Protocol for Real-Time RT-PCR

This protocol describes the detailed experimental procedure for real-time RT-PCR using SYBR Green I as mentioned in Xiaowei Wang and Brian Seed (2003) A PCR primer bank for quantitative gene expression analysis. Nucleic Acids Research 31(24): e154; pp.1-8. Please refer to this paper and the PrimerBank Help page for more background information. This protocol is also available in Microsoft Word format here. Here is a one-page succinct protocol. The procedure begins with reverse transcription of total RNA. The cDNA is then used as template for real-time PCR with gene specific primers. You may need to modify this protocol if you use different reagents or instruments for real-time PCR.

ime required

cDNA synthesis: 2 hours. 　　real-time PCR: 2 hours. 　　Dissociation curve analysis: 0.5 hour.

Reagents and Equipments

Oligonucleotide Primers. Gene specific primers are retrieved from PrimerBank .These primers are ordered from the MGH DNA Core facility . All the primers are desalted and both UV absorbance and capillary electrophoresis are used to assess the quality of primer synthesis. 　　 Mouse total liver RNA (Stratagene). 　　Mouse total RNA master panel (BD Biosciences / Clontech). 　　 SYBR Green PCR master mix, 200 reactions (Applied Biosystems). 　　 Optical tube and cap strips (Applied Biosystems). 　　 SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). 　　 25 bp DNA ladder (Invitrogen). 　　 ABI Prism 7000 Sequence Detection System (Applied Biosystems). 　　ABI Prism 7000 SDS software (Applied Biosystems). 　　 3% ReadyAgarose Precast Gel (Bio-Rad). 　　Agarose gel electrophoresis apparatus (Bio-Rad).

Detailed procedure

Reverse Transcription

Reverse Transcription is carried out with the SuperScript First-Strand Synthesis System for RT-PCR. The following procedure is based on Invitrogen’s protocol.

1. Prepare the following RNA/primer mixture in each tube: 　　Total RNA 5 mg 　　random hexamers (50 ng/ml) 3 ml 　　10 mM dNTP mix 1 ml 　　DEPC H2O to 10 ml

2. Incubate the samples at 65°C for 5 min and then on ice for at least 1 min.

3. Prepare reaction master mixture. For each reaction: 　　10x RT buffer 2 ml 　　25 mM MgCl2 4 ml 　　0.1 M DTT 2 ml 　　RNAaseOUT 1 ml

4. Add the reaction mixture to the RNA/primer mixture, mix briefly, and then place at room temperature for 2 min.

5. Add 1 ml (50 units) of SuperScript II RT to each tube, mix and incubate at 25°C for 10 min.

6. Incubate the tubes at 42°C for 50 min, heat inactivate at 70°C for 15 min, and then chill on ice.

7. Add 1 ml RNase H and incubate at 37°C for 20 min.

8. Store the 1st strand cDNA at -20°C until use for real-time PCR.