5.2@ RT-PCR

Yuji Hiwatashi

 

Introduction

The reverse transcription (RT) reaction is performed using either random hexamers, oligo (dT) primers, or gene-specific primers. An aliquot of the RT reaction is then transferred to another tube containing thermostable DNA polymerase, DNA polymerase buffer, and PCR primers) for PCR. To avoid contamination by genomic DNA, RNA should be treated with DNaseI.@

 

@

1. RNA preparation

Procedure

1) Mix each reagent (see below) in a microtube.

1 µl @~1.5 µg/µl total RNA

1 µl @10x reaction buffer@ *reaction buffer: 200 mM Tris-HCl (pH 8.4), 500 mM KCl

1 µl @25 mM MgCl2

1 µl @RNase-free DNase I@ (GIBCO-BRL, amplification-grade)

5.5 µl DEPC-H2O

@Total 9.5 µl

 

2) Incubate at room temperature for 15 min.

3) Add 1 µl of 20 mM EDTA to stop the reaction.

4) Incubate at 65˚C for 15 min, then on ice.

 

Solution required

E 10x reaction buffer (200 mM Tris-HCl (pH 8.4), 500 mM KCl)

E DNaseI (GIBCO-BRL, amplification-grade)

E 20 mM EDTA

E DEPC-treated H2O

 

 

2. First strand synthesis

Solution required

E Primer (oligo-dT, gene-specific primer)

E 25 mM MgCl2

E 10 mM dNTP

E 0.1 M DTT

E Superscript II (Invitrogen)

E RNase H (Invitrogen)

 


Procedure

1) Mix each reagent (see below) in a microtube.

Ca. 0.5 µg @DNase I-treated total RNA

1 µl @@@@@2.5 pmol/µl@ oligo dT primer or GSP

@ + H2O

@@@@ @<total 15 µl>

 

2) Heat at 70˚C for 10 min, then cool on ice for 1 min.

3) Add 2.5 µl of 10x reaction buffer, 3 µl of 25 mM MgCl2, 1 µl of 10 mM dNTPmix, and 2.5 µl of 0.1 M DTT in the denatured RNA solution and mix gently.

4) Incubate at 42˚C for 2 min.

5) Add 1 µl of 200 units/µl Superscript II in the mixture and mix by pipeting gently.

6) Incubate at 42˚C for 1 hr.

7) Incubate at 51˚C for 30 min.

8) Incubate 65˚C for 15 min.

9) Spin down briefly and transfer on 55˚C.

10) Add 1 µl of 2 units/µl RNaseH to the mixture and mix gently.

11) Incubate at 55˚C for 10 min and then cool on ice.

 

3. PCR

Procedure

1) Mix each reagent (see below) in a 0.2 ml PCR tube.

1.0 µl@@@ first strand cDNA

2.5 µl@@@ 10x Ex Taq buffer

2.5 µl@@@ 2.5 mM dNTPmix

1 µl@@@@ 10 pmol/µl GSP primer

1 µl@@@@ 10 pmol/µl GSP primer

0.125 µl@ 5 u/µl ExTaq

16.875 µl@ dH2O

 

2) Perform the following PCR cycle.

94˚C, 2 min

94˚C, 30 sec@„Ş„Ş„­

(tm-5)˚C, 30 sec@@ x 30

72˚C, min/kb@„Ş„Ş„Ž

72˚C, 5 min

4˚C

 

3) Prepare the appropriate agarose gel while the PCR is cycling.

4) After cycling is complete, add loading dye to samples, load sample on gel, and run to desired distance.

5) Stain the gel with EtBr.

 


Solution required

1) cDNA

2) 10X Ex Taq buffer

3) 2.5 mM dNTP mixture

4) 10 pmol/µl Gene specific primers

5) 5U/µl Ex Taq

6) Nuclease-free water

 

Key points

1) Successful RT-PCR requires a high quality, intact RNA template. @@@

E To minimize the activity of RNases that are released during cell lysis, include RNase inhibitors in the lysis mix or use methods that simultaneously disrupt cells and inactivate RNases.

E Take steps to eliminate all potential sources of RNase contamination from glassware, plasticware, reagents, etc.

2) For avoiding DNA contamination in RT-PCR

E Make a control tube without reverse transcriptase.@ Presence of a product by PCR in the absence of RT enzyme indicates DNA contamination.@

E Use intron-spanning primers. @Make each forward and reverse primer located on two adjacent exons.@ If @contaminating DNA is present, you get a larger band than expected due to the presence of the intron.

E Design primers to anneal at a splice junction. @Such primers should not anneal properly to genomic DNA.

3) Nested PCR

E A second PCR with nested primers increases specificity and sensitivity of the PCR reaction.

4) Semi-quantitative PCR

E Scale up the reaction volume and run PCR.@ Recover the reaction solution at several successive points during the PCR cycling.@ The number of cycles should be determined empirically to find the linear range of amplification.@ If you are not good at recovering the reaction solution in a very short time, it is better to prepare many tubes.

5) RT-PCR Southern

E To confirm the specific amplification of the target gene, perform DNA-gel blot analysis against RT-PCR products with the target gene as a probe.