Analysis of gene expression by real-time PCR

RNA Isolation from tomato

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.

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

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

CAUTION:

RNases ARE EVERYWHERE!

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 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

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

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)

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

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

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

Next Week

RNA Electrophoresis cDNA Synthesis