3.5.2. RT-qPCR Analysis to Test Gene Expression

Lyse the cells in 1 mL of TRIzol per well according to the manufacturer’s instructions, and transfer it into a 1.5 mL microcentrifuge tube (see Note 12).

Pipette the mixture up and down until no pellet is visible at the bottom of the tube (see Note 13).

Incubate the homogenized sample at RT for 5 min.

Add 0.2 mL of chloroform and cap the sample tubes securely. Shake the tubes vigorously by hand for 15 s and incubate at RT for 2–3 min.

Centrifuge the samples at no more than 12,000 × g for 15 min at 4 °C (now the mixture separates into three phases, the upper aqueous phase contains RNA).

Transfer the aqueous phase to a fresh tube and precipitate the RNA by mixing it with 0.5 mL of isopropyl alcohol.

Incubate the samples at RT for 10 min and centrifuge at no more than 12,000 × g for 10 min at 4 °C.

Remove the supernatant, wash the RNA pellet with 1 mL of 75% ethanol, vortex the sample, and centrifuge at no more than 7500 × g for 5 min at 4 °C (keep it at −20 °C until use).

Briefly dry the RNA pellet, dissolve the RNA in RNase-free water, and incubate for 10 min at 55–60 °C. Quantify the RNA by Nanodrop spectrophotometer.

Prepare cDNA using a first-strand cDNA synthesis kit according to the manufacturer’s instructions.

Assess for gene expression using a SYBR Green PCR Master Mix together with designed primers. The genes involved in sarcomere structures (Actc1, TnnT2, Myh6, Myh7), ion channels (Pln, Slc8a1, Scn5a), and cell junctions (Gja1, Kcna5, Cacba1c) could be evaluated. Successful cardiac reprogramming shows significant upregulation of those genes and downregulation of genes represented as fibroblast markers including Col1a1 and Col3a1. GAPDH expression should be detected simultaneously as a housekeeping gene for normalization.