Quantitative real-time PCR

-For each sample prepare 10ul gDNA digestion reaction mix according to SuperScript IV VILO Master Mix protocol (see pdf attached or substeps below).

Digest gDNA for 0h 2m 0s min at 37°C .

Place the tubes on ice.

Add SuperScript IV VILO Master Mix and Nuclease-free water.

Gently mix and incubate at 25°C for 0h 10m 0s .

Then at 50°Cfor 0h 10m 0s .

Inactivate enzyme by incubation at 85°C for 0h 5m 0s.

Perform Pre-amplification according to TaqMan PreAmp Master Mix protocol (see pdf attached or substeps below).

-TaqMan PreAmp Master Mix 25ul, pooled assay mix (0.2X) 12.5ul, cDNA 2ul, nuclease free water, total 50ul.   

Run reaction settings: 95°C for 0h 10m 0s then 95°C for 0h 0m 15s, 60°Cfor 0h 4m 0s (10 cycles), inactivate enzyme 99°C for 0h 10m 0s, hold at 4°C

-Dilute each reaction 10 times.

-PCR reaction mix: Gene Expression Assay (20X) 1ul, Preamplified cDNA product 5ul, TaqMan Fast Advanced Master Mix (2X) 10ul, nuclease free water 4ul, total 20ul.

-Run the reactions: Using incubation 50°C for 0h 2m 0s then enzyme activation-95°C for 0h 0m 20s, Denature 95°C for 0h 0m 1s, Anneal/Extend 60°C for 0h 0m 20s 40 cycles.

Experimental Ct values were normalized to hprt values using the following

formula: ΔCt = Ct (gene of interest) − Ct (hprt). The final expression levels were shown as ΔCt

values.