Gene Expression Approach Using Residual Cells Feasible for Future CLL Studies

A technique for isolating and preparing RNA for gene expression profiling (GEP) using a small quantity of residual leukemic cells from patients treated for chronic lymphocytic leukemia (CLL) was published in the International Journal of Laboratory Hematology.

A team of researchers aimed to generate an effective gene expression technique to analyze residual leukemic cells, as minimal residual disease (MRD) analysis has value in predicting relapse in CLL. However, studies on residual leukemic cells obtained just prior to relapse have been hindered by the challenges around evaluating small quantities of tumor cells. The analytical approach described in the report involved RNA samples obtained from cell quantities as small as 1 x 10⁴ cells.

The researchers sorted leukocytes from patients with leukemia using a 6-color panel flow cytometry assay and then tested RNA extractions from these cells using either the RNeasy Micro Kit (QIAGEN) or the RNeasy Mini Kit (QIAGEN).

When extracting RNA from a sample of 5 x 10⁵ cells, a significantly greater mean RNA yield was found with the RNeasy Mini Kit than with the RNeasy Micro Kit (P <.01). When extracting from 1 x 10⁴ cells, the RNeasy Micro Kit trended toward providing a greater mean yield of RNA. However, the difference in yield between kits with this smaller quantity of cells was not statistically significant (P =.97).

The researchers also identified a 2-step workflow that showed good performance in providing complementary DNA (cDNA) for gene array analysis, even when RNA was extracted from a small number of cells (1 x 10⁴ cells). This process involved RNA extraction using the RNeasy Micro Kit, followed by cDNA preparation using an Ovation Pico WTA kit (NuGEN Technologies Inc.). The researchers also noted that this process was suitable with samples from both fresh and frozen cellular sources.

“Our results illustrate difficulties and advantages in working with a reduced number of cells and describe the steps of an appropriate protocol to analyze GEP studies from leukemic B cells in the range of 1 x 10⁴ to 5 x 10⁵,” the researchers wrote in their report. They concluded that the processes used in this study could have utility in evaluating MRD in CLL.

Reference

Mora A, Bosch R, Cuellar-García C, et al. Gene expression workflow to analyze residual leukemic cells in chronic lymphocytic leukemia [published online April 25, 2020]. Int J Lab Hematol. doi: 10.1111/ijlh.13215

This article originally appeared on Hematology Advisor