Last class Overall goal for labs 7-9 How to pick candidate miRNA targets Overview: “Validate” target prediction

Use qPCR to quantify expression level 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 ? ? ? http://www.thermoscientificbio.com/uploadedImages/Products/Nucleic_Acid_Purification/Kits_-_Genomics/D_RNA_cleanup_and_concentration_protocol.jpg

Lab session Each group will start with either miRNA or Control Make total RNA (freeze half!) Remaining: Split into TWO tubes Keep one, swap one with another group

Lab session Group B Group A miRNA Control Control RNA miRNA RNA

Quantifying DNA/RNA: qPCR Start with 1 molecule Start with 10 molecules 1 cycle 2 cycles 3 cycles 30 cycles What would happen with 1000 vs 10,000 molecules?

EtBr Gels… PCR data: BEST conclusion? http://www.sabiosciences.com

EtBr Gels (continued)… PCR data: BEST conclusion? http://www.sabiosciences.com

Solution: qPCR http://www.abbottmolecular.com

“Threshold” cycle http://www.sabiosciences.com

“Threshold” cycle http://www.sabiosciences.com

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

Quantifying PCR products Template Template Primer Primer dNTPs dNTPs Product Product

After picking a target… What controls do you need to include? Transfection / RNA introduction affected transcription DNA contamination? Amount of sample ? Endogenous expression? Change in levels due to miRNA targeting?

Controls RT RT RT PCR PCR PCR Product Product Product

Controls -RT -RT -RT PCR PCR PCR Product Product

qPCR – Things to think about… qPCR data: Conclusion? 1 2 3 Fluorescence units Wildtype miR-23b overexpression

After picking a target… What controls do you need to include?

qPCR – Things to think about… qPCR data: Conclusion? 1 2 3 Normalized fluorescence Wildtype miR-23b overexpression

Controls Transfection / RNA introduction affected transcription DNA contamination? Amount of sample ? Endogenous expression? Change in levels due to miRNA targeting?

“Threshold” cycle http://www.sabiosciences.com

Calculations DDCT = DCT(control) - DCT(miR) DCT(control) = CT(target-control) - CT(endogenous control-control) DCT(miR) = CT(target-miR) - CT(endogenous control-miR) Expression fold change = 2DDCT

Use qPCR to quantify expression level 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