Transcript Dr. Escobar

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 (Cab1b) 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