Selected article for: "cdna synthesis and total volume"
Author: Sultani, Mozhdeh; Mokhtari Azad, Talat; Eshragian, Mohammadreza; Shadab, Azadeh; Naseri, Maryam; Eilami, Owrang; Yavarian, Jila
Title: Multiplex SYBR Green Real-Time PCR Assay for Detection of Respiratory Viruses
  • Document date: 2015_8_1
    • ID: 1dvj60yk_9
    Snippet: RNA was extracted from 200 µL of each sample using high pure viral nucleic acid kit (Roche, Germany). For synthesis of cDNA, 16 µL master mix (Fermentas, Germany) consisted of RT-PCR buffer, dNTPs, random hexamer, reverse transcriptase enzyme and RNase inhibitor was mixed with 24 µL of extracted RNA. Reverse transcription was performed at 37°C for one hour. Three multiplex SYBR Green real-time PCR assays were developed for simultaneous detect.....
    Document: RNA was extracted from 200 µL of each sample using high pure viral nucleic acid kit (Roche, Germany). For synthesis of cDNA, 16 µL master mix (Fermentas, Germany) consisted of RT-PCR buffer, dNTPs, random hexamer, reverse transcriptase enzyme and RNase inhibitor was mixed with 24 µL of extracted RNA. Reverse transcription was performed at 37°C for one hour. Three multiplex SYBR Green real-time PCR assays were developed for simultaneous detection of 12 respiratory RNA viruses. Each multiplex set was designed for detection of four viruses as follows: the first set was respiratory syncytial virus (RSV), human metapneumovirus (HMPV), rhinovirus (RV), and enterovirus (EV); the second set was parainfluenza viruses 1-4 (PIV1-4) and the third set was coronaviruses NL63, 229E, SARS, OC43. The next step was primer designing and the most important factor was the Tm of PCR products which must be different in each primer pair for specific detection of each virus according to the melt curve. Table 1 shows the properties of the primers. In all the sets, cDNA was amplified by a real-time PCR using Power SYBR Green PCR Master Mix Kit (ABI, USA) in OneStep ABI instrument (ABI, USA). Each reaction had a total volume of 25 µL, including 12.5 µL SYBR Green master mix, 200 nmol of each forward and reverse primers, 5 µL cDNA plus 7.1 µL ddH 2 O. The cycling conditions included an initial denaturation step of 10 minutes at 94°C, followed by 40 cycles of 15 seconds at 95°C, one minute at 55°C and one minute at 60°C. Fluorescent detection was at the end of each cycle. Melting curve analysis program was used for identification of specific PCR products. After the last cycle, the temperature was increased to 94°C, then decreased to 75°C and slowly increased to 94°C at a rate of 0.1°C per second, with continuous fluorescence monitoring. Positive and negative controls were added in each set. To prevent contamination, RNA samples and PCR master mixes were prepared under biosafety hoods in different rooms.

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