qRT-PCR
Cells expanded on microcarriers in ultra-low attachment 6-well plates
were detached as previously described. Cell suspensions were centrifuged
and excess medium was removed prior to storage at
-80oC until further analysis. Pellets were first
re-suspended in RLT Buffer (QIAGEN) before being homogenised in
Qiashredder Columns (QIAGEN) according to the manufacturer’s
instructions. Synthesis of complementary deoxyribonucleic acid (cDNA)
and removal of genomic DNA was performed using the QIAGEN RT-PCR kit
following the manufacturer’s instructions. Genomic DNA was eliminated
using gDNA Wipeout Buffer (QIAGEN) with up to 1 µg template RNA and
RNAse free water in a total reaction volume of 14 µL. The entire 14 µL
reaction volume was subsequently mixed with a MasterMix (Bio-Rad):
template RNAs or no template for NTC (no template control), RT Primer
Mix Quantiscript RT Buffer and Quantiscript Reverse Transcriptase or
with RNA free water for no-RT (No reverse transcriptase control), in a
total volume of 20 µL. The qPCR reactions were performed in Bio-Rad
Hard-Shell Low Profile Thin-Wall 96 Well Skirted PCR plates on the
Bio-Rad CFX 96 Connect Real-Time PCR System using the Quantitect SYBR
Green PCR Kits (QIAGEN) according to the manufacturer’s instructions.
For each reaction Quantitect Mastermix Buffer, Quantitect pre-validated
primer assay (Table 3), water and cDNA were combined to make final
reaction volumes of 25 µL. On each 96-well plate samples were analysed
in triplicate with β-actin as an internal reference control and standard
controls No-RT and NTCs to check for cross contamination. The 2-ΔΔCt
method was used to analyse the data.