Reverse Transcriptase PCR

Qiagen ®

1) Thaw template RNA, primer solutions, dNTP Mix, 5x QIAGEN OneStep RT-PCR Buffer, and RNase-free water, and place them on ice. It is important to mix the solutions completely before use to avoid localized differences in salt concentration.

2) Prepare a master mix according to the table. The master mix typically contains all the components required for RT-PCR except the template RNA. Prepare a volume of master mix 10% greater than that required for the total number of reactions to be performed. A negative control (without template RNA) should be included in every experiment.

Componentx   		Volume/reactionx		Final concentration   
---------------------------------------------------------------------------
    Master mix
RNase-free water 		Variable		-
(provided)           		    		       	 
5x QIAGEN OneStep 		10.0 μl			1x
RT-PCR Buffer       		         		 
dNTP Mix 			2.0 μl			400μl of each dNTP
(10 mM of each dNTP)    	        		 
Primer A                    	Variable    		0.6 μM†
Primer B                    	Variable    		0.6 μM†
QIAGEN OneStep 			2.0 μl			-
RT-PCR Enzyme Mix    		        		 
RNase inhibitor 		Variable		5–10 units/reaction
(optional)‡
---------------------------------------------------------------------------            		    		 
    Template RNA
Template RNA,			Variable 		1 pg–2 μg/reaction
added at step 4
---------------------------------------------------------------------------       		    		 
Total volume                    50.0 μl        		–

3) Mix the master mix thoroughly, and dispense appropriate volumes into PCR tubes. Mix gently, for example, by pipetting the master mix up and down a few times.

4) Add template RNA ( 2 µg/reaction) to the individual PCR tubes.
The QIAGEN OneStep RT-PCR Kit can be used with total RNA, messenger RNA, or viral RNA.

5) When using a thermal cycler with a heated lid, do not use mineral oil. Proceed directly to step 6. Otherwise, overlay with approximately 50 µl mineral oil.

6) Program the thermal cycler according to the program and start the RT-PCR program while PCR tubes are still on ice. Wait until the thermal cycler has reached 50°C. Then place the PCR tubes in the thermal cycler.

PCR program:

50°C for 30 minutes
 
95°C for 15minutes
25-40 cycles:
94°C for 30-60seconds  
50-68°C for 30-60seconds 
72°C for 1 minute
72°C for 10 minute 

4°C on hold

7) After amplification, samples can be stored overnight at 2–8°C, or at –20°C for longer-term storage.