Thermoscript rt pcr system for first strand cdna synthesis kit

Total RNA of frozen liver tissues (50 mg) was isolated with a RNeasy Lipid Tissue Mini Kit (Qiagen, cat. no. 74804) using the Qiacube robot (Qiagen, Hilden, Germany). In order to prevent genomic contamination, RNA was treated with DNase I. Quality and concentrations of RNA were verified using spectrophotometer (Nanodrop) and RNA gel electrophoresis, respectively. Subsequently, cDNA was synthesised using the ThermoScript™ RT-PCR System for First-Strand cDNA Synthesis Kit (Invitrogen) in accordance with the manufacturer's protocol. All cDNA was kept at −20 °C until use.

RNA was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA) as per manufacturer’s instructions from 50 to 100 mg of prostate tissue. All the experimental work was carried out under RNase-free environment using RNA-grade labware and reagents. The first-strand cDNA was synthesized from 2 μg of total RNA using the ThermoScript™ RT-PCR System for First-Strand cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA) as per manufacturer’s instructions. The primers for the PSP94 transcript, MSMB1, were as mentioned by Pathak et al. (2010 (link)) and primers for the gene of the house-keeping protein β-actin, (ACTB) were as mentioned by Cai et al. (2007 (link)). These primers were used for amplification of the cDNA by real-time PCR in duplicate reactions using SYBR green chemistry in a CFX96 qPCR system (BioRad, Hercules, CA, USA). The difference in the C_t values of MSMB1 in the test (BPH or PCa) and the calibrator sample (healthy) was noted as ΔC_t (MSMB1, Healthy—BPH or PCa). Similarly, the ΔC_t (ACTB, Healthy—BPH or PCa) was also noted for all samples. E_MSMB1 and E_ACTB, the amplification efficiencies of the qPCR reaction were calculated and the relative gene expression was determined as a ratio, by the Pfaffl method.

The eight influenza gene segments were first amplified using the RT-PCR protocol published by Deng et al. (2015) (link). Briefly, cDNA was made using uni-12 primer [5′-AGCAAAAGCAGG] with ThermoScript RT-PCR system for first-strand cDNA synthesis kit (Invitrogen) as per the manufacturer’s instructions. Then 2 μl cDNA was added in 17 separate PCR reactions with Platinum Taq DNA polymerase high fidelity kit (Invitrogen) using gene specific primers that tagged with M13 universal sequences to the 5′ end. The PCR program used were as follows: 2 min at 94°C, then 35 cycles of 30 s at 94°C, 30 s at 55°C, and 1 min at 68°C, with a final extension at 68°C for 2 min. PCR amplicons were visualized by E-gel (Invitrogen), followed by ExoSAP IT (GE Healthcare) purification and used for sequencing with the forward and reverse M13 primers with Big Dye Terminator Reaction Mix (Applied Biosystems). The products were purified by Big Dye XTerminator Purification Kit (Applied Biosystems) and run on ABI 3500 XL Genetic Analyzer (Applied Biosystems). Sequencing results were analyzed using the DNASTAR Lasergene 9 package. The A/New Zealand/316/2014 full genome sequence is publically available from the GISAID EpiFlu database¹ (accession numbers EPI587441-EPI587448).