Analysis of gene expression by real-time PCR

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Analysis of gene expression by real-time PCR

• RNA Isolation from tomato

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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.

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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
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Todays 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

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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

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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

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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)

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Guanidinium Thiocyanate RNA Extraction

• Step 3 Phenolchloroformisoamyl 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
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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

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Theoretical Basis of UV Spectrophotometry for
Quantitating Amount and Purity of RNA
Lambert Beer law Aebc c concentration b
path length (1 cm) e 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

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Next Week

• RNA Electrophoresis

cDNA Synthesis