RNA isolation and qRT-PCR
Narayana Yadavalli, Shawn M. Ferguson
Abstract
This protocol describes the RNA isolation from cultured cells and quantitative RT PCR.
Attachments
Steps
RNA isolation and qRT-PCR
Aspirate media from cells and rinse cells with PBS On 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.
The iScript cDNA was diluted 1:10 by using sterile water.
Combine 10µL SYBR Green Master Mix (BioRad) with 6.78µL Sterile Water 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 2 technical replicates were ran for each sample.
Pipette 2µL of diluted RNA from step 4 in well with SYBR Green Master Mix.
Cover plate with Optical Adhesive Covers (Applied Biosystems).
Spin down plate in tabletop centrifuge.
Run qPCR in CFX96 Real-Time System (BioRad) using the following PCR steps.
PCR cycle.
Analysis of qRT PCR data
Calculate the ΔCT values by subtracting respective gene CT value from housekeeping gene GAPDH value.
Followed by calculating 2ΔCTvalues, normalize the mRNA expression levels for genes of interest to GAPDH and represented relative to control.
