectrofluorometric thermal cycler (7700 PRISM; Applied Biosystems). PCR reactions were performed in triplicate as follows: 95°C for 10 min, followed by 45 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. All samples were normalized to the amount of β-actin/GAPDH transcript present in each sample. The primers used in the study are listed in Table 2.