Total RNA was extracted with RNeasy Mini Kit followed by DNase I treatment.
Label
SYBR GREEN
Label protocol
One microgram of RNA was reverse transcribed to cDNA using the Biorad iScript cDNA Reaction Kit (per kit’s instructions). Using Biorad’s SYBR Green Master Mix reagent, 4 uL of cDNA was placed into 36 uL of water, master mix, and primers and the quantitative PCR reaction run in triplicate reactions of 10 uL each on an Applied Biosystem QuantStudio3 cycler with the standard protocol of 95C and 60C over 40x cycles.
Hybridization protocol
n/a
Scan protocol
n/a
Description
DONOR A EV CONTROL
Data processing
Normalization of the data was performed by the manufacturer's website (https://geneglobe.qiagen.com/us/analyze) For the normalization it uses the average of five housekeeping genes: B2M, HPRT1 , RPL13A, GAPDH, ACTB. Target gene signals normalized to housekeeping genes; 2^-deltaCt, where deltaCt = (Ct_Target − Ct_HKG)] The web-based software package automatically performs all deltadeltaCt based fold-change calculations from the uploaded raw threshold cycle data. Fold Change file reports test/control (i.e., AMPKg2/EV) ratios.
