Fam dye labeled taqman mgb probe

Total RNA was isolated from PANC-1 or Capan-1 cells untreated or treated with TSA. RNA was extracted with TRIzol (Life Technologies) according to the manufacturer’s instructions. One microgram of total RNA was DNase treated (Promega, Madison, Wis.) and reverse transcribed into cDNA using a commercial kit (Qiagen, Hilden, Germany). FAM dye-labeled Taqman MGB probes and unlabeled PCR primers for human NDRG1, VEGF, LOX (lysyl oxidase), GLUT1 (glucose transporter 1), and CAIX (carbonic anhydrase 9) were purchased from Applied Biosystems (Warrington, UK). Internal ribosomal RNA 18S was used as an internal positive control with a VIC dye-labeled Taqman MGB probe (Applied Biosystems). Real-time PCR was performed using the ABI PRISM 7000 Sequence Detector System (Applied Biosystems). cDNA was amplified for 40 cycles with denaturation at 95°C for 15 seconds and a combined annealing-extension step at 60°C for 1 minute. Mean cycle threshold (C_t) values were calculated for 18S and the reporter gene. C_t values for NDRG1, VEGF, LOX, GLUT1, and CAIX were normalized against the 18S control probe to calculate ∆C_t values. ∆∆C_t values were calculated by subtracting the ∆C_t value of control cells under normoxia from the ∆C_t values of the treated (differentiated) cells under normoxia or control or treated cells under hypoxia. Fold increases were calculated using the formula 2^-(∆∆Ct).

Quantitative polymerase chain reaction (qPCR) was performed using an ABI StepOne Plus Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). FAM dye-labeled TaqMan MGB probes as well as PCR primers for human PTEN (Hs00171132_m1) and β-actin (Hs01060665_g1) were purchased from Thermo Fisher Scientific.

The RNA isolation kit was purchased from Ambion, Inc. The methods of RNA extraction have been described previously.¹⁹ (link) Briefly, ten 10-μm sections were cut from the fresh frozen tissue blocks of the residual sample from the kidney biopsy using a microtome, pooled in a 1.5-mL microcentrifuge tube, and homogenized and dissolved in digestion buffer.²⁰ (link) Urine samples were centrifuged at 3,000g for 30 minutes and 13,000g for 5 minutes at 4 °C. Specimens were then stored at −80 °C until use.
Tissue and urinary miR-21 levels were quantified using real-time quantitative polymerase chain reaction (RT-QPCR) using the ABI Prism 7900 Sequence Detection System (Applied Biosystems). Commercially available Taqman primers and probes, including 2 unlabeled PCR primers and 1 FAM dye-labeled TaqMan MGB probe, were used (all from Applied Biosystems). RNU48 (Applied Biosystems) was used as housekeeping genes.²¹ (link)^,22 (link) All RT-QPCR experiments were performed in triplicate. Results were analyzed using Sequence Detection Software, version 2.0 (Applied Biosystems). The ΔΔCT method for relative quantitation was used. Urinary miR-21 level represents the total amount excreted in an 8-hour overnight urine specimen, as compared with that of the housekeeping gene.