Abi prism 7000 sequence detection system and software

Total RNA was isolated by RNeasy Plant Mini Kit (Qiagen, Germany) according to the manufacturer protocol followed by on-column DNase-I treatment for removal of genomic DNA contamination. For real time PCR analysis, first strand cDNA was synthesized with 2μg of total RNA using the High Capacity cDNA Archive kit (Applied Biosystems, USA) and 200nM of each primer mixed with SYBR Green PCR Master Mix (Applied Biosystems) for real-time PCR reactions, using the ABI Prism 7000 Sequence Detection System and Software (PE Applied Biosystems) according to the manufacturer’s instruction. Primer Express 2.0 (Applied Biosystems^®) was used for designing primers from a unique region, specific to the gene and each pair was confirmed by BLAST program in NCBI and TAIR database (S1 Table). The relative mRNA levels in different RNA samples were normalized with respect to internal control gene, Actin. The Ct (threshold cycles) values were averaged for two biological replicates and three technical replicates.

For PCR, cellular DNA was extracted from MARC-nsp11 cells using QIAamp DNA kit (Qiagen) and PCR was performed to determine the nsp11 gene integration. For reverse transcription (RT), total cellular RNA was extracted using Trizol (Invitrogen) and was treated with RQ1 RNase-free DNase I (Promega) followed by RT using the nsp11-specific reverse primer and PCR using the primer set as described above. Quantitative (q) PCR was performed using ABI Prism 7000 Sequence Detection System and Software (Applied Biosystems) in a final volume of 25 μL containing 2.5 μL of cDNA synthesized from the RT reaction, 2.5 pmol of each primer, 12.5 μL of SYBR Green Master Mix (Applied Biosystems), and 5 μL of water. The primer sequences were designed using Primer 5.0 Software (Invitrogen) or obtained from previous reports (Table 2). The amplification parameters were 40 cycles of two steps, each step comprised of heating at 95°C and extension at 60°C. The final mRNA levels of target genes were normalized using GAPDH as a house keeping gene.

Real-time PCR was performed using ABI Sequence Detector System (ABI Prism 7000 Sequence Detection System and software; Applied Biosystems) in a final volume of 25 μl containing 2.5 μl of cDNA from the reversed transcribed reaction, primer mix (2.5 pM each of sense and antisense primers), 12.5 μl of SYBR Green Master Mix (Applied Biosystems) and 5 μl of distilled water. The oligonucleotide primers were designed using Vector NTI software (Invitrogen) or obtained from literatures (S1 Table). The amplification parameters were 40 cycles of two steps each cycle comprised of heating to 95°C and 60°C. The final mRNA levels of the genes were normalized using GAPDH.