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? B. Regulate developmental programs A. Processed from longer precursors 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 Screenshot date:

Using miRNA target databases Screenshot date:

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 - T m - 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 T m 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 T m 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 exon E. F-primer binds 4 different sites in genome

Using Primer3Plus Screenshot date:

OligoCalc Screenshot date:

Primer design What important parameter have we still not checked? B. Primer-primer dimers A. T m C. GC-Content D. Primer self-complementarity E. Primer specificity

PrimerBLAST Screenshot date:

Primers designed: Now what? Enter in Google Drive Spreadsheet DO NOT EDIT someone else’s already existing data! Lab Section Group number Group members Transcript Gene Reason