Quantitect sybr green rt pcr kit

Total RNA from RAP treated HCEC's was extracted using the RNeasy Mini Kit (Qiagen). The following PCR primers were used: AXIN2, forward, 5′-TTCCCGAGAACCCACCGCCT-3′, reverse, 5′-GCCCCCTCCCGCGAATTGAG-3′; NKD1, forward 5′- CATGCACGACATGAAAATCC-3′, reverse 5′-TTGCCTGGATCTTGGAAAAC-3′; IRS1, forward 5′-CAAGACCATCAGCTTCGTGA-3′, reverse, 5′-AGAGTCATCCACCTGCATCC-3′. Real-time PCR was performed using the QuantiTect SYBR Green RT-PCR kit (Qiagen).
Reverse transcription of mRNA and real-time PCR were performed using the one step QuantiTect SYBR Green RT-PCR kit (Qiagen) according to the manufacturer's protocol. Relative expression of AXIN2, NKD1 and IRS1 were assessed and normalized with housekeeping gene U6.

Cells grown in three wells of a 24-well plate were pooled for RNA isolation with Trizol according to manufacturer’s protocol. One-step quantitative RT-PCR for ZIKV and the ribosomal housekeeping gene S7 RNA was performed using specific primers (Supplementary Table S2), SYBR Green master mix (QuantiTect SYBR Green RT-PCR Kit, Qiagen, Germany) and a LightCycler 480 (Roche, Basel, Switzerland) according to manufacturer’s instructions. Results were analyzed using the ∆∆C_T method normalized to S7 housekeeping gene.
One-step quantitative RT-PCR for SFV was used for relative quantification with efficiency correction using the LightCycler 480 software (version 1.5.1.62; Roche, Basel, Switzerland), specific primers (Supplementary Table S2), SYBR Green master mix (QuantiTect SYBR Green RT-PCR Kit, Qiagen, Germany) and a LightCycler 480 (Roche, Basel, Switzerland) according to manufacturer’s instructions. Standard curves were created using serial dilutions of purified PCR products (SFV and S7 as reference).

Validation of the microarray data was performed using a real-time RT-PCR assay for EP300 and cyclin-dependent kinase inhibitor 1A (CDKN1A) mRNAs using real-time RT-PCR. The primer sequences for EP300 forward and reverse were 5′-CTC CAA CTT TCT GCC ACA ACA-3′ and 5′-CCA GAA TCC CTT CCC TTT CG-3′, respectively. For CDKN1A, the primer sequences forward and reverse were 5′-GGA CCT GGA GAC TCT CA-3 and 5′-CCT CTT GGA GAA GAT CAG-3, respectively. The transcript level of each gene was determined using the QuantiTectTM SYBR Green RT-PCR Kit (Qiagen) and ABIprism 7000 SDS. GAPDH mRNA was also used as an internal control to normalize the mRNA expression levels.

Total RNA was extracted using an Isogen-LS RNA extraction kit (Nippon Gene, Tokyo, Japan). Genomic DNA was removed from RNA preparations using the DNase I recombinant RNase-free (Roche, Germany) prior to RT-PCR. After quantification of RNAs using the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies), RNA samples were stored at −20°C, until needed for no more than 2 days. Single-strand cDNAs were synthesized from mRNAs and quantified using the QuantiTectTM SYBR Green RT-PCR Kit (Qiagen) and ABI Prism 7000 sequence detection system (Applied Biosystems). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an internal control to normalize the cDNA expression levels. GAPDH primer sequences for forward and reverse were 5′-TGA TGA CAT CAA GAA GGT GGT GAA G-3′ and 5′-TCC TTG GAG GCC ATG TGG GCC AT-3′, respectively. For each target gene, 1 µl of cDNA was amplified in a total volume of 20 µl containing 2 × QuantiTect SYBR Green RT-PCR master mixes supplemented with 300 nM of each primer. The data were analysed using ABIprism 7000 SDS software (Applied Biosystems). For all the samples, crossing points (CP) normalized expression of the target gene versus GAPDH for each treatment or control sample were calculated [26 ] using the following formula: 2^{−(ΔCP treatment sample–ΔCP control sample)}. Each sample was assayed three times.

Inflammatory cytokines and cytokine receptor genes were analyzed with primers specific for rat genes, with a QuantiTect SYBR Green RT-PCR kit (both Qiagen, Valencia, CA) on a DNA Engine Optican 2 System (MJ Research, Waltham, MA). An mRNA pool for each group was tested three times. Relative levels of mRNA were calculated and normalized to the level of GAPDH and acidic ribosomal protein mRNA.

Whole-genome DNA was extracted using Tiangen magnetic universal genomic DNA kit (catalog no. DP705). To determine the copy number of the tmexCD1-toprJ1 gene cluster, qPCR was conducted using NEB Luna universal qPCR master mix (catalog no. M3003S). The primers are listed in Table S4 in the supplemental material. To determine the expression level of tmexCD1-toprJ1 in isolates continually isolated from the same patients, whole-genome RNA was extracted using Tiangen RNAprep pure cell/bacteria kit (catalog no. DP430), and RT-qPCR was also conducted using Qiagen QuantiTect SYBR green RT-PCR kit (catalog no. 204243). The PCR amplification consisted of the initial denaturation for 3 min at 95°C, followed by 35 cycles of denaturation for 30 s at 95°C, annealing for 30 s at 54°C, and extension for 30 s at 72°C, and then the final extension for 5 min at 72°C. We used the 16S rRNA gene as an internal control in PCR analysis.