miRNA targets
Using undergraduate molecular biology labs to
discover targets of miRNAs in humans
Adam Idica, Jordan Thompson, Irene Munk Pedersen, Pavan Kadandale
miRNAs
Which of the following is NOT true about miRNAs?
A. Processed from longer precursors
B. Regulate developmental programs
C. miRNAs are transcriptionally regulated
D. 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.cgi
Screenshot 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?
A. RT-PCR
B. qPCR
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
A. Primer has GC content of ~62%
B. Primer sequence is AGGCGAGTGTTCGCCT
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:
A. F-primer has GC content of ~78%
B. Predicted Tm is 72°C for R-primer
C. F-primer binds unique sequence in intron
D. R-primers binds unique sequence in exon
E. F-primer binds 4 different sites in genome
Using Primer3Plus
http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi
Screenshot date: 3-30-2015
OligoCalc
http://www.basic.northwestern.edu/biotools/OligoCalc.html
Screenshot date: 3-30-2015
Primer design
What important parameter have we still not checked?
A. Tm
B. Primer-primer dimers
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
Lab Section
Group number
Group members
Transcript
Gene
Gene Reason
DO NOT EDIT someone else’s already existing data!