Crude biological derivatives competent for nucleic acid detection

Abstract

The invention relates generally to the fields of making biological unit lysates or admixtures of body fluids and of RNA analysis. More specifically, it relates to direct methods for the detection of a specific sequence of RNA in a biological unit, for example a virus, cell or tissue sample, or a body fluid, for example saliva, sputum, blood plasma, etc. More generally, the invention may be used to enzymatically manipulate and protect the RNA in lysate or bodily fluids for a number of applications.

Description

[0001] The government may own rights in the present invention pursuant to grant number R44 HL69718 from National Institutes of Health National Heart, Lung, and Blood Institute.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present invention relates generally to the fields of making biological unit lysates or admixtures of body fluids for RNA analysis. More specifically, it teaches a more direct method for the detection of a specific sequence of RNA in a biological unit, for example a virus, cell or tissue sample, or a body fluid, for example saliva, sputum, blood plasma, etc. More generally, the invention may be used to enzymatically manipulate and protect the RNA in lysate or bodily fluids for a number of applications. [0004] 2. Description of Related Art [0005] There are many molecular biology techniques that can be used to analyze RNA or RNA-containing samples. For example, reverse transcription followed by the polymerase chain reaction (RT-PCR) is one of...

AI Technical Summary

Benefits of technology

[0022] A further advantage of the present invention is that, in some embodiments, it allows for the processing of samples with high cell concentrations. This ability to use high cell concentrations can provide advantages in any of the procedures discussed above or known to those of skill in the art. For example, many prior RT-PCR techniques are limited to the use of samples containing less than 1, 1-10, 30, 40, 50, 60, 75, or 100 cells/μl of buffer. When a sample is added to a reverse transcriptase reaction, the concentration of cellular equivalents of RNA components in the reaction mixture is even less. For example, Gaynor et al. (1996) teach that one can use the 1 to 1000 cells in a 20 μl RT reaction, i.e., 0.05 to 50 cells per μl of reaction. Given standard dilutions that occur in making cell sample lysates or other RNA containing admixtures into reverse transcriptase reaction, most prior techniques begin with reaction mixtures comprising the RNA of less than one to about 50 cells. The buffers and techniques of the present invention certainly work in the context of low cell concentrations. However, they also allow for higher concentrations of cells to be used. For example, using the methods and buffers of the invention, it is possible to make cellular lysates of 5000 cells per μl, 2000 cells per μl, 1500 cells per μl, 1000 cells per μl, 900 cells per ρl, 800 cells per μl, 750 cells per μl, 700 cells per μl, 650 cells per μl, 600 cells per μl, 550 cells per βl, 500 cells per μl, 450 cells per μl, 400 cells per μl, 350 cells per μl, 300 cells per μl, 250 cells per μl, 200 cells per μl, 175 cells per μl, 150 cells per μl, 125 cells per μl, 100 cells per μl, 90 cells per μl, 80 cells per μl, 75 cells per μl, 70 cells per μl, 65 cells per μl, 61 cells per μl, 55 cells per μl, 51 cells per μl, 50 cells per μl, 45 cells per μl, 41 cells per μl, 40 cells per μl, 35 cells per μl, 31 cells per μl, 30 cells per μl, 25 cells per μl, 21 cells per μl, 20 cells per μl, 18 cells per μl, 16 cells per μl, 15 cells per μl, 14 cells per μl, 12 cells per μl, 11 cells per μl, 10 cells per μl, 9 cells per μl, 8 cells per μl, 7 cells per μl, 6 cells per μl, 5 cells per μl, 4 cells per μl, 3 cells per μl, 2 cells per μl, 1 cell per μl, 0.9 cell per μl, 0.8 cell per μl, 0.7 cell per μl, 0.6 cell per μl, 0.5 cell per μl, 0.4 cell per μl, 0.3 cell per μl, 0.25 cell per μl, 0.20 cell per μl, 0.15 cell per μl, 0.1 cell per μl, 0.05 cell per μl, and/or of any concentration range defined by any of these points, or any lower cell concentration. Further, it is possible, according to the invention to make RT-PCR reaction mixtures comprising concentrations of cellular RNA equivalent to 1500 cells per μl, 1000 cells per μl, 900 cells per μl, 800 cells per μl, 750 cells per μl, 700 cells per μl, 650 cells per μl, 600 cells per μl, 550 cells per μl, 500 cells per μl, 450 cells per μl, 400 cells per μl, 350 cells per μl, 300 cells per μl, 250 cells per μl, 200 cells per μl, 175 cells per μl, 150 cells per μl, 125 cells per μl, 100 cells per μl, 90 cells per μl, 80 cells per μl, 75 cells per μl, 70 cells per μl, 65 cells per μl, 61 cells per μl, 55 cells per μl, 51 cells per μl, 50 cells per μl, 45 cells per μl, 41 cells per μl, 40 cells per μl, 35 cells per μl, 31 cells per μl, 30 cells per μl, 25 cells per μl, 21 cells per μl, 20 cells per μl, 18 cells per μl, 16 cells per μl, 15 cells per μl, 14 cells per μl, 12 cells per μl, 11 cells per μl, 10 cells per μl, 9 cells per μl, 8 cells per μl, 7 cells per μl, 6 cells per μl, 5 cells per μl, 4 cells per μl, 3 cells per μl, 2 cells per μl, 1 cell per μl, 0.9 cell per μl, 0.8 cell per μl, 0.7 cell per μl, 0.6 cell per μl, 0.5 cell per μl, 0.4 cell per μl, 0.3 cell per μl, 0.25 cell per μl, 0.20 cell per μl, 0.15 cell per μl, 0.1 cell per μl, 0.05 cell per μl, 0.01 cell per μl, and/or of any concentration range defined by any of these points, or any lower cell concentration. The ability to employ higher cellular concentration has many advantages. For example, if one can use 2, 3, 4, 5, 10, 20, 50, 100, 500, or even 1000 times the concentration of cells and therefore obtain 2, 3, 4, 5, 10, 20, 50, 100, 500, or even 1000 times the concentration of RNA in the RT-PCR reaction mixture, then this can provide a tremendous advantage in terms of speed, lower numbers of cycles, sensitivity, tolerance, and/or robustness of the RT-PCR reaction. These advantages could be significant to overcome any situations where the lysates and/RT-PCR reaction mixtures of the invention might not be as stable, from an RNA standpoint or as efficient from an RT-PCR standpoint, as prior lysates and/or RT-PCR reactions using lower cell concentrations.

