MiRNA targets Using undergraduate molecular biology labs to discover targets of miRNAs in humans Adam Idica, Jordan Thompson, Irene Munk Pedersen, Pavan.

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Presentation transcript:

miRNA targets Using undergraduate molecular biology labs to discover targets of miRNAs in humans Adam Idica, Jordan Thompson, Irene Munk Pedersen, Pavan Kadandale

Labs 7-9 flow chart Pick target Design primers Isolate RNA from cells Make cDNA using RT-PCR Use qPCR to quantify expression level Repeat

RNA Isolation: Overview ? Lyse cells BindWashElute ??

RT-PCR What are components for RT-PCR? - RNA template - Primer: which? - dNTPs - RT - Buffer

Controls Product RTPCR RT PCR RT PCR

Controls Product -RTPCR -RT PCR -RT PCR

Quantifying DNA/RNA: qPCR 1 cycle 2 cycles 3 cycles 30 cycles Start with 1 molecule 2 molecules 4 molecules 8 molecules ~1 billion molecules Start with 10 molecules 20 molecules 40 molecules 80 molecules ~10 billion molecules

Same starting material… Looks different!

Different starting material… Looks the same!

Solution: qPCR

“Threshold” cycle

How can we quantify DNA in PCR? DNA molecules in PCR - Template - Primer - dNTPs - Product How to quantify ONLY product?

TemplatePrimerdNTPs Quantifying PCR products TemplatePrimerdNTPsProduct

qPCR – Things to think about… qPCR data: Conclusion? B. “X” is not target A. “X” is target C. Need a control Fluorescence units Wildtype miR-128 overexpression

qPCR controls Normalize amount of starting material? Could normalize total RNA Better method?

qPCR – Things to think about… qPCR data: Conclusion? B. “X” is not target A. “X” is target C. Need a control Normalized fluorescence Wildtype miR-128 overexpression

qPCR controls Normalize for overexpression of miR-128? Positive control

“Threshold” cycle

Calculations  C T =  C T (control) -  C T (miR)  C T (miR) = C T (target-miR) - C T (endogenous control-miR)  C T (control) = C T (target-control) - C T (endogenous control-control) Expression fold change = 2  C T