High capacity cdna reverse transcriptase kit

LNCaP cells were treated for 24 h with solvent control or SAL. AR (Abcam, ab19066) or IgG (goat IgG, Abcam, ab6885) antibodies were added to protein G Dynabeads (Invitrogen) and incubated at 4 °C for 1 h. Antibody-bound beads were incubated with cytosolic and nuclei extracts for 2 h at 4 °C. After three washing steps on magnetic rack, RNA extraction was performed using peqGOLD TriFast.Glycogen (Invitrogen). cDNA was obtained by High Capacity cDNA reverse transcriptase kit (Invitrogen).

Total RNA was isolated from cultured podocytes using TRIzol reagent (Invitrogen). Two micrograms of RNA was reverse transcribed using a High Capacity cDNA Reverse Transcriptase Kit (Invitrogen) according to the manufacturer’s protocol. The following primers were used for quantitative real-time PCR: GAPDH cDNA, 5′-CGGAGTCAACGGATTTGGTCGTAT-3′ (sense) and 5′-AGCCTTCTCCATGGTGGTGAAGAC-3’ (antisense); and WAVE1 cDNA, 5′-GGGAACGAGCCTTATTCCGT-3′ (sense) and 5′-AGTGGGAAGGGGTTCTGCTA-3′ (antisense). Quantitative real-time PCR was performed using a SYBR Green PCR Master Mix Kit (Invitrogen). The cycling conditions included denaturation at 95 °C for 10 min followed by 35 cycles of annealing at 95 °C for 15 s and extension at 64 °C for 1 min. The relative mRNA expression was normalized to GAPDH expression and calculated using the delta-delta method from the threshold cycle numbers. Based on the exponential amplification of both the target gene and the calibrator, the relative expression of a target gene at a particular threshold cycle is represented by 2^−∆∆Ct.

Total RNA was isolated using RNeasy Mini Kit (QIAGEN, Valencia, CA). cDNA was synthesized from total RNA (1 μg) with the High Capacity cDNA Reverse Transcriptase Kit (Invitrogen) which uses the random primer scheme. The primers used for FcRn are as follows: sense 5′-TGA CCT GTG CTG CTT TCT CCT-3′, antisense 5′-CAG CAA TGA CCA TGC GTG GAA-3′. Real-time PCR was performed with the use of the AppIied Biosystems Step One Plus Real-Time PCR System (Life Technologies, Carlsbad, CA). The expression of a target gene in relation to a reference gene was calculated using a comparative cycle threshold (Ct) method.

RNA was extracted from the lungs, kidneys, and reproductive tract tissues using Trizol reagent (Invitrogen Canada Inc., Burlington, ON, Canada) following the manufacturer’s instruction. Briefly, the tissues (50 mg) were homogenized using a microtube homogenizer (Ambion, Invitrogen Canada Inc, Burlington, ON, Canada) in 1 mL of Trizol reagent. Then, 200 μL of chloroform was added. The tubes were spun at 12,000× g for 15 min at 4 °C. They were then incubated for 10 min at room temperature following the transfer of the upper phase of the solution containing RNA into a separate tube and adding isopropanol (500 μL). The tubes were spun again at 12,000× g for 11 min (4 °C) and the supernatant was carefully discarded. Next, 1 mL of 75% ethanol was added and the tubes were spun at 7500× g for 5 min. After the supplement was discarded, the pellet was air-dried, suspended in 20 μL of RNA free water, and incubated for 10 min at 56 °C. The quantity of RNA was measured using a Nanodrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The extracted RNA was used to synthesize cDNA using RT random primers (high capacity cDNA reverse transcriptase kit, Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s guidelines.

The tubes containing OP and CL swabs were vortexed and 250 μL of the medium was used to extract the RNA using Trizol LS^® reagent (Ambion, Invitrogen Canada Inc., Burlington, ON, Canada) according to manufacturer’s guidelines. Total RNA was also extracted from the trachea, lung, kidney, reproductive tract (uterus, isthmus and magnum), cecal tonsils, and proventriculus using Trizol reagent (Ambion, Invitrogen Canada Inc., Burlington, ON, Canada) following the manufacturer’s protocol. The amount of the extracted RNA was quantified using Nanodrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) at 260 nm wavelength. The quality of the RNA was determined based on the 260/280 absorbance ratio. Approximately 1000 ng from swab samples and 2000 ng of tissue samples were used to synthesize cDNA using RT random primers (High capacity cDNA reverse transcriptase kit, Invitrogen Life Technologies, Carlsbad, CA, USA) according to manufacturer’s guidelines.