C DNA SYNTHESIS Yaprak Dönmez December, 2009

1.2% agarose, 70 V, 90 min RNA Ladder W3 G2 F2 4 EN/DA F/ITTH3 EC2 B/A 1 Y RNA Ladder

 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.

Th3EC2Başak1Y OD OD OD260/ µg/mL µg5µg3.32µL2.32µL2.37µL2,77µL3.38µL

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!

Random hexamer o Hybridizes somewhere along the mRNA so that all mRNA segments are represented in the cDNA: Gene specific primers o Adv: specific mRNA o 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 RNA100 ng - 5 µg Poly (A) + RNA ng Specific RNA0.01 pg µg Oligo dT µg (100 pmol) Random hexamer 0.2 µg (100 pmol) Gene specific primers pmol Protocol for First-strand cDNA Synthesis Mix and briefly centrifuge all components after thawing, keep on ice. DEPC treated dH 2 O 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 dH 2 O2.5µL

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 RNA3.32µL 2.32µL 2.37µL Primer (oligo dT) 0.5 µg/µL 1µL 1µL 1µL DEPC dH 2 O6.68µL 7.68µL 7.63µL 70° for 5 min, chill on ice. 5X Reaction buffer4 µL dNTP mix 10mM2µL DEPC dH 2 O2.7µL 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: =mcb.section.1611# Kok J B et.al. Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes. Laboratory Investigation 2005; 85, 154– or_qPCR.pdfl Farrell R E (2005). RNA Methodologies ISBN ,