Stratagene mx4000 real time qpcr system

The total DNA was isolated from the spinal cords and vaginal tissues preserved in RNAlater (Thermo Fisher Scientific, Waltham, MA, USA) using an RNA/DNA Extracol kit (Eurx, Gdansk, Poland). HSV-2 was detected using a HSV-2 probe labeled with FAM in a real-time PCR instrument, the Stratagene MX4000 Real-Time qPCR System (Agilent Technologies, Santa Clara, CA, USA), as described by Namvar et al. in 2005 [24 (link)]. A plasmid vector, pCR 2.1, containing envelope glycoprotein (gB) gene fragment was constructed and purified by the Institute of Biochemistry and Biophysics, Polish Academy of Sciences (Warsaw, Poland). The standard curve analysis was based on Ct values and the serial of 10-fold dilutions of the plasmid standard with an initial concentration of 2.62 × 10⁶ HSV-2 genome copy numbers per reaction. A standard curve was included in each PCR run. The amplification efficiency (E) was calculated from the standard curves, using the formula E = 10(−1/a) − 1, where a is the slope. Data are expressed as the HSV-2 copy number per ng of the total DNA in the tissue.

The total RNA was isolated from the spinal cords and the vaginal tissues preserved in RNAlater (Sigma Aldrich) using a Universal RNA Purification Kit (Eurx). Transcripts of IFN-α, IFN-γ, CXCL9, CXCL10, and GAPDH were quantified using Taqman(R) Gene Expression Assays (Thermo Fisher Scientific). All of the PCR reactions were carried out with the QuantiFast Probe RT-PCR Kit (Qiagen, Hilden, Germany), using a real-time PCR instrument, Stratagene MX4000 Real-Time qPCR System (Agilent Technologies), according to the manufacturer’s protocol. The 2^−∆∆Ct method was used for calculating the relative ratio to the control uninfected tissue.

Total DNA was isolated from trigeminal ganglia and brains preserved in RNA later (Thermo Fisher Scientific, MA, USA) using RNA/DNA Extracol kit (EURx, Gdansk, Poland). HSV-1 was detected using a HSV-1 probe labeled with FAM in a real-time PCR instrument Stratagene MX4000 Real-Time qPCR System (Agilent Technologies, USA) as described by Namvar et al. [27 (link)] and Orłowski et al. [28 (link)]. A plasmid vector pCR 2.1 containing an envelope glycoprotein (gB) gene fragment was constructed and purified by the Institute of Biochemistry and Biophysics Polish Academy of Sciences (Warsaw, Poland). Standard curve analysis was based on Ct values and serial of 10-fold dilutions of the plasmid standard with an initial concentration of 2.62 × 10⁶ HSV-1 genome copy numbers per reaction. A standard curve was included in each PCR run. The amplification efficiency (E) was calculated from the standard curves, using the formula E = 10(−1/a) − 1, where a is the slope. Data are expressed as the HSV-1 copy number per ng of the total DNA in the tissue.