Gene expression analysis on a photodiode array-based bioluminescence analyzer by using sensitivity-improved SRPP.
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.
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ABSTRACT: In pyrosequencing chemistry, four cascade enzymatic reactions with the catalysis of polymerase, adenosine triphosphate (ATP) sulfurylase, luciferase, and apyrase are employed. The sensitivity of pyrosequencing mainly depends on the concentration of luciferase which catalyzes a photoemission reaction. However, the side-reaction of adenosine 5' phosphosulfate (APS, an analogue of ATP) with luciferase resulted in an unavoidable background signal; hence, the sensitivity cannot be much higher due to the simultaneous increase of the background signal when a larger amount of luciferase is used. In this study, we demonstrated a sensitive pyrosequencing using a large amount of ATP sulfurylase to lower the concentration of free APS in the pyrosequencing mixture. As the complex of ATP sulfurylase and APS does not react with luciferase, a large amount of luciferase can be used to achieve a sensitive pyrosequencing reaction. This sensitivity-improving pyrosequencing chemistry allows the use of an inexpensive light sensor photodiode array for constructing a portable pyrosequencer, a potential tool in a point-of-care test (POCT).Analytical Chemistry 03/2011; 83(9):3600-5. DOI:10.1021/ac2000785 · 5.83 Impact Factor
- ChemBioChem 04/2011; 12(6):845-9. DOI:10.1002/cbic.201100023 · 3.06 Impact Factor
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ABSTRACT: Streptococcus suis (S. suis for short) can cause a variety of infections in pigs, and the infections have brought about great losses in the swine industry and some cases of deaths in human beings. In order to rapidly diagnose and control the infections of S. suis, we designed a pyrosequencing-based assay to identify the serotypes of S. suis. In the assay, pyrosequencing is used to genotype most of the pathogenic serotypes of S. suis by detecting five informative regions on the Chaperonin 60 (cpn60) gene and one species-specific region on the 16S rRNA gene, and further a few undistinguished serotypes by pyrosequencing were finely discriminated by multiplex PCR of serotype-specific fragments on the cps gene as well as species-specific fragments on the 16S rRNA gene. Through carefully designing the dispensing order of dNTP for each pyrosequencing reaction, the serotypes of S. suis could be discriminated by four pyrosequencing reactions within three hours. Five reference serotypes and three clinical strains were successfully detected and genotyped by our assay. The results indicated that our assay is a reliable, information-rich diagnostic method for the accurate detection ofS. suis serotypes.Analytical methods 11/2011; 3(11):2517-2523. DOI:10.1039/C1AY05285C · 1.94 Impact Factor