TempO-Seq human tissue
Changes to the protocol
None.
Tissue
Human, stored in PFA 4% in Pathology. Tissue was taken from storage and processed into paraffin embedded tissue via the pathology unit.
Non-diabetic, donor.
Tissue was not sectioned, instead pieces were taken and fed into the protocol as normal. The protocol was followed as if 20µm2 of tissue was obtained from a section (doubling the lysis buffer).
Amplification (cDNA library preparation)
Both ct curves are from the same template amplified in 2 different experiments. The first had a PCR set up of 30 cycles whilst the second 35.
PCR
37°C 10 minutes
95°C 1 minute
n cycles of 95°C 10 sec; 65°C 30 sec; 68°C 30 sec
68°C 2 minutes
Hold at 25°C
ct curves
