High throughput method for extraction of high quality of total RNA from human lung epithelial cells for Nanopore RNA sequencing?

13 January 2023 1 2K Report

I want to extract good amount of high quality RNA from human lung epithelial cells. I have pellets of nearly 1X10^7 cells per tube. I need very high purity total RNA that can be successfully used for subsequent Nanopore RNA sequencing. Can anyone suggest a good method please. Thanks.

Nicolò M. Villa

Dear Asif Muhammad ,

There are several methods that can be used to extract high quality total RNA from human lung epithelial cells. Some popular options include:

Trizol-based extraction: Trizol reagent is a popular choice for extracting high quality total RNA from various types of cells. It can be used to isolate both small and large RNA molecules, including mRNA, rRNA, and miRNA.

RNeasy Mini Kit: This kit is a spin-column based method that can be used to purify high quality total RNA from a wide variety of samples, including lung epithelial cells. It uses silica-based spin columns to selectively bind and purify RNA while removing contaminants such as proteins, DNA, and polysaccharides.

NucleoSpin RNA Plus Kit: This kit uses silica-based spin columns and a special lysis buffer that allows for efficient and selective isolation of high-quality total RNA from lung epithelial cells.

QIAzol Lysis Reagent : QIAzol Lysis Reagent is a versatile, one-step lysis reagent for the isolation of high-quality total RNA from various samples, including lung epithelial cells.

After extraction, it is recommended to purify your RNA using a poly(A) tail purification step and/or poly(A) tail selection in order to increase the percentage of mRNA in the sample.

It is also important to quality control the RNA samples by measuring the concentration, purity and integrity using techniques such as spectrophotometry and gel electrophoresis before proceeding with downstream applications such as nanopore sequencing.

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