Relative quantification of mRNA transcript levels by qPCR

Culture the HeLa-M cells at 37°C in 5% CO₂ and DMEM containing 10% FBS, 100U/ml penicillin, 100mg/mL streptomycin, and 2millimolar (mM) L-glutamine (all from Gibco).

For any given experiment, plate the cells at such density so as to be approximately 90% confluent at the time of lysis.

For experiments using siRNA, transfect 60 pmols of the indicated siRNA using 6µL Lipofectamine RNAiMax (ThermoFisher) in Opti-MEM (Gibco) per well according to manufacturer protocol. Lyse the cells 72h 0m 0s after siRNA transfection.

Aspirate media from cells and rinse cells with PBS 37On ice.

Isolate RNA using RNeasy Micro Plus kit (Qiagen) according to manufacturer’s protocol.

Generate cDNA from 1µg purified RNA using iScript cDNA synthesis Kit (Bio-Rad) according to manufacturer’s protocol.

Dilute the iScript reaction to a total of 400µL Sterile Water (American Bio).

Combine 10µL SYBR Green Master Mix (BioRad) with 6.78µL Sterile Water (American Bio) per sample.

Combine 16.78µL diluted SYBR Green Master Mix with 0.61µL each of 10micromolar (µM) forward and reverse primers per sample. Pipette this mixture into wells of 96-well qPCR plate. Perform at least two technical replicates for each sample.

Pipette 2µL of diluted RNA from step 7 in well with SYBR Green Master Mix.

Cover plate with Optical Adhesive Covers (Applied Biosystems).

Spin down plate in table top centrifuge.

Run qPCR in CFX96 Real-Time System (BioRad) using the following protocol:

A	B	C
95 ˚C	3 min
95 ˚C	10 sec	Repeat 39x
55 ˚C	10 sec
72˚C	30 sec
95 ˚C	10 sec
65 ˚C	5 sec
95 ˚C	5 sec

Subtract the housekeeping gene (b-actin) mean threshhold cycle (Ct) values from transcript of interest mean Ct values to calculate ΔCt.

Subtract the ΔCt of the control sample from each sample ΔCt to calculate the ΔΔCt value.

Calculate relative expression using the 2^−ΔΔCt method.