Functional Genomics Research Stream. Research Meeting: June 19, 2012 SYBR Green qpcr, Research Update

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Charity Carr
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1 Functional Genomics Research Stream Research Meeting: June 19, 2012 SYBR Green qpcr, Research Update

2 Updates Alternate Lab Meeting Fridays 11:30-1:00 WEL Welcome to attend either one Lab Log thanks Ashley New Normalization Primers New Yeast Transformation Protocol

3 qpcr Quantitative PCR also known as RT-PCR (real time) quantitate levels of input template based on PCR output PCR amplification measured at each cycle Two general reporter methods double-stranded DNA-binding dyes fluorescent probe

4 SYBR Green double-stranded DNA-binding dye absorbs blue light and emits green light much less mutagenic than ethidium bromide

5 Power SYBR Green RNA-to-CT 1-Step Kit Kit from Life Technologies Perform reverse transcription and qpcr in a single tube Novel formulation reduces false positive results caused by primer-dimers

6 Power SYBR Green RNA-to-CT 1-Step Kit Kit includes: Power SYBR Green RT-PCR Mix (2x) Buffer, SYBR Green, dntps, hotstart DNA polymerase, RXO dye (passive reference) RT Enzyme Mix (125x) Reverse transcriptase, RNase inhibitor

7900HT FAST Real-Time PCR System Reaction Setup T Prepare the RNA

and set up the experiment System software RT-PCR Prepare the

Analyze the experiment Amplification plot http://www3.

7 7900HT FAST Real-Time PCR System Reaction Setup T Prepare the RNA template and or ABI PRISM 6100 Nucleic Acid PrepStation Design and set up the experiment System software RT-PCR Prepare the RT-PCR reactions or Run the experiment Real-time PCR instrument Analyze the experiment Amplification plot

8 Reaction Setup Component Power SYBR Green RT-PCR Mix (2 ) Volume for One Reaction 10 µl 20 µl 50 µl 5.0 µl 10.0 µl 25.0 µl Forward primer (100 to 200 nm final) Variable Variable Variable Reverse primer (100 to 200 nm final) Variable Variable Variable RT Enzyme Mix (125 ) 0.08 µl 0.16 µl 0.4 µl RNA template (up to 100 ng) Variable Variable Variable RNase-free H 2 O to 10 µl to 20 µl to 50 µl Total Volume 10.0 µl 20.0 µl 50.0 µl

9 Example Output Data

10 Example Output Data

11 What will we do? Do dilution series for each sample Do each sample in triplicate Compare stressed vs. non-stressed samples Compare cycle threshold (CT) of known upregulated gene and normalization gene

12 Primer Design Primer design Overview Using Primer Express Software General Primer Design Guidelines Using Primer Express Software, design primers to amplify the target sequence. Design the primers using Primer Express Software as described in the Primer Express Version 3.0 Getting Started Guide and Software Help. The optimal primer length is 20 bases. Keep the GC content in the 30to 80% range. Avoid runs of identical nucleotides. If repeats cannot be avoided, there must be fewer than 4 consecutive G bases. Important: Keep the T m between 58 to 60 C. Make sure the last 5nucleotides at the 3 end contain no more than two G and/or C bases. If you cannot find acceptable primer sequences, you may need to examine the sequence and select another amplicon site or screen for more sites.

Technical Review. Real time PCR

Technical Review. Real time PCR Technical Review Real time PCR Normal PCR: Analyze with agarose gel Normal PCR vs Real time PCR Real-time PCR, also known as quantitative PCR (qpcr) or kinetic PCR Key feature: Used to amplify and simultaneously