Taqman universal pcr master

The 661W photoreceptor cell line was generously provided by Dr Muayyad
Al-Ubaidi (Department of Cell Biology, University of Oklahoma Health
Sciences Center, Oklahoma City, OK, USA). These cells were cultured in
Dubelcco's modified Eagle's medium (Invitrogen) supplemented with
10% heat-inactivated fetal bovine serum and 1%
penicillin/streptomycin (Invitrogen), at 37 °C in a
humidified atmosphere with 5% CO₂. Cells (10⁶)
were seeded in tissue culture 100 mm Petri dish and were allowed to
attach for 15 h. The cells were then washed with PBS twice (pH 7.4)
followed by the addition of 10 ml serum-free medium. The control
group contained only media, whereas the experimental group was treated with
Tn (10 μg/ml) for 1 or 8 h. After incubation
at different time points cells were harvested, washed with PBS and collected
for RNA isolation. cDNA preparation was described previously. qRT-PCR was
performed using 66 ng of cDNA, TaqMan Universal PCR master (Applied
Biosystems) and the StepOnePlus Real Time PCR system (N=4).
We detected Il-1R, Il-1β and Il-6 genes including
Gapdh as a control. The fold change was calculated by dividing
the mean of the RQs for the control by the mean RQ of the Tn treatment at
each time point. The result were analyzed by one-way ANOVA using GraphPad
Prism software.

Expression of miR-132 was measure using a TaqMan microRNA assay, a real-time PCR. Total RNA was extracted as per the instruction of the mirVana™ isolation kit (Invitrogen, USA). Reverse transcription was performed with miRNA specific stem loop RT primers and the TaqMan microRNA reverse transcription kit (Applied Biosystems, USA). The miR-132 expression was detected using the TaqMan Universal PCR Master (Applied Biosystems, USA). Fold changes of target miRNA were calculated relative to the expressions in those of untreated cells, and U6 was used as the endogenous control.

MiRNA was extracted from human NSCLC tissues and cells using mirVana miRNA Isolation Kit (Ambion, Carlsbad, CA, USA). The TaqMan microRNA assay and TaqMan universal PCR master (Applied Biosystems, Carlsbad, CA, USA) mix were used to examine the expression of miRNAs, and normalized by U6 small nuclear RNA. Profiling of miRNA was performed using miScript miRNA PCR Array (obtained from Qiagen in Valencia, CA), and the data was analyzed online (http://pcrdataanalysis.sabiosciences.com/mirna/arrayanalysis.php). Total RNA was extracted using TRIzol reagent (Invitrogen). First-strand cDNA was synthesized using Maxima First Strand cDNA Synthesis Kit (Thermo Scientific, Rockford, IL, USA). The gene expression was validated by real-time PCR using GoTaq qPCR Master Mix with SYBR green (Promega) with GAPDH as an internal control. The sequences of the sense (S) and antisense (AS) primers used in SYBR green quantitative PCR were as follows: MYO10-S: 5′-AAGTGGGGCAGGTAAAACCG-3′, AS: 5′-GCTCGTTCAACACAGGATGTC-3′; GAPDH-S: 5′-A ACAGCGACACCCACTCCTC-3′ and AS: 5′-CATACC AGGAAATGAGCTTGACAA-3′.