Brilliant 3 ultra fast sybr green qpcr master mix

qRT-PCR was performed using a two-step protocol as in (Ramos-Montanez et al., 2008 (link), Kazmierczak et al., 2009 (link)). Specifically, cDNA was synthesized from 100 ng of total RNA and random primers using the qScript Flex cDNA Kit (Quanta BioSciences). RT-PCR was performed using the Brilliant SYBR Green qPCR Master Mix (Stratagene), the Brilliant III Ultra-Fast SYBR Green qPCR Master Mix (Agilent), or the FastStart Universal SYBR Green Master Mix (Roche) and appropriate primers (see Table S6) as in (Kazmierczak et al., 2009 (link), Ramos-Montanez etal., 2008). Reactions were performed in duplicate and normalized to 16S rRNA amounts. The 16S rRNA was quantified using the same cDNA samples except that the samples were diluted 100-fold further. Data were collected on an MX3000P thermocycler (Stratagene) or on a CFX96 thermocycler (Bio Rad) and analyzed with the SYBR Green (with dissociation curve) program associated with each machine. Four dilutions of cDNA from S. pneumoniae strains wild-type for tprA and phrA (either IU1781 or Spn049) were used to generate standard curves for each primer set. Normalized transcript amounts were compared as indicated by performing pairwise unpaired two-tailed t tests.

qRT-PCR was carried out on genomic DNA and RNA samples to determine relative copy number and relative RNA expression levels of transgenes, respectively. Genomic DNA was extracted from approximately 5.0 × 10⁶ cells using GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific). RNA was extracted from a minimum of 1.0 × 10⁶ cells using TRIzol™ Reagent (Thermo Fisher Scientific), followed by DNase treatment to remove contaminating DNA (TURBO DNA-free™ DNase Treatment and Removal Reagents, Thermo Fisher Scientific). cDNAs were synthesized from 1 μg of total RNAs using Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Fisher Scientific). The qRT-PCR was performed in an Mx3005P qPCR System (Agilent Technologies) using Brilliant III Ultra-Fast SYBR^® Green QPCR Master Mix (Agilent Technologies) as described previously^{23 (link)}. Target regions were amplified using primers listed in Supporting Table S3 that were validated by melting curve analysis and agarose gel electrophoresis. Fold changes were calculated using the ∆∆CT method. Vinculin was used as normalizer for genomic DNA quantification^{23 (link)} and β-actin (ACTB) was used for RNA quantification since the expression of β-actin RNA/protein was considered constant and was not affected by temperature in CHO cells^{50 (link)}.