5.2@ RT-PCR
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
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.
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
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)
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
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@ŞŞ
(tm-5)˚C,
30 sec@@ x 30
72˚C, min/kb@ŞŞŽ
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.
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
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.