One-Step RT-PCR Protocol

Acc.no: MF6M41 | Published: 2008-12-04 by Unknown user

Keywords: one-step, RT-PCR, reverse transcription, amplification, RNA

Synthesize cDNA from RNA, then amplify the cDNA using the polymerase chain reaction.

Coupled reverse transcription and polymerase chain reaction (RT-PCR) allows exponential amplification of an RNA template in vitro. The RNA is first converted to cDNA using a reverse transcriptase, then the cDNA is amplified by PCR. In one-step RT-PCR, all reaction components are added at the same time, then the reaction is incubated at the appropriate temperature for reverse transcription, followed immediately by thermal cycling for PCR.

Required Materials

template DNA
downstream PCR primer
upstream PCR primer
AMV Reverse Transcriptase, GPR , supplied with the Access RT-PCR System
Tfl DNA Polymerase , supplied with the Access RT-PCR System
AMV/Tfl 5X Reaction Buffer , supplied with the Access RT-PCR System
Magnesium sulfate , 25 mM, supplied with the Access RT-PCR System
PCR Nucleotide Mix, 10mM , supplied with the Access RT-PCR System
Positive Control RNA with Carrier , supplied with the Access RT-PCR System
Upstream Control Primer , supplied with the Access RT-PCR System
Downstream Control Primer , supplied with the Access RT-PCR System
RNase free water , supplied with the Access RT-PCR System
dNTP kit (10 mM)
nuclease-free light mineral oil , if using a thermal cycler without a heated lid

Main steps

Prepare the reaction mix by combining the indicated volumes of Nuclease-Free…
Overlay the reaction with one or two drops (20-40?l) of nuclease-free mineral…
Place the tubes in a thermal cycler equilibrated at 45°C, and incubate…
Heat the reverse transcription reactions at 94°C for 2 minutes to inactivate…
Proceed directly to thermal cycling the reactions for second-strand cDNA…
These conditions work well to detect the 323bp PCR product generated from…

Instructions

Prepare the reaction mix by combining the indicated volumes of Nuclease-Free Water, AMV/Tfl 5X Reaction Buffer, dNTP Mix, 25mM MgSO₄ and specific upstream and downstream primers in a thin-walled 0.5ml reaction tube on ice. Mix the components by pipetting. Add the AMV Reverse Transcriptase and Tfl DNA Polymerase to the reaction. Gently vortex the tube for 10 seconds to mix the components.

Component	Volume	Final Concentration
Nuclease-Free Water (to a final volume of 50?l)	X?l
AMV/Tfl 5X Reaction Buffer	10?l	1X
dNTP Mix, 10mM each dNTP	1?l	0.2mM each
AMV Reverse Transcriptase (5unit/?l)	1?l	0.1unit/?l
25mM MgSO4	2?l	1mM
Tfl DNA Polymerase (5unit/?l)	1?l	0.1unit/?l
Downstream PCR primer	50pmol	1?M
Upstream PCR primer	50pmol	1?M
Template RNA*	Y?l

*We recommend 10³ -10⁶ copies of a specific target template or 1pg-1?g total RNA. For the positive control reaction, use 2?l of the Positive Control RNA with Carrier (2.5 attomoles or 1 × 106 copies) and 50pmol each of the Upstream and Downstream Control Primers.

Overlay the reaction with one or two drops (20-40?l) of nuclease-free mineral oil to prevent condensation and evaporation. Mineral oil addition is not necessary if you are using a thermal cycler with a heated lid.
Place the tubes in a thermal cycler equilibrated at 45°C, and incubate for 45 minutes.
Heat the reverse transcription reactions at 94°C for 2 minutes to inactivate the AMV reverse transcriptase and denature the RNA/cDNA hybrid.
Proceed directly to thermal cycling the reactions for second-strand cDNA synthesis and amplification (Table 1).

Table 1 Second-Strand cDNA Synthesis and PCR
1. 40 cycles: 94°C for 30 seconds denaturation; 60°C for 1 minute annealing; 68°C for 2 minutes extension
2. 1 cycle 68°C for 7 minutes final extension
3. 1 cycle 4°C soak
These conditions work well to detect the 323bp PCR product generated from the Positive Control RNA using the Upstream and Downstream Control Primers provided with the Access RT-PCR System. We recommend optimizing RT-PCR parameters for each target RNA. Information about RT-PCR optimization can be found at: www.promega.com/paguide/chap1.htm. Additional information about the Access RT-PCR System can be found in the Access RT-PCR System Technical Bulletin at: http://www.promega.com/tbs/tb220/tb220.html