Mesa blue qpcr kit for sybr assay

HFFF cells infected at MOI 3 were acid washed 1 h after infection to inactivate unpenetrated virus, then harvested at 2 or 16 h after infection. DNA was harvested using the DNeasy blood and tissue kit (Qiagen), and qPCR assays were carried out in a LightCycler96 system (Roche), using MESA BLUE qPCR kit for SYBR assay (Eurogentec) according to the manufacturer’s instructions with primers for 18S (see Table 2) and HSV1 UL48 gene.

RNA was isolated from cells using an RNeasy minikit (Qiagen), and 400 ng to 1 μg of RNA was then DNase I treated according to the manufacturer’s protocol (Thermo Fisher Scientific). Superscript III (Invitrogen) was used with a random primer mix to make cDNA according to the manufacturer’s instructions in a Veriti 96-well thermal cycler (Applied Biosystems). All quantitative PCRs (qPCRs) were assembled using the Mesa Blue qPCR kit for SYBR assay (Eurogentec) according to the manufacturer’s instructions, with the primer sets listed in Table S4. The qPCRs were carried out on a LightCycler96 system (Roche).

HeLa cells were reverse transfected with 20 nM siRNA duplexes and incubated for 48 h before infection with HSV1 Sc16 at an MOI of 2, including a control of untransfected cells infected in the presence of 100 ng/ml cytosine arabinoside (AraC). After 1 h, cells were subjected to a gentle acid wash to inactivate any virus that had not entered the cells. After a total of 2 or 24 h of infection, DNA was harvested using the DNeasy blood and tissue kit (Qiagen). The qPCR assays were carried out in a LightCycler96 system (Roche) using the Mesa Blue qPCR kit for SYBR assay (Eurogentec) according to the manufacturer’s instructions with primers for 18S (Table S4) and the HSV1 UL48 gene.