The Development of Circadian Rhythms in Human Infants

2.8 Gene expression measurement
Sample collection was conducted in the home setting by the parents who were trained
in Buccal cell collection using a foam tip Catch All sample collection swab
(Epicentre, Madison Wisconsin) (see appendix 2). The cells were preserved in
RNAlater to prevent RNA degradation. Samples were labelled with date and time of
collection. They were then stored in the household freezer at -20 degrees Celsius until
transportation in dry ice and long term storage at -80 degrees at University of
Leicester Genetics department until analysis.
The samples were processed in a three step process. Firstly the RNA was extracted.
Secondly, the mRNA was converted to cDNA by reverse transcriptase. The last step
involved qPCR which was used to examine clock gene expression.
A hemocytometer was used to estimate the cell yield from a series of adult samples
for comparison with the samples taken from the infant population. An average was
taken for adult samples. The sample size from an adult buccal swab was estimated to
be 50 000 to 100 000 cells. The yield from an infant swab was 5000 to 15 000 cells.
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