Gene expression analysis on a photodiode array-based bioluminescence analyzer by using sensitivity-improved SRPP

Qinxin Song; Hua Jing; Haiping Wu; Guohua Zhou; Tomoharu Kajiyama; Hideki Kambara

doi:10.1039/C0AN00012D

Gene expression analysis on a photodiode array-based bioluminescence analyzer by using sensitivity-improved SRPP

Qinxin Song,^a^b Hua Jing,^b Haiping Wu,^a Guohua Zhou,*^a^b^c Tomoharu Kajiyama^d and Hideki Kambara^d

* Corresponding authors

^a Huadong Research Institute for Medicine and Biotechnics, Nanjing, China
E-mail: ghzhou@nju.edu.cn
Fax: +86-25-84514223
Tel: +86-25-84514223

^b China Pharmaceutical University, Nanjing, China

^c Medical School, Nanjing University, Nanjing, China

^d Central Research Laboratory, Hitachi, Ltd., 1-280 Higashi-Koigakubo, Kokubunji-shi, Japan

Abstract

Most methods used for gene expression analysis are based on dye-labeling, which requires costly instruments. Recently a dye-free gene expression analysis method-SRPP (Sequence-tagged reverse-transcription polymerase chain reaction coupled with pyrosequencing) was developed to compare relative gene expression levels in different tissues, but the throughput of the SRPP assay is very limited due to the use of a photomultiplier tube (PMT)-based pyrosequencer for the detection. To increase the throughput of the SRPP assay, an inexpensive photodiode (PD) array-based bioluminescence analyzer (termed as “PD-based pyrosequencer”) was coupled to SRPP; however the low sensitivity of PD limited the wide application of SRPP. To enable SRPP analyzing low abundance genes in clinical samples, sequence-tagged gene-specific primers instead of sequence-tagged poly (T)_n primers were used for reverse-transcription, and the SRPP sensitivity was thus improved more than 10 times. This improvement compensates the sensitivity loss due to the use of PD in a pyrosequencer. The accurate determination of the expression levels of ten prognostic marker genes (AL080059, MMP9, EXT1, ORC6L, AF052162, C9orf30, FBXO31, IGFBP5, ESM1, and RUNDC1) differing between normal tissues and tumor tissues of breast cancer patients demonstrated that SRPP using gene-specific RT primers coupled with the PD array-based bioluminescence analyzer is reliable, inexpensive, and sensitive in gene expression analysis.

Supplementary files

Publication details

The article was received on 18 Jan 2010, accepted on 04 Mar 2010 and first published on 24 Mar 2010

Article type: Paper

DOI: 10.1039/C0AN00012D

Citation: Analyst, 2010,135, 1315-1319

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Gene expression analysis on a photodiode array-based bioluminescence analyzer by using sensitivity-improved SRPP

Q. Song, H. Jing, H. Wu, G. Zhou, T. Kajiyama and H. Kambara, Analyst, 2010, 135, 1315
DOI: 10.1039/C0AN00012D
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Gene expression analysis on a photodiode array-based bioluminescence analyzer by using sensitivity-improved SRPP

Abstract

Supplementary files

Publication details

Gene expression analysis on a photodiode array-based bioluminescence analyzer by using sensitivity-improved SRPP

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