18s rrna vic

The ABI PRISM 7500HT Sequence Detection System (Applied Biosystems, NY) was used for qt-RT-PCR analysis. Primer-probes sets for TLR1, TLR2, TLR4 and PU.1 (all FAM^TM) and 18S rRNA VIC® were obtained predesigned from Applied Biosystems and tested for primer efficacy (gene expression assays: Hs99999901_s1 18S VIC®, Hs00413978_m1 TLR1, Hs01872448_s1 TLR2, Hs00152939_m1 TLR4, Hs00162150_m1 PU.1). Multiplex amplification was carried out in a total volume of 20 μl for 40 cycles of 3 seconds at 95°C, 30 seconds at 60°C. Initial denaturation was performed for 3 min at 95°C. Target gene expression was normalized to 18s rRNA house keeping gene expression. Normalized target gene expression was analysed by the comparative ΔΔ-CT method and calculated as x-fold expression.

RNA was extracted from tissue and cells using TRI-reagent (Invitrogen). RNA quality was determined by visualization on a 1.5% agarose gel and quantified using a nanodrop. Reverse transcription was conducted using 500 ng RNA that was incubated with 250 μM random hexamers, 5.5 mM MgCl₂, 500 μM dNTPs, 20 units RNase inhibitor 63 units multiscribe reverse transcriptase, and 1× reaction buffer. Reverse transcription was performed using a thermocycler set at the following conditions: 25 °C for 10 min and 37 °C for 120 min before the reaction was terminated by heating to 85 °C for 5 min qPCR was performed in a 384-well plate in single-plex format. Primers and probes were purchased as Assay on Demand (FAM) products (Applied Biosystems). Total reaction volumes used were 10 μl containing Taqman Universal PCR mix (Applied Biosystems). All Ct values were normalized to 18s rRNA (VIC) (Applied Biosystems). The real-time PCR reaction was performed under the following protocol: 95 °C for 10 min, then 40 cycles of 95 °C for 15 s, and 60 °C for 1 min using an ABI7500 system. Data were collected as Ct values and used to obtain deltaCt (dCt) values.

Total RNA was extracted from skeletal muscle tissue using TRI-reagent (Invitrogen). RNA quality was determined by visualisation on a 1.5% agarose gel and quantity was measured by nanodrop absorbance at 260 nm. Reverse transcription was conducted using 1 μg RNA not DNAse treated that was incubated with 250uM random hexamers, 5.5 mM MgCl₂, 500uM dNTPs, 20 units RNase inhibitor 63 units multi-scribe reverse transcriptase and 1x reaction buffer. Reverse transcription was performed on a thermocycler set at the following conditions: 25 °C 10 min, 48 °C for 30 min before the reaction was terminated by heating to 98 °C for 5 min. cDNA levels were determined using an ABI7500 system (Applied Biosystems), reactions were conducted in a 384-well plate in single-plex format. Primers and probes were purchased as Assay on Demand (FAM) products (Applied Biosystems), predesigned to cross exon boundaries to preclude genomic DNA measurement. Total reaction volumes used were 10ul containing Taqman Universal PCR mix (Applied Biosystems). All reactions were normalised to 18 s rRNA (VIC) (Applied Biosystems). The real-time PCR reaction was performed at the following conditions: 95 °C for 10 min then 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Data were collected as Ct values and used to obtain deltaCt (dCt) values and expressed as fold change ±standard error of the mean (SEM).