CDNA SYNTHESISYaprak DnmezDecember, 2009
1.2% agarose, 70 V, 90 minRNA LadderRNA LadderW3 G2 F2 4 EN/DA F/IT TH3 EC2 B/A 1 Y RNA Ladder
Determination of the RNA Concentration[RNA] g/ml = A260 x dilution x 40.0whereA260 = absorbance (in optical densities) at 260 nmdilution = dilution factor (200)40.0 = average extinction coefficient of RNA.
cDNA SynthesisTemplate:Total RNA Poly (A)+ RNASpecific RNA
PrimingOligo 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 hexamerHybridizes somewhere along the mRNA so that all mRNA segments are represented in the cDNA:
Gene specific primersAdv: specific mRNADisadv: lower yields
Reverse Transcriptases (RTs)The reverse transcriptase from the avian myoblastosis virus (AMV-RT): Temperature optimum of activity at 45-50C. Highly thermostable, can be used at temperatures up to 60C. 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.
Protocol for First-strand cDNA SynthesisMix and briefly centrifuge all components after thawing, keep on ice.DEPC treated dH2O to 11 L
Primer:Total RNA100 ng - 5g Poly (A)+ RNA10 - 500ngSpecific RNA0.01pg - 0.5gOligo dT 180.5g (100 pmol) Random hexamer0.2g (100 pmol) Gene specific primers15-20pmol
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 4LdNTP mix 10mM1mM final conc, so add 2LDEPC dH2O2.5L
Heat in 37 for 5 min, chill for a few minutes. This will anneal primer to complementary RNA.Add 0.3 L 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 BaakRNA3.32L 2.32L 2.37LPrimer (oligo dT) 0.5g/L1L 1L 1LDEPC dH2O6.68L 7.68L 7.63L70 for 5 min, chill on ice.5X Reaction buffer4 L dNTP mix 10mM2LDEPC dH2O2.7L37 for 5 min, chill on ice.M-MuLV Reverse Transcriptase0.3L (total rxn V= 20 L)42 for 60 min72 for 10 min
NormalizationMost 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 isolationSample concentration, RNA integrity, the reagents used, presence of contaminants reverse transcriptionHousekeeping genes such as -actin, -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.pdfhttp://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=cDNA,Synthesis&rid=mcb.section.1611#1619http://www.fermentas.com/catalog/modifyingenzymes/m_mulvrt.htmlKok J B et.al. Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes. Laboratory Investigation 2005; 85, 154159.http://www.dna.ohiou.edu/literature/qRT_pCR/Normalization_Methods_for_qPCR.pdflFarrell R E (2005). RNA Methodologies ISBN 0122496965, 9780122496967.