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In vivo experimentation of pharmacological products mandates an accurate analysis of the cellular function and gene expression to determine efficacy and safety. The expression of a particular gene is often an indicator ofthe efficacy ofthe drug product. PCR was originally disclosed and claimed by Mullis et al.

Since that time, considerable advances have been made in the reagents, equipment and techniques available for PCR. These advances have increased both the efficiency and utility ofthe PCR reaction, leading to its adoption in an increasing number of different scientific applications and situations.

PCR was used to determine if a given DNA sequence was present in any quantity at all or to obtain sufficient quantities of a specific nucleic acid sequence for further manipulation. Only in recent years has quantitative PCR come to the forefront of nucleic acid research. There are many steps in the pathway leading from DNA to protein and all of them can in principle be regulated.

A cell controls the proteins its makes by: Molecular Biology of the Cell, 3 rd Ed. Although all of these steps involved in expressing a gene can in principle be regulated, for most genes, transcriptional controls are paramount and the initiation of RNA transcription is the most important point of control. Therefore, mRNA is purified and cDNA clones produced to measure gene expression in the experimentation of pharmacological products. The two-step protocol is prone to contamination because ofthe need to expose the samples to air while adding reagents.

RNA degrades when exposed to heat or high pH. RNA degradation by alkaline hydrolysis is accelerated by heat. If RNA is degraded, an inaccurate analysis may result. Hence, maintaining RNA at a low temperature minimizes degradation.

When this occurs, the yield and specificity of PCR is decreased at least partially due to the priming or mis-priming of sequences. Hence, premature taq polymerase activity provides inaccurate results in the analysis of genetic expression. Hence, following purification of the mRNA or DNA, an automated liquid handling device is often used to add reagents to the biological sample receptacle reactions to maintain accuracy and eliminate repetitive injury to researchers.

Scientific Tango Liquid Handling System. These devices are able to transfer reagents from one location to another according to a pre-programmed pattern. However, the refrigerated table is not satisfactory for maintaining the sample at a sufficient temperature to preserve the activity of the enzyme and avoid degradation of mRNA.

The racks are not designed to maintain low temperature. Therefore, the cooling effect ofthe refrigerated table is dissipated and certain enzymes added to the sample receptacles lose activity. In addition, most available sample racks are not designed for use on an automated liquid handling device. This type of device is simply a cooler and is not subject for use in an automated liquid handling device. Even with the cumbersome equipment set up, heat transfer is likely to occur through the well containing the sample receptacle.

Still other devices such as centrifuge rotors require a refrigeration system that maintains the entire chamber at a given temperature United States Patent No. Each well has an open cylindrical upper end and a closed conical lower end. Each well is design to accommodate a biological sample receptacle.

The receptacle has substantially the same shape as the well, thereby maintaining the temperature of a biological sample in the receptacle during sample set up and prior to polymerase chain reaction. Use of the metal block in an automated liquid handling device provides an improvement to liquid handling systems currently available. In a nucleic acid amplification device, the sample is then caused to undergo reverse transcriptase polymerase chain reaction to determine the presence of RNA or DNA.

The metal block is first chilled and then fixed into position on an automated liquid handling device. The metal block and the liquefied biological sample temperature is. The typical handling device is adapted to transfer, dispense and aspirate liquid from one location to another automatically and is capable of a wide range of bioanalytical procedures including sample pipetting, serial dilution, reagent additions, mixing reaction timing and similar known manual procedures.

The typical handling device includes table for supporting microtiter plates and other biological sample receptacles, a pod for transferring fluid to a well located on the table and a means for moving the pod relative to the table between selected locations on said table. The improvement to the liquid handling device is use of the metal block having a plurality of wells, each well having an open cylindrical upper end and a closed conical lower end.

Each well accommodates a biological sample receptacle having substantially the same shape as the well and the temperature of a biological sample in the receptacle during sample set-up and prior to polymerase chain reaction analysis is maintained. FIGURE 4 is a perspective view of a liquid handling device suitable for use in connection with the subject invention. Each well 12 has an open cylindrical upper end 14 and a closed conical lower end Each well 12 is designed to accommodate a biological sample receptacle The receptacle 18 has substantially the same shape as the well, thereby maintaining the temperature of a biological sample in the receptacle during sample set up and prior to polymerase chain reaction.

Use of the metal block with an automated liquid handling device 20 and for genetic analysis of biological samples provides an improvement to liquid handling systems currently available.

Here, the biological sample is inserted into the biological sample receptacle 18 as held by the wells 12 of the metal block 10 in the automated liquid handling device Subsequently, reverse transcriptase polymerase chain reaction is used to determine the presence of RNA or DNA in the sample via a nucleic acid amplification machine. The handling device 20 controls dispensing, aspirating and transferring of liquid from a first microtiter plate well or other biological sample receptacle to a second microtiter plate well or other second biological sample receptacle.

