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Reverse Transcriptase PCR

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by

Ruth Bishop

on 27 March 2015

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Transcript of Reverse Transcriptase PCR

Gene Identification
Given QTL coordinates on male and female chromosome 3
4 QTLs/sex
Gene Selection Criteria (Relative order of importance):
Closeness to peak region
Expression level/location
Function/identity
Identified approximately 20 genes/QTL
Primer Optimization
Primer optimization is the process of determining the optimal temperature at which a primer functions

Run PCR plate with cDNA
Add 1 primer per column
Run temperature gradient
Project Overview
The Pipeline
3rd instar larvae-mRNA
Gene expression
Dark Pupae
Pupal weight data
We are trying to identify genes from the pupal weight QTL that modulate pupal weight phenotype and show diet-specific expression
RT-PCR
Ingredients:
Thermocycler
Master mix
Taq polymerase
dNTPs
nuclease-free H2O and buffer
primers
SYBR Green
-binds to the double-stranded DNA of the PCR products, it will emit light upon excitation. The intensity of the fluorescence increases as the PCR products accumulate.
Results
Calculate the
∆∆Cq
Overall, ∆∆Cq yields a normalized, relative gene expression value. This is accomplished by normalization of a gene target with experimental treatment to an endogenous reference gene(s) whose expression should remain unchanged by the treatment.
Subsequently, this value is normalized to the targeted gene’s expression detected in a separate control sample.
Calculation
∆Cq1= Expression of sample-Expression of Beta tubulin
∆Cq2=Expression of pool-Expression of Beta tubulin
∆∆Cq=
∆Cq1 - ∆Cq2
Primer Design
Primer-strand of nucleic acid that serves as a starting point for DNA synthesis

Most primers are designed to be between 20 and 30 basepairs long, desired product size is 120 bp

Design Considerations
Complementary to the region of the template flanking the target to be amplified
Geneious
Splice site-spanning

Region that is chosen for primer binding occurs only once within the template DNA
NCBI BLAST (E-value)
Base composition GC=55%
Forward and Reverse primers have similar melting temperatures (Tms), 60 degrees
Low self-dimer temperatures (hairpins)
UNAfold
Conversion of mRNA to cDNA
Extract mRNA from larvae samples
Use
Reverse Transcriptase
to convert mRNA to cDNA
To mRNA samples, add reverse transcriptase, oligoDT primers,RNAse inhibitor,buffers, MGCl2, and dNTPs= and like MAGIC you have cDNA! Way to go RT!
Central Dogma
-Transcripts=mRNA

-Transcriptome-set of ALL RNA
molecules (mRNA, rRNA, tRNA,
and other non-coding RNA
produced
-Unlike the genome, the
transcriptome can vary with
the environment

**The transcriptome reflects
the GENES that are being actively expressed at any given time**

QTL
Genotype by Diet QTL (quantitative trait loci)
Quantitative trait loci (QTLs) are stretches of DNA containing or linked to the genes that underlie a quantitative trait.
Quantitative traits refer to phenotypes (characteristics) that vary in degree and can be attributed to polygenic effects, i.e., product of two or more genes, and their environment
30 larvae samples (2 tubes)
Metabolomics
RNA-----> cDNA -------> RT-PCR
primer
Temp. gradient
What we hope to find
Identifying pupal weight-modulating
genes interacting with diet in Drosophila melanogaster

Conclusion
Full transcript