miRNA targets

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

miRNAs

Which of the following is NOT true about miRNAs?

B. Regulate developmental programs

A. Processed from longer precursors

C. miRNAs are transcriptionally regulatedD. miRNAs are conserved

E. Different miRNAs can be separated on a gel

Doing real science!

miR-NNN is involved in cancers

What might we want to know?

How to identify miR-NNN targets?- Does target need to be completely complementary?- Are all complementary sequences targets?

How to identify miR-128 targets?

Using computer algorithms for miRNA targets

Different algorithms to identify targets (Why?)Which one is best?

Common targets better?

So, what use are they?

Using miRNA target databases

http://www.targetscan.org/Screenshot date: 3-30-2015

Using miRNA target databases

http://pictar.mdc-berlin.de/cgi-bin/new_PicTar_vertebrate.cgiScreenshot date: 3-30-2015

Using miRNA target databases

Database will give you list

How will you pick target?

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

After picking a target…

Pick a target – then what?

How will you “validate” target?

What controls do you need to include?

DNA contamination?

Amount of sample?

Change in levels due to miRNA targeting?

Finding targets

Which of the following is NOT required to find miR targets?

B. qPCR

A. RT-PCR

C. Clone target gene

D. Isolate total mRNA

E. Make cDNA

How to design primers?

What do you need to know?- Sequence of transcript

What are important considerations?- Length

- Tm

- Primer dimer

- Primer self-complementarity

- Primer specificity

- Sequence runs & GC- content

Primer design

If true, which of the following would make you discard a primer

B. Primer sequence is AGGCGAGTGTTCGCCT

A. Primer has GC content of ~62%

C. Predicted Tm is 62°C

D. Primer has AT content of 66%

E. Primer binds one location on the genome

Primer design

You design primers for a gene; using these primers in a PCR yields 1 band. Using the primer in an RT-PCR gives no band. Which of the following is not a valid reason:

B. Predicted Tm is 72°C for R-primer

A. F-primer has GC content of ~78%

C. F-primer binds unique sequence in intron D. R-primers binds unique sequence in exonE. F-primer binds 4 different sites in genome

Using Primer3Plus

http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgiScreenshot date: 3-30-2015

OligoCalc

http://www.basic.northwestern.edu/biotools/OligoCalc.htmlScreenshot date: 3-30-2015

Primer design

What important parameter have we still not checked?

B. Primer-primer dimers

A. Tm

C. GC-Content

D. Primer self-complementarity

E. Primer specificity

PrimerBLAST

http://www.ncbi.nlm.nih.gov/tools/primer-blast/Screenshot date: 3-30-2015

Primers designed: Now what?

Enter in Google Drive Spreadsheet

DO NOT EDIT someone else’s already existing data!

Lab SectionGroup numberGroup membersTranscriptGene GeneReason