The automated liquid handling device is capable of functioning with test tubes, freezing vials, reservoirs and other wet chemistry containers. The improvement to the liquid handling device comprises use ofthe metal block 10 comprising a plurality of wells 12 where each well 12 has an open cylindrical upper end 14 and a closed conical lower end Each well 12 accommodates a biological sample receptacle 18 having substantially the same shape as the well The biological sample and reagents are pipeted into the receptacle 18 and the temperature of a biological sample during sample set-up and prior to polymerase chain reaction analysis is maintained.

RNA laboratory is provided. Such method includes the steps of cl illing the metal block, inserting the biological sample receptacle into the metal block, positioning the metal block onto an automated liquid handling device and transferring the biological sample into biological sample receptacle in the metal block for polymerase chain reaction analysis. Any material with having high thermal conductivity may be suitable for use in the present invention.

Biological sample receptacles may be made of plastic or glass. Frequently, biological sample receptacles are plastic and are made of polypropylene or polycarbonate. Thin- walled tubes and plates are preferred as they allow rapid and consistent heat transfer. Tube volume capacity may range from approximately 0.

Volume capacities of individual microplate tubes vary from approximately 0. The tissue from which the RNA originated may include, but are not limited to, epithelial, connective, muscular, and nerve tissues. It is important that RNA purification is done by a method that mimmizes degradation.

The researcher analyzing the results of gene expression must collect and analyze animal tissues as quickly as possible, beginning at the time the animal is euthanized and the organs harvested. Other devices for purification include but are not limited to the Qiagen BioRobot or RNA may also be purified through precipitation reactions using phenol based products, isopropyl alcohol and lithium chloride.

Also, available is a product known as Nucleopin by BD Biosciences. As an alternative, some thermostable DNA polymerases such as Thermus thermophilus DNA polymerase have reverse transcriptase activity in the presence of manganese, allowing for the use of only one enzyme for reverse transcription and polymerase chain reaction.

If bicine buffer with manganese is used, intermediate additions between reverse transcription and amplification are not needed and stability at elevated temperatures is not a concern. However the presence of manganese may reduce the fidelity of nucleotide incorporation. Therefore, this method is not suitable for a high throughput RNA analysis.

As described in more detail below, other reagents may include, but are not limited to, ohgonucleotide primers, a thermostable DNA polymerase and an appropriate reaction buffer such as mM KC1, mM Tris-HCL 0. These devices are able to transfer reagents from one location to another according to a preprogrammed pattern.

The refrigerated table designed to maintain sample temperature table is not satisfactory for maintaining the sample at a sufficient temperature to preserve the activity ofthe enzyme. In this liquid handling system, microtiter plates, tip support plates, and troughs are supported in a table attached to the laboratory workstation base.

Movement of the table is provided by a motor means causing the table to reciprocally move in at least one axis. A modular pod suspended above the table has an arm attached at one end for movement up and down a vertically extending tower rising from the base of the workstation. The pod is capable of motion along the arm in at least a second axis that is perpendicular to the first axis of movement of the support table.

The arm moves up and down in a third direction perpendicular to both the first and second directions. Fluid is dispensed using interchangeable modules of one or more nozzles. The nozzles have pipettor tips affixed to them that are automatically picked up and ejected by the pod. The table 24 acts as a surface for supporting the metal block, biological sample receptacles, reagent reservoirs and pipettor tips.

The pod 28 is capable of movement horizontally and vertically. The temperature of the table 24 is controllable and is achieved through the use of one or more circulating water baths. The device software allows the user to specify the location of the aspiration, dispensation and mixing, what type of labware the liquid is being aspirated from and into and the volume and height of the aspiration and dispensation. The Qiagen BioRobot is a nucleic acid purification and liquid handling workstation.

It has robotic handling, automated vacuum and a buffer delivery system. Sample receptacles and reagent troughs are present on a platform and an 8 channel pipetting system performs high-speed dispensing. The Qiagen BioRobot is an automated liquid handling and sample processing workstation. It allows the integration of other hardware, such as cyclers or spectrophotometers. It has fully automated plate processing by transferring labware to various positions on and off of the worktable, as well as temperature control, small volume liquid handling and customizable processing parameters.

The Qiagen BioRobot is an automated workstation for nucleic acid purification, reaction set-up, PCR product cleanup, agarose-gel loading and sample rearray and has a worktable and programmable pipetting mechanism. The Gilson Constellation Liquid Handler has a bed that can hold up to 12 microplates, a robotic gripper arm, capability to dispense nanoliter volumes and an optional heating and cooling recirculator.

The Zymark Sciclone ALH Workstation has a 20 position deck, bulk dispensing capabilities to microplates by syringe or peristaltic pump and can pipet using a single channel, 8 channel, 12 channel or 96 channel head. The Robbins Scientific Tango Liquid Handling System comprises a worktable and automated aspiration and dispensing of liquid in a 96 or well format. These devices are able to transfer reagents from one location to another according to a pre-programmed pattern and may be suitable for use in connection with the present invention.

The metal block 10 having been previously refrigerated or frozen is fixed into position on an automated hquid handling device Biological sample receptacles 18 are then inserted into the metal block As the temperature of the liquefied biological sample is maintained, reagents are added to the liquid biological sample for polymerase chain reaction analysis. Reagents are added into the biological sample receptacles 18 by the automated liquid handling device.