All in one first strand cdna synthesis kit

The total RNA from E. coli cells grown for 24 h in shake flasks was isolated using an RNA extraction kit (Dongsheng Biotech, Guangzhou, China), following the manufacturer's instructions. The first-strand cDNA was synthesized using an All-in-One™ First-Strand cDNA Synthesis kit (GeneCopoeia, Guangzhou, China). The qRT-PCR was perfor1med with the All-in-One™ qPCR Mix kit (GeneCopoeia) on an iCycler iQ5 Real Time PCR system (Bio-Rad Laboratories, California, USA). The template was 100 ng of cDNA. The PCR conditions were as follows: 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 20 s, and extension at 72°C for 15 s. The primers for qRT-PCR are presented in Supplementary Table 1. The data were analyzed by the 2^−ΔΔCt method described by Livak and Schmittgen (2001 (link)) and normalized by cysG gene expression.
Gene copy numbers were measured by qPCR on genomic DNA isolated from the appropriate CIChE strains. qPCR was performed as described above. The primers QPt1F/QPt1R and QHF/QHR (Supplementary Table 1) were used to measure the copy number of Pt1 and HMGS, respectively.

Total RNA from 16 pairs of OS tissue specimens and cell lines was isolated by TRIzol (Invitrogen, USA). The RNA was reverse-transcribed into cDNA using an All-in-one First-Strand cDNA Synthesis Kit (GeneCopoeia, Rockville, MD, USA). Quantitative reverse-transcription polymerase chain reaction (RT-qPCR; ABI 7500, Applied Biosystems, Foster City, CA, USA) was performed using Power SYBR^® Green PCR Master Mix (Applied Biosystems). GAPDH and U6 were used as internal references. RNA expression was quantified and fold-changes in expression were determined using the 2^–△△Ct method. All experiments were performed in triplicate. The primer sequences used in this study are listed in Additional file 1: Table S1.