Download (A) + RNA

CDNA
SYNTHESIS
Yaprak Dönmez
December, 2009
TH3 EC2 B/A 1
1.2% agarose, 70 V, 90 min
Y
RNA Ladder
4 EN/DA F/IT
RNA Ladder
RNA Ladder
W3 G2 F2
Determination
of the RNA
Concentration
[RNA] μg/ml = A260 x dilution x 40.0

where
 A260 = absorbance (in optical densities) at 260 nm
 dilution = dilution factor (200)
 40.0 = average extinction coefficient of RNA.

Th3
EC2
Başak
1
Y
OD260
0.188
0.27
0.264
0.226
0.185
OD280
0.98
0.145
0.142
0.136
0.113
OD260/280
1.92
1.86
1.86
1.67
1.64
µg/mL
1504
2160
2112
1808
1480
5µg
3.32µL
2.32µL
2.37µL
2,77µL
3.38µL
cDNA Synthesis
 Template:
-
Total RNA
Poly (A)+ RNA
Specific RNA

Priming

Oligo dT 18)

Adv: You can synthesize complete cDNAs, beginning at the poly
A+ tail and ending at the 5 end of the mRNA.
Disadv: The reverse transcriptases used to synthesize cDNAs
have an average length of 1 to 2 kb, whereas mRNAs can easily
be 10 kb long. The protein coding portion of interest is usually in
the vicinity of the 5 end!


o

o
o
Random hexamer
Hybridizes somewhere along the mRNA so that all mRNA
segments are represented in the cDNA:
Gene specific primers
Adv: specific mRNA
Disadv: lower yields

Reverse Transcriptases (RTs)

The reverse transcriptase from the avian myoblastosis virus
(AMV-RT):
Temperature optimum of activity at 45-50°C.
Highly thermostable, can be used at temperatures up to 60°C.
Demonstrates DNA exonuclease and RNase activities.
The reverse transcriptase from the Moloney murine leukemia
virus (MMLV-RT):
The optimal working temperature is about 37C, with a maximum
of 42C.
Weaker RNase activity.
o
o
o

o
o
Protocol for First-strand cDNA Synthesis
Mix and briefly centrifuge all components after thawing, keep on ice.
Template:
Primer:
Total RNA
100 ng - 5 µg
Poly (A)+ RNA
10 - 500 ng
Specific RNA
0.01 pg - 0.5 µg
Oligo dT 18
0.5 µg (100 pmol)
Random
hexamer
0.2 µg (100 pmol)
Gene specific
primers
15-20 pmol
DEPC treated dH2O to 11 µL
 Heat
in 70° for 5 min, chill for a few minutes. This
will disrupt secondary structures formed in the
RNA to make it rather linear for the primer to pair
to it.
 Add:
5X Reaction
buffer for
Reverse
Transcriptase
1X final
concentration,
so add 4µL
dNTP mix
10mM
1mM final
conc, so add
2µL
DEPC dH2O
2.5µL
 Heat
in 37° for 5 min, chill for a few minutes. This
will anneal primer to complementary RNA.
 Add 0.3 µL M-MuLV Reverse Transcriptase.
 Incubate at 42° for 60 min. Extension of primer
will occur via RT.
 Heat-inactivate reverse transcriptase at 72° for
10 min.
Th3
EC2
Başak
RNA
3.32µL
2.32µL 2.37µL
Primer (oligo dT)
0.5 µg/µL
1µL
1µL
DEPC dH2O
6.68µL
7.68µL 7.63µL
1µL
70° for 5 min, chill on ice.
5X Reaction buffer
4 µL
dNTP mix 10mM
2µL
DEPC dH2O
2.7µL
37° for 5 min, chill on ice.
M-MuLV Reverse
Transcriptase
0.3µL (total rxn V= 20 µL)
42° for 60 min
72° for 10 min
 Normalization





Most gene expression assays are based on the comparison of
two or more samples and require uniform sampling conditions
for this comparison to be valid.
Many factors can contribute to variability in the analysis of
samples, making the results difficult to reproduce between
experiments:
Sample degradation, extraction efficiency, contamination →
RNA isolation
Sample concentration, RNA integrity, the reagents used,
presence of contaminants → reverse transcription
Housekeeping genes such as ß-actin, ß-tubulin, GAPDH, and
18S ribosomal RNA have often been used as reference genes
for normalization, with the assumption that the expression of
these genes is constitutively high and that a given treatment
will have no effect on the expression level.
References:
 http://hominid.uchicago.edu/ProtocolPDFs/OligodTcDNASynthesis.pdf
 http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=cDNA,Synthesis&rid
=mcb.section.1611#1619
 http://www.fermentas.com/catalog/modifyingenzymes/m_mulvrt.html
 Kok J B et.al. Normalization of gene expression measurements in tumor
tissues: comparison of 13 endogenous control genes. Laboratory
Investigation 2005; 85, 154–159.
 http://www.dna.ohiou.edu/literature/qRT_pCR/Normalization_Methods_f
or_qPCR.pdfl
 Farrell R E (2005). RNA Methodologies ISBN 0122496965,
9780122496967.