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Real Time Quantitative PCR (for Suspension Cells)
Published: Feb 5, 2011 DOI: 10.21769/BioProtoc.27 Views: 12435
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Abstract
Real-time quantitative PCR (qPCR) is an efficient method to detect the levels of gene expression. This protocol provides a complete and detailed procedure for qPCR, including mRNA extraction, genomic DNA removal, cDNA synthesis and targeted gene amplification steps.
Materials and Reagents
- Suspension cells (such as K562 cells)
- Chloroform
- Isopropyl alcohol
- 75% DEPC-ETOH
- RNase-free H2O
- E.coli RNase H
- Trizol (Life Technologies, Invitrogen™)
- Turbo DNA-free kit (Life Technologies, Ambion®)
- Superscript III first-strand synthesis supermix (Life Technologies, Invitrogen™)
- SYBRgreen qPCR Supermix universal (Life Technologies, Invitrogen™)
Equipment
- CFX96TM or CFX384TM real-time PCR detection system (Bio-Rad)
- NanoDrop
Procedure
Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
Category
Molecular Biology > RNA > qRT-PCR
Molecular Biology > RNA > Transcription
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