[0023] Another advantage of some embodiments of the invention is that they can allow for highly efficient RT-PCR reactions using the methods and buffers of the invention, without the need for RNA isolation or sample preparation steps that require temperature variations and/or protein addition, DNA and/or protein precipitation in the sample, or long incubation times. One manner of measuring the efficiency of a real-time PCR reaction involves the determination of “Ct values.” Ct refers to “Cycle Threshold.” In real-time PCR, the amount of fluorescent signal is monitored after each cycle of PCR. Once the signal reaches a certain level, it has reached the “threshold.” The Ct is the number of cycles of PCR that it took to reach that threshold of fluorescent signal. Thus, the lower the Ct value, the greater the concentration of nucleic acid target. For example, in the TaqMan® assay, typically each cycle almost doubles the amount of PCR product and therefore, the fluorescent signal should double if there is no inhibition of the reaction and the reaction was nearly 100% efficient with purified nucleic acid. In practice, if the Ct value produced by an RT plus reaction is at least 3 Ct values less than the Ct value from an RT minus reaction, the gDNA contribution to the Ct is less than ˜12%. The lower the Ct value, the greater the signal. Thus, if the RT step is contributing cDNA in much greater excess than the gDNA, then you should observe a lower Ct value in the RT plus r

Problems solved by technology

Most other RNA isolation procedures lead to some loss of genomic DNA,