Rt2 preamp cdna synthesis kit

RNA extraction was performed using the Qiagen RNeasy FFPE kit and RNA was converted to cDNA and pre-amplified for qPCR using the Qiagen RT² PreAMP cDNA synthesis kit. In the initial qPCR experiment, because of the smaller than expected amount of RNA collected, RNA samples could not be quantified to ensure there was equal RNA input amounts in the reverse transcription reaction for each group, thus qPCR data were normalized only to the geometric mean of the reference genes PPIA and RPL10A (Becker et al., 2010 (link)). These genes showed a close pattern of expression changes across the groups. In the follow-up LCM study, in which a greater number of RGCs was captured, the concentration and integrity of RNA could be quantified using the Agilent RNA 6000 Pico Kit run on an Agilent 2100 Bioanalyzer prior to housekeeper normalization in qPCR; 1 ng total RNA from each sample was reverse transcribed. Although there was still RNA degradation (RNA Integrity Numbers < 8.0) from fixation and the unavoidable and extensive tissue processing prior to laser capture, the amount of RNA was sufficient to perform cDNA synthesis and qPCR. In both the pilot and second follow-up experiment, cDNA was pre-amplified using the Qiagen RT² PreAMP cDNA synthesis kit for eight cycles.

Neutrophils from lungs or prostate lobes were isolated from sham male and castrated males and were pooled with 5–6 mice per group. RNA was extracted using an miRNA extraction kit (miRNeasy, Qiagen, 217004). Synthesis of cDNA was performed using the RT² First Strand Kit (Qiagen, 330401) and cDNA was amplified using the RT² PreAMP cDNA synthesis kit (Qiagen, 330451) and AR pathway primer mix (Qiagen, PBM-142Z) for neutrophils. The RT² Profiler PCR Array Mouse Androgen Receptor Signaling Targets (Qiagen, 330231, PAMM-142ZD-2) plates were run on a CFX96 Touch Real-Time PCR Detection System (BioRad). All steps were performed according to the manufacturer’s protocol.

Venous blood samples were drawn directly into Tempus™ Blood RNA Tube (Life Technologies, Austin, USA), and immediately vortex for 10 s. Then stabilized blood was transferred to 50-mL sterile conical tubes, and total RNA was isolated, purified, and stored by Tempus™ Spin RNA Isolation Kit (Life Technologies, Austin, USA) following the user guide. Extracted RNA was converted to cDNA by using the RT2 PreAMP cDNA Synthesis Kit (Qiagen). cDNA was synthesized from total RNA using HiScript II 1st Strand cDNA synthesis kit (Vazyme, China). The primers for qRT-PCR were: 5′-CGTGGAGAAGAATTGGGC-3′ (Forward) and 5′-CGTGGAGAAGAATTGGGC-3′ (Reverse) for FGD5-AS1; 5'-AACGGATTTGGTCGTATTG-3' (Forward) and 5'-GGAAGATGGTGATGGGATT-3' (Reverse) for GAPDH. qRT-PCR was performed with FastStart Universal SYBR Green Master (Rox) (Roche, Indianapolis, USA) at a 7500 Real-Time PCR System (Applied Biosystems, Waltham, USA). Data were analyzed by the 2^−ΔΔCt method normalized relative to the amount of GAPDH.