The protocol of real-time reverse transcription-PCR (RT-PCR) analysis was slightly modified from that performed by Porro and colleagues (27 (link)). Total RNA of blood samples were isolated with the Trizol reagent, respectively (Invitrogen, Carlsbad, CA), and reverse transcribed with β-actin and telomere-specific oligonucleotides using HiScript SuperMix for qPCR kit (Vazyme Biotech Co., Ltd, Nanjing, China). cDNA was amplified using the SYBR green master mix (Vazyme Biotech Co., Ltd, Nanjing, China) with the primers listed in Supplementary Table 4 and analyzed using the Rotor gene Q 2000 (QIAGEN, Hilden, Germany) with the following thermal cycling profile: Stage 1: 2 minutes at 95°C; Stage 2: 40 cycles of 15 seconds at 98°C, 20 seconds at 60°C, 1 minute at 74°C with signal acquisition.
Rotor gene q 2000
Manufactured by Qiagen
Sourced in Germany
The Rotor-Gene Q 2000 is a real-time PCR thermal cycler designed for nucleic acid amplification and detection. It features a compact design and a 72-well rotor format for high-throughput sample processing. The instrument provides precise temperature control and robust data acquisition capabilities for sensitive and reliable real-time PCR analysis.
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2 protocols using rotor gene q 2000
1
Real-Time RT-PCR Analysis of Blood RNA
2
Quantifying mRNA Expression of DNA Repair Genes
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The mRNA expression of BRCA1, RAD51B, and RAD51C were examined by qPCR. The total RNA was reverse-transcribed into cDNA with a reverse transcription system (Promega, Madison, WI, USA) consistent with the manufacturer’s protocol. The objective sequences of genes to be observed were amplified with SYBR green master mix (QIAGEN, Germany) using the Rotor gene Q 2000 (QIAGEN, Germany). The primers were listed in Table S1 . The level of GAPDH served as control for the data analysis.
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