c dna synthesis yaprak dönmez december, 2009. 1.2% agarose, 70 v, 90 min rna ladder w3 g2 f2 4...
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1.2% agarose, 70 V, 90 min
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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
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!
Random hexamero Hybridizes somewhere along the mRNA so that all mRNA
segments are represented in the cDNA:
Gene specific primerso Adv: specific mRNAo Disadv: lower yields
Reverse Transcriptases (RTs) The reverse transcriptase from the avian myoblastosis virus
(AMV-RT): o Temperature optimum of activity at 45-50°C. o Highly thermostable, can be used at temperatures up to 60°C. o Demonstrates DNA exonuclease and RNase activities. The reverse transcriptase from the Moloney murine leukemia
virus (MMLV-RT):o The optimal working temperature is about 37C, with a maximum
of 42C.o Weaker RNase activity.
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
Protocol for First-strand cDNA SynthesisMix and briefly centrifuge all components after thawing, keep on ice.
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 1µL
DEPC dH2O 6.68µL 7.68µL 7.63µL
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_for_qPCR.pdfl
Farrell R E (2005). RNA Methodologies ISBN 0122496965, 9780122496967.