Methods of assaying RNA from fetal extravillous trophoblast cells isolated from a maternal endocervical sample

Patent metadata

Field	Value
Publication number	US-10330680-B2
Application number	US-201515517882-A
Country	US
Kind code	B2
Filing date	Oct 12, 2015
Priority date	Oct 10, 2014
Publication date	Jun 25, 2019
Grant date	Jun 25, 2019

Abstract

Methods of isolating and assaying fetal extravillous trophoblast cells, including assays of RNA of the fetal extravillous trophoblast cells according to aspects of the disclosure include obtaining a maternal endocervical sample containing fetal extravillous trophoblast cells from a pregnant subject; fixing the maternal endocervical sample in an aldehyde fixative, removing fetal extravillous trophoblast cells from the maternal endocervical sample thereby producing isolated extravillous trophoblast cells, isolating and assaying RNA from the fetal extravillous trophoblast cells.

First claim

The invention claimed is:

1. A method of assaying RNA from fetal extravillous trophoblast cells, comprising:
obtaining a maternal endocervical sample from a pregnant subject;
removing fetal extravillous trophoblast cells from the maternal endocervical sample, producing isolated fetal extravillous trophoblast cells;
fixing fetal extravillous trophoblast cells by treatment with an aldehyde fixative producing aldehyde fixed fetal extravillous trophoblast cells;
washing the aldehyde fixed fetal extravillous trophoblast cells or washing a lysate of the isolated aldehyde fixed fetal extravillous trophoblast cells to promote removal of the crosslinks introduced by the aldehyde fixative, producing washed fetal extravillous trophoblast cells or washed lysate;
extracting fetal extravillous trophoblast cell RNA from the washed fetal extravillous trophoblast cells or washed lysate, thereby isolating the fetal extravillous trophoblast cell RNA from DNA; and
assaying the fetal extravillous trophoblast cell RNA to determine one or more characteristics of the fetal extravillous trophoblast cell RNA.

2. The method of claim 1 , wherein the treatment with the aldehyde fixative is performed prior to and/or following removing fetal extravillous trophoblast cells from the maternal endocervical sample.

3. The method of claim 1 , wherein the maternal endocervical sample is fixed immediately after obtaining the sample from the pregnant subject.

4. The method of any of claim 1 , wherein the maternal endocervical sample is fixed in an alcohol fixative immediately after obtaining the sample from the pregnant subject and prior to fixing fetal extravillous trophoblast cells by treatment with the aldehyde fixative producing aldehyde fixed fetal extravillous trophoblast cells.

5. The method of any of claim 1 , wherein the maternal endocervical sample is fixed by treatment with the aldehyde fixative immediately after obtaining the sample from the pregnant subject.

6. The method of any of claim 1 , wherein assaying the fetal extravillous trophoblast cell RNA comprises sequencing, PCR, quantitative PCR, real-time PCR or a combination of any two or more thereof.

7. The method of claim 1 , wherein the pregnant subject is human.

8. The method of claim 1 , wherein removing fetal extravillous trophoblast cells from the maternal endocervical sample comprises contacting the fetal extravillous trophoblast cells with an antibody specific for the fetal extravillous trophoblast cells, wherein the antibody does not bind to maternal cells in the maternal endocervical sample.

9. The method of claim 8 , wherein the antibody specific for the fetal extravillous trophoblast cells is attached to a support.

10. The method of claim 9 , wherein no Protein A or Protein G is attached to the support.

11. The method of claim 9 , wherein the support is a plurality of magnetic particles.

12. The method of claim 11 , wherein removing fetal extravillous trophoblast cells from the maternal endocervical sample comprises exposure of the magnetic particles to a magnet.

13. The method of claim 1 , wherein the maternal endocervical sample is not treated with a mucolytic agent.

Assignees

• Univ Wayne State

Inventors

• Armant D Randall
• Drewlo Sascha

Classifications

• G01N33/54326

  Magnetic particles · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• G01N33/56977

  HLA or MHC typing · CPC title

• G01N2001/305

  Fixative compositions · CPC title

• C12Q1/6883

  for diseases caused by alterations of genetic material · CPC title