Cryopreserved human B cells as an alternative source for single cell mRNA analysis

Area of Science:

• Immunology
• Molecular Biology
• Cell Biology

Background:

• Single-cell reverse transcription-polymerase chain reaction (RT-PCR) is crucial for analyzing mRNA in B-lymphocyte subpopulations.
• Longitudinal studies necessitate the use of cryopreserved cells for consistent B-cell analysis.

Purpose of the Study:

• To evaluate the suitability of cryopreserved viable B cells for single-cell RT-PCR analysis.
• To compare the efficacy of RT-PCR on fresh versus cryopreserved B cells for detecting specific mRNA transcripts.

Main Methods:

• Human peripheral blood B cells (fresh and cryopreserved) were analyzed using four-color flow cytometry and fluorescence-activated cell sorting (FACS).
• Individual viable CD19+CD27+ memory B cells were isolated and subjected to nested RT-PCR to detect GAPD, CXCR4, and IgVH gene transcripts.

Main Results:

• B-cell subpopulations were reliably stained and separated from both fresh and cryopreserved samples, with minor reductions in fluorescence intensity for some markers in cryopreserved cells.
• RT-PCR analysis demonstrated comparable yields of cells expressing GAPD, CXCR4, and IgVH transcripts from both fresh and cryopreserved B cells, indicating preserved mRNA integrity.
• The study confirmed the feasibility of using cryopreserved B cells for single-cell RT-PCR, with a recommendation to re-optimize FACS and RT-PCR protocols for post-thawed cells.

Conclusions:

• Optimally cryopreserved B cells serve as a viable alternative source for single-cell RT-PCR analysis, particularly beneficial for longitudinal studies.
• While cryopreservation maintains mRNA integrity for key genes, adjustments to FACS and RT-PCR protocols may be necessary for cryopreserved samples.