Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample

被引：270

作者：

Marone M. ^{[1
]}

Mozzetti S. ^{[1
]}

De Ritis D. ^{[1
]}

Pierelli L. ^{[2
]}

Scambia G. ^{[1
]}

机构：

[1] Department of Gynecology, Catholic University, 00168 Rome

[2] Department of Hematology, Catholic University, 00168 Rome

来源：

Biological Procedures Online | 2001年 / 3卷 / 1期

关键词：

bcl-2; Gene expression; mRNA; RT-PCR; Technique;

D O I：

10.1251/bpo20

中图分类号：

学科分类号：

摘要：

We describe a semiquantitative RT-PCR protocol optimized in our laboratory to extract RNA from as little as 10,000 cells and to measure the expression levels of several target mRNAs from each sample. This procedure was optimized on the human erythroleukemia cell line TF-1 but was successfully used on primary cells and on different cell lines. We describe the detailed procedure for the analysis of Bcl-2 levels. Aldolase A was used as an internal control to normalize for sample to sample variations in total RNA amounts and for reaction efficiency. As for all quantitative techniques, great care must be taken in all optimization steps: the necessary controls to ensure a rough quantitative (semi-quantitative) analysis are described here, together with an example from a study on the effects of TGF-β1 in TF-1 cells. © 2001 Biological Procedures Online. All rights reserved.

引用

页码：19 / 25

页数：6

共 24 条

[1]

PCR Technology: Principles and Applications for DNA Amplification, (1989)

[2]

Carding S.R., Lu D., Bottomly K.A., A polymerase chain reaction assay for the detection and quantification of cytokine gene expression in small number of cells, J. Immunol. Methods, 151, pp. 277-287, (1992)

[3]

Marone M., Pierelli L., Mozzetti S., Masciullo V., Bonanno G., Morosetti R., Rutella S., Battaglia A., Rumi C., Mancuso S., Leone G., Scambia G., High Cyclin-dependent kinase inhibitors and p53 levels in Bcl-2 and Bcl-xL-expressing primitive proliferating hematopoietic progenitors, Br. J. Haematol., pp. 654-662, (2000)

[4]

Pierelli L., Marone M., Bonanno G., Battaglia A., Mozzetti S., Rumi C., Mancuso S., Leone G., Scambia G., Modulation of bcl-2 and p27 in primitive, proliferating hematopoietic progenitors by autocrine TGF-beta1 is a cell cycle independent effect and influences their hematopoietic potential, Blood, 95, pp. 3001-3010, (2000)

[5]

Marone M., Ferrandina G., Macchia G., Mozzetti S., De Pasqua A., Benedetti-Panici P., Mancuso S., Scambia G., Bcl-2, bax, bcl-xL and bcl-xS expression in neoplastic and normal endometrium, Oncology, 58, pp. 161-168, (2001)

[6]

Marone M., Scambia G., Mozzetti S., Ferrandina G., Iacovella S., De Pasqua A., Benedetti-Panici P., Mancuso S., Bcl-2, bax, bcl-x<sub>l</sub> and bcl-x<sub>s</sub> expression in normal and neoplastic ovarian tissues, Clinical Cancer Res., 4, pp. 517-524, (1998)

[7]

Marone M., Scambia G., Giannitelli C., Ferrandina G., Masciullo V., Bellacosa A., Benedetti-Panici P., Mancuso S., Cyclin e and cdk2 alterations in ovarian cancer: Amplification and overexpression, Int. J. Cancer, 74, pp. 390-395, (1997)

[8]

Kitamura T., Tange T., Terasawa T., Chiba S., Kuwaki T., Miyagawa K., Piao Y.F., Miyazono K., Urabe A., Takaku F., Establishment and characterization of a unique human cell line that proliferates dependently on GM-CSF, IL-3, or erythropoietin, J. Cell. Physiol., 140, pp. 632-634, (1989)

[9]

Sambrook J., Fritsch E.F., Maniatis T., Molecular Cloning. A Laboratory Manual, (1989)

[10]

Wang T.T.Y., Phang J.M., Effects of estrogen on apoptotic pathways in human breast cancer cell line MCF-7, Cancer Res, 55, pp. 2487-2489, (1995)

← 1 2 3 →