Method and formulation for lyophilizing cultured human cells to preserve RNA and DNA contained in cells for use in molecular biology experiments

#Not applicable.

#The present invention relates to methods for lyophilizing eukaryotic cells and isolating intact nucleic acids from such cells, and kits for using the same.

#Methods of lyophilizing or “freeze-drying” viruses, biologically active molecules, and bacteria are routinely used in laboratories. Typically, the cells or molecules to be lyophilized are suspended in a solution that allows recovery of the desired activity after the freezing process. Ideal formulations for lyophilization solutions should provide a stabilizing environment for a finite time before lyophilization; provide good thermal and freezing properties during lyophilization; and cryoprotect the desired activity.

#FIG. 1 shows a picture of an ethidium bromide stained gel with RNA isolated from cell samples that were lyophilized in DEPC treated 1×PBS. Lanes N1 and N2 contain RNA from HeLa S3 samples stored at −20° C. for 3 months; lanes P1 and P2 contain RNA from SAM-1 samples stored at −20° C. for 6 months; and lanes P3 and P4 contain RTNA from SAM-1 samples stored at 25° C. for 2 days, which is equivalent to storage for 2 years at −20° C. (see stressed stability calculation below). This gel was scanned using a densitometer to determine the ratio of the 28S rRNA band to the 18S rRNA band.

#FIG. 2 shows a diagram of PCR primers used for amplification of the BCR/ABL fused mRNA, β-2 microglobulin mRNA, and CAT mRNA.

#I. Introduction

#This invention provides for a nucleic acid stable preparation of lyophilized cell for diagnostic kits. When cells are lyophilized in an isotonic solution, the nucleic acid is expected to be protected from all nucleases because the cell membrane reportedly maintains its integrity in the isotonic solution. We discovered that the nucleic acid rapidly degrades in cells lyophilized using the isotonic solutions of the prior art. Contrary to expectations, endogenous cellular nucleases are not significantly degrading the cellular nucleic acid. We surprisingly discovered that despite the use of an isotonic solution, the cells were rendered permeable to exogenous nucleases just prior to or during the short, cold lyophilization procedure. This problem of exogenous nucleases in the lyophilization buffer was solved by using lyophilization solutions that are nuclease free.

#Pathologists often use molecular biological approaches to aid in the diagnosis of diseases such as cancer, genetic diseases, and infectious diseases. In addition, investigators in the field of forensics also use DNA probe and molecular analysis for criminal investigations and paternity determinations. This molecular analysis often takes the form of DNA or RNA isolation from eukaryotic cells, typically followed by amplification and gel electrophoresis. A major problem for these experiments is the lack of a standard cellular source for positive and negative nucleic acid controls. This invention provides lyophilized eukaryotic cells for use as controls in diagnostic and forensic analysis. These cells are lyophilized in an isotonic solution that is nuclease free because it has been treated with a nuclease inhibitor, or because it contains a nuclease inhibitor. This method of lyophilization provides a means of long term storage of the cells and allows isolation of intact nucleic acid from the cells, e.g., for use as controls in molecular biological assays.