Method of preparing cell lysate
AssigneesHitachi Chemical Company, Ltd., Hitachi Chemical Research Center, Inc.
InventorMasato Mitsuhashi
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Abstract
The entire process of reverse transcription-polymerase chain reaction (RT-PCR) is simplified by using oligonucleotide-immobilized microplates made of, e.g., polypropylene, to which oligonucleotides are securely immobilized and which can be subjected to thermal cycles of PCR. RT-PCR is preferably conducted in solid-phase. Capturing of mRNA and RT-PCR can be conducted in the same plates. The cDNA synthesized from the mRNA captured on the microplates can be used more than once. Further, in combination with the microplates, a filter plate is used for the preparation of cell lysates wherein target cells are placed on the filter plate, and a lysis buffer is passed through the cell layer on the filter to transfer cell lysate directly to the microplate via well-to-well communication.
Claims2
A method of preparing cell lysate from target cells comprising nuclei, said lysate comprising cytosolic RNA, but substantially free of nuclear mRNA, said method comprising:
- transferring the target cells to a filter plate provided with a membrane having a pore size such that the target cells are trapped, but cytosolic mRNA present in the cells can pass therethrough, wherein the target cells placed on the membrane form a cell layer;
- and
- passing a lysis buffer through the cell layer on the membrane to obtain the cell lysate of the target cells, wherein the lysis buffer comprises a mild detergent for destructing cell membranes but maintaining nuclei to be intact and a reagent for inhibiting RNase activity or inactivating RNase, said lysis buffer having a pH and salt concentration for hybridization.
Dependencies are inferred from the claim text.