Analysis of gene expression by real-time PCR

1. Analysis of gene expression by real-time PCR RNA Isolation from tomato

2. Broad and Long Term Objective To characterize the expression of the genes encoding Ribulose 1-5 bisphosphate carboxylase oxygenase-3 (RBCS3) and Chlorophyll A/B binding protein-1b (Cab-1b) in the leaves of tomato plants grown under continuous light or subjected to 24 hr, 48 hr, or 72 hr in complete darkness.

3. Research Plan RNA Isolation from tomato leaves (continuous light or 24-72 hr darkness) RNA Electrophoresis cDNA synthesis RBCS3 and Cab-1b transcript quantitation by real time PCR Analysis of real time PCR data

4. Today’s Laboratory Objectives 1. To isolate high quality total RNA from leaves of dark- and light-grown tomato plants 2. To quantitate the amount and purity of RNA isolated 3. To become familiar with the nuances of handling RNA

5. CAUTION: RNases ARE EVERYWHERE! Control of endogenous RNases • Keep tissue frozen during disruption and cell lysis • Thaw tissue in an extraction buffer containing strong protein denaturants that will inactivate RNases • Once RNA is out of the extraction buffer, keep RNA on ice or frozen • Work quickly and carefully Control of exogenous RNases • Wear gloves and practice sterile technique • Use disposable plastics or baked glassware • Treat solutions with chemicals that will inactivate RNAses (DEPC, detergents, etc) • Always keep RNA on ice or frozen

6. Guanidinium Thiocyanate RNA Extraction Step 1: Tissue is frozen and ground into a fine powder in a liquid nitrogen-cooled mortar Function: Tissue disruption and cells lysis while endogenous nucleases are temporarily inactivated by low temperatures

7. Guanidinium Thiocyanate RNA Extraction Step 2: Resuspend tissue powder in extraction buffer (guanidium thiocyanate, sarkosyl, ß-mercaptoethanol) Function: A. Solubulization of cell membranes (sarkosyl) B. Inactivation of RNAses by denaturation (sarkosyl disrupts hydrophobic interactions in proteins, guanidium thiocyanate is a strong protein denaturant, ß-mercaptoethanol reduces disulfide bonds and prevents phenolic compounds from crosslinking with RNA)

8. Guanidinium Thiocyanate RNA Extraction Step 3: Phenol:chloroform:isoamyl alcohol extraction Function: A. Separation of proteins from nucleic acid (proteins precipitate and collect at interface or remain in organic phase) B. Separation of DNA from RNA (at acid pH, most DNA remains in organic phase, while RNA is in aqueous phase) Aqueous Phase: RNA Interface (precipiated protein) Phenol Phase (pH4.3): Lipids, protein, DNA

9. Guanidinium Thiocyanate RNA Extraction • Step4: RNA precipitation #1. isopropanol + sodium acetate, • #2. LiCl • Function: Concentrate RNA, further purification of RNA • First precipitation (isopropanol + sodium acetate)- concentrates • RNA, allows removal of RNA from extraction buffer • Second precipitation- (2M LiCl) selective precipitation of RNA • (remaining DNA, protein, carbohydrate, and small RNA molecules • remain in solution) • 70% ethanol wash- removes remaining LiCl • associated with the RNA

10. Theoretical Basis of UV Spectrophotometry for Quantitating Amount and Purity of RNA Lambert Beer law: A=εbc c = concentration b = path length (1 cm) ε = extinction coefficient (for RNA = 0.025 [ng/ul]-1 cm-1) To quantify your RNA sample*: A260 x Dilution Factor x 40 = concentration of RNA (ng/ul) in a sample using a 1 cm pathlength Also determine total yield and yield/gram tissue To estimate the purity of your sample*: A260/A280= ratio of nucleic acids/protein A260/A280= 1.8-2.1 is optimal for RNA

11. Next Week RNA Electrophoresis cDNA Synthesis