Related Experiment Video
Updated: Aug 5, 2026
Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples
Published on: December 22, 2014
Enabling methanol fixation of pediatric nasal wash during respiratory illness for single cell sequencing in
Khaled A Ismail1, Malini Mukherjee2, Michael S Kareta2,3,4
1Environmental Influences on Health and Disease Group, Sanford Research, Sioux Falls, SD, USA.
Insights
Methanol fixation preserves pediatric nasal wash samples for single-cell RNA sequencing. This method allows for flexible processing and multicenter collaboration in studying respiratory infections.
Area of Science:
- Immunology
- Molecular Biology
- Pediatrics
Background:
- Lower respiratory tract infections (LRTI) are a major global cause of mortality, especially in children.
- The immune response at the upper respiratory tract mucosa, the initial site of infection, is not well understood.
- Obtaining lower respiratory tract samples is invasive, unlike easily collected nasal wash (NW) samples.
Purpose of the Study:
- To develop and validate a protocol for single-cell RNA sequencing (scRNA-seq) using methanol-fixed nasal wash samples from children with LRTI.
- To assess the feasibility of using NW samples for detailed immune profiling in pediatric respiratory infections.
Main Methods:
- A pilot study was conducted using nasal wash samples from hospitalized children diagnosed with LRTI.
- A protocol was developed to perform scRNA-seq on methanol-fixed NW samples.
- Qualitative and quantitative parameters of scRNA-seq were compared between methanol-fixed and fresh NW samples.
Main Results:
- No significant differences were observed in scRNA-seq qualitative and quantitative parameters between methanol-fixed and fresh NW samples.
- Methanol fixation effectively preserves the integrity of NW samples for scRNA-seq analysis.
Conclusions:
- A novel, effective protocol enables scRNA-seq analysis of methanol-fixed pediatric nasal wash samples.
- This method overcomes challenges associated with clinical sample collection, offering processing flexibility.
- The protocol facilitates multicenter collaboration for studying pediatric respiratory tract infections.
Background:
Lower respiratory tract infection (LRTI) including pneumonia, bronchitis, and bronchiolitis is the sixth leading cause of mortality around the world and leading cause of death in children under 5 years. Systemic immune response to viral infection is well characterized. However, there is little data regarding the immune response at the upper respiratory tract mucosa. The upper respiratory mucosa is the site of viral entry, initial replication and the first barrier against respiratory infections. Lower respiratory tract samples can be challenging to obtain and require more invasive procedures. However, nasal wash (NW) samples from the upper respiratory tract can be obtained with minimal discomfort to the patient.
Method:
In a pilot study, we developed a protocol using NW samples obtained from hospitalized children with LRTI that enables single cell RNA sequencing (scRNA-seq) after the NW sample is methanol-fixed.
Results:
We found no significant changes in scRNA-seq qualitative and quantitative parameters between methanol-fixed and fresh NW samples.
Conclusions:
We present a novel protocol to enable scRNA-seq in NW samples from children admitted with LRTI. With the inherent challenges associated with clinical samples, the protocol described allows for processing flexibility as well as multicenter collaboration.
Impact:
There are no significant differences in scRNA-seq qualitative and quantitative parameters between methanol fixed and fresh Pediatric Nasal wash samples. The study demonstrates the effectiveness of methanol fixation process on preserving respiratory samples for single cell sequencing. This enables Pediatric Nasal wash specimen for single cell RNA sequencing in pediatric patients with respiratory tract infection and allows processing flexibility and multicenter collaboration.
