Analysis of gene expression by real-time PCR RBCS3 and Cab-1b transcript quantitation by real time PCR

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

Today’s Laboratory Objectives 1.To set up and run a series of real time PCR reactions with Cab-1b- and RBCS3-specific primers 2.To understand the theoretical basis of real time PCR and its quantitative applications

PCR 10X buffer Primers Taq DNA polymerase dNTPs template DNA After 30 thermocycles, a DNA molecule initially present in a single copy is present in > 1 billion copies

Standard PCR is non-quantitative PCR cycle # DNA amount (arbitrary units) 100 White tube= 1000 copies Green tube= 10 copies Primers/dNTPs exhausted electrophoresis Equal amounts of PCR product Post-PCR DNA quantitation

Real time PCR is quantitative Fluorescence (arbitrary units) = 6.6 cycles = 100 White sample is 100-fold more abundant than green sample “real time” DNA quantitation Fluorescence threshold 10X buffer Primers Taq DNA polymerase dNTPs template DNA fluorescent “marker” of dsDNA C t = 10.0 C t = 16.6 White tube= 1000 copies Green tube= 10 copies

Output data from real time PCR PCR cycle # Fluorescence (arbitrary units)

Fluorescence-based chemistries for quantitation of dsDNA SYBR green I cyanine dye (abs. 480nm, em. 520nm) binds almost exclusively to dsDNA (minor groove) fluorescence increases >1000-fold when bound to DNA sensitive, but nonspecific TaqMan bp ssDNA probe, complementary to template DNA sequence 5’ fluorescent dye, 3’ fluorescence quencher Taq 5’ exonuclease activity cleaves ssDNA probe, releasing free dye into solution (fluorescence) sensitive, sequence specific, multiplex PCR possible

Molecular beacons bp ssDNA probe, the central region of sequence is complementary to template DNA 5’ fluorescent dye, 3’ fluorescence quencher 5’ and 3’ end of sequence are self complementary, form stem loop structure which promotes fluorescence quenching during annealing phase of PCR, the central part of the probe hybridizes with the complementary template DNA sequence, separating fluorescent dye and quencher, resulting in fluorescence Fluorescence-based chemistries for quantitation of dsDNA For all chemistries, fluorescence and dsDNA content are directly proportional (i.e. fluorescence doubles after each thermocycle). Therefore, DNA content in the PCR reaction can be quantified after each cycle by measuring fluorescence

Real time PCR hardware Optical quality PCR tubes or 96 well plate BioRad icycler Illumination: laser, LED, tungsten halogen bulb excitation emission * Light filter Detector: photomultiplier

Experimental Design Time Series ► RNA Extractions Performed on tomato plants grown in the light or in darkness for 24, 48, or 72 hrs ► Each sample assayed in triplicate in 96-well format Reaction Mix cDNA (diluted 1:100) 5.0 μl Sybr Green Super Mix 7.1 μl Primer RB1 or CA1 (20 μM) 0.3 μl Primer RB2 or CA2 (20 μM) 0.3 μl dH2O 12.3 μl total volume per reaction 25 μl 8 total reactions per group (e.g. 3 RB1/RB2 + light cDNA, 3 RB1/RB2 + dark cDNA, 2 RB1/RB2 + water)

Real Time RT PCR Cycling Parameters Polymerase activation95° C 10 min 40 cycles Denaturation95° C 10 sec Primer Annealing60° C 30 sec Extension72° C 30 sec Melt Curves Denaturation95° C 1 min Renaturation55° C 1 min DenaturationRamp 0.5° C every 10 sec

Alternative methods for transcript quantitation Northern Blot RNase protection assay

Next Week Analysis of real time PCR data

RNA gels RNA ladder Class gels Expected result (plant RNA)