Housekeeping gene at different larval stages

Model Organism: Worms Methods & Reagents
1

Hi everyone,

           I am looking for the expression of certain genes at different stages of the worm (L1-L4). Does the actin levels or any other housekeeping

genes changes at different stages of the worm?

I used act-1 and pmp-3 as housekeeping genes but they are not the same at different stages of the worm. If I am using the right ones then what
should be the acceptable range of CT values between different larval stages? Is there a better one?

Rakesh

2

Are you trying to compare the expression of a single gene at different points in development or comparing conditions (i.e. Wt vs mutant) at each larval stage? If it’s the former, then you can just normalize to a standard housekeeping gene. If it’s the latter, you should extract RNA from your experimental conditions in triplicate and generate cDNA (from the same volumes). Then take a panel of candidate reference genes (commonly used ref genes are act-1, tub-1, ama-1, cdc-42, php-3, and F45F10D.4). Run qRT-PCR to find the Cq values for these genes and find the standard deviation. A good reference gene should give a std. dev of less than 0.5. Anything higher indicates that it’s too variable across your conditions.

The MIQE guidelines are helpful in designing a properly controlled qPCR experiment.
http://www.ncbi.nlm.nih.gov/pubmed/19246619

Some validated primer pairs for potential reference genes:
act-1 GCTGGACGTGATCTTACTGATTACC GTAGCAGAGCTTCTCCTTGATGTC 114 bp Taubert et al., 2008 PLoS Geneticas
cdc-42 GACAATTACGCCGTCACAG CGTAATCTTCCTGTCCAGCA 81 bp From Hoogewijs et al., 2008 BMC Mol Biol.
pmp-3 ATATGGAACTTAGAGTCAAGGGTC CAGTTCGACTAGCTATCGAGG 80 bp From Hoogewijs et al., 2008 BMC Mol Biol.
Y45F10D.4 TTCACTGTTCAATGCTCGC CTTAGGCCTTCTTAGTCTGCT 80 bp From Hoogewijs et al., 2008 BMC Mol Biol.
tba-1 GCTGCGTACACTCCACTGAT CTCGAAGCAACTGTTGGTGA 84 bp Ward et al., PLOS ONE (in press)
ama-1 CCTACGATGTATCGAGGCAAA CCTCCCTCCGGTGTAATAATG 129 bp Taubert et al., 2008 PLoS Geneticas

3

Thanks for your reply. I am actually looking at single gene at different points during larval development. I used act-1 and pmp-3.
Both these housekeeping genes are variable at different points in development. I have attached the Ct values at different time points.
Can you have a look at it?

L0: 0 hrs (after overnight hatching in K medium)
L1: 6 hrs (6 hrs on food plate)
L2: 27 hrs(27 hrs on food plate)
L3: 40 hrs(40 hrs on food plate)
L4: 50 hrs(50 hrs on food plate)

Thanks
Rakesh

4

I am not able to attach the file. Can I send it to you through email?

Thanks again for your time
Rakesh

5

Sure, send to jordan.ward@ucsf.edu