5.2@ RT-PCR
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 of 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 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 in the mixture and mix gently.
11) Incubate at
55˚C for 10 min and then 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 Genes specific primers
5) 5U/µl
Ex Taq
6) Nuclease-free
water
1) Successful
RT-PCR requires a high quality, intact RNA template.@@@@@ Ľ @@@@
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
locating on two adjacent exons.@ If DNA
is contaminated, you can get larger band with expected size and intron
size.@
E Design primers to anneal
at a splice junction. @Such primers
should not anneal properly to genomic DNA.
3) Nested PCR
E The 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 a few points of the PCR cycling.@ The number of cycle should be determined empirically to find the
linear range of amplification.@ If you
are not good at to recover the reaction solution in 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.