Quantitect sybr green rt pcr kit

The primers for real time reverse transcription-
polymerase chain reaction (RT-PCR) were newly
designed using GenBank (http://www.ncbi.nlm.nih.gov)
and synthesized at CinnaGen Company (Iran) (Table 1).
The housekeeping gene (ß-actin) was used as an internal
control. After cDNA synthesis, we performed real time
RT-PCR with an Applied Biosystems real-time thermal
cycler according to the QuantiTect SYBR Green RTPCR
kit (Applied Biosystems, UK). For each sample, the
reference gene and the target genes (αV and ß3
integrins,
IL-1R, and LIFR) were amplified in the same run and
melting curve analysis was used to confirm the amplified
product. The real-time thermal condition included a
holding step: 95°C 10 minutes and cycling step: 95°C
15 seconds, 60°C 1 minute was continued by a melting
curve step: 95°C 15 seconds, 60°C 1 minutes and 95°C 15
seconds . The relative quantification of target genes was
determined using the Pfaffl method (31 (link)). All experiments
were repeated three times.

The primers for real-time reverse transcriptasepolymerase chain reaction (RT-PCR) were formulated
(Table 1) utilizing GenBank (http://www.ncbi.nlm.nih.
gov) and Primer3 software, then synthesized by Generary
Biotech Company (China).
RT-PCR was carried out by the Applied Biosystems
(UK) real-time thermal cycler as indicated by QuantiTect
SYBR Green RT-PCR Kit (Applied Biosystems, UK). The
housekeeping gene, β-ACTIN, was considered as internal
control. For each specimen, the house keeping gene and
the target genes were amplified in the same round. One
microliter of cDNA, 1 μl of the mixture of forward and
reverse primers and 10 μl SYBR Green Master Mix were
used per 20 μl of the reaction volume. After each PCR
run, melt curve was analyzed to determine amplification
specificity. Real-time heating condition included holding
step at 95˚C for 5 minutes, cycling steps (35-40 cycles) at
95˚C for 15 seconds, 58˚C for 30 seconds and 72˚C for 15
seconds which was continued by a melt curve analysis at
95˚C for 15 seconds, 60˚C for 1 minutes and 95˚C for 15
seconds. Then the relative quantification of target genes
was calculated by the Pfaffl formula (27 (link)). The real-time
RT-PCR experiments were performed duplicate for each
specimen in at least a three biological repeats.

RPE1 cells were transfected with siRNAs targeting SMC6 or non-targeting controls using Lullaby transfection reagent. 56 h later, RNA was isolated from RPE1 cells using the RNeasy^®-Kit (Qiagen). Reverse transcription and Real-time PCR was performed using QuantiTect SYBR Green RT-PCR Kit and the Applied Biosystems 7500 Fast System Real-time cycler. Primers are described in Supplementary Table S1. We analysed data using the 2^−ΔΔCT-method with GAPDH as internal standard. Gene expression was normalized to gene expression in the control siRNA-transfected samples. Mean and S.D. of three experiments were determined.

After extraction of total RNA and cDNA synthesis, according to QuantiTect SYBR Green RT-PCR kit (Applied Biosystems, UK), real-time RT-PCR was performed. Thermal conditions for the process included 3 steps; holding stage (95ºC for 5 min), step 2 was performed at 95ºC for 15 s, 58ºC for 30 s, and 72ºC for 30 s, and the last step (melt curve step) was continued at 95ºC for 15 s, 60ºC for 1 min, and 95ºC for 15 s. Then, the relative quantification of target genes to housekeeping genes was determined.

After the designated treatments with EPZ015666 or transfected with siRNA for 48 hrs and then treated with TNF‐α or IL‐1β for 12 hrs, total RNAs were extracted using TRIzol (Sigma‐Aldrich) and were reverse‐transcribed to cDNA using miScript Reverse Transcription Kit (TaKaRa Biomedical Technology, Kusatsu, Japan ). The mRNA expression of PRMT5, IL‐6 and IL‐8 was analysed by real‐time quantitative polymerase chain reaction (qPCR) which was performed on cDNA using QuantiTect SYBR Green RT‐PCR Kit on StepOnePlusTM Instantaneous analyse PCR System (Applied Biosystems, Foster City, CA, USA). The expression of relative mRNA was more normalized to the GAPDH. The used RT‐PCR primers were listed in Table S1.

Total RNA was extracted using TRIzol (Sigma) and reverse transcribed to cDNA using a miScript Reverse Transcription Kit. qRT-PCR analysis for the expression of Brd2, Brd3, Brd4, and Brdt was performed on cDNA using a QuantiTect SYBR Green RT-PCR Kit on a StepOnePlus TM Real-Time PCR System (Applied Biosystems). Relative mRNA expression was normalized to the expression of GAPDH. The RT-PCR primers used are listed in supplementary Table 2 (STable 2).