# Simple, streamlined, cost-effective cDNA synthesis method from cell cultures

## TL;DR

This paper introduces a simple and low-cost method for extracting and analyzing mRNA from 96-well cell culture plates, which is useful for applications like drug development.

## Contribution

A novel, cost-effective cDNA synthesis method is proposed, validated against commercial kits with improved performance and reduced variability.

## Key findings

- The method reduced Ct values by 2.4 ± 1.3 compared to the 'Cells-to-cDNA' kit.
- It showed a 1.4 ± 0.5 reduction in Ct values compared to RNA purification kits.
- The method exhibited lower variability in gene expression measurements.

## Abstract

Applications like drug development need simple and streamlined methods to process samples from 96-well cell culture plates for gene expression measurements. Unfortunately, current options are expensive for such processing. Therefore, our aim was to develop a method that would allow streamlined analysis of mRNA from 96-well cell culture plates while being relatively cheap and simple. We developed a method based on the qPCR ‘Cells-to-cDNA’ approach and validated it against commercially available kits using the same approach or spin columns-based RNA purification. For this purpose, we conducted a series of comparisons of gene expression from peripheral blood mononuclear cells, SK-HEP-1 and U-87 cell cultures in 96-well plates. Our final method involved lysing cells with 25–100 µl solution of 0.5% SDS, 10 mM DTT, 1 mg ml−1 proteinase K dissolved in water, 1 h incubation at 50°C, followed by proteinase K inactivation at 90°C for 5 min and lysate neutralization with 1 : 1 dilution by 20% Tween 20 solution. Reverse transcription and qPCR were carried out using standard methods. This method showed a mean reduction of Ct ± s.d. value by 2.4 ± 1.3 compared with the ‘Cells-to-cDNA’ kit and by 1.4 ± 0.5 compared with the RNA purification kit with lower variability.