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1 ISSN CN / Q http / / cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology ~ 388 PCR htert mrna * human telomerase reverse transcriptase htert SKOV3 htert mrna htert LV4-pGLV-EF1a-EGFP-hTERT SKOV3 htert SKOV3 h SKOV3 b SKOV3 PCR 50 Ct P < 0 05 SKOV3 h htert mrna SKOV3 b SKOV3 P < 0 05 SKOV3 htert mrna htert PCR htert Q291 Q78 Detection of mrna Expression of htert at Single Cell Level by Real-time PCR LI Nan 1 HUANG Li 2 HE Bing 2 ZHONG Yan-Ping 3 LIN Ruo-Yun 1 LI Li 1 XUE Lin-Tao 2 WANG Shi-Kai 2 1 LI Dan-Rong 3 * 1 Department of Gynecologic Oncology Affiliated Cancer Hospital Guangxi Medical University Nanning 2 Center for Reproductive Medicine and Genetic The People s Hospital of Guangxi Zhuang Autonomous Region Nanning 3 Scientific Experiment Center Guangxi Medical University Nanning China China China Abstract The expression of human telomerase reverse transcriptase htert defines the cellular activity of telomerase A number of studies have shown that telomerase closely related to tumorigenesis and cell immortalization To detect the htert mrna expression at single cell level SKOV3 cells infected with lentiviral vector LV4-pGLV-EF1a-EGFP-hTERT SKOV3h was used and compared with empty vector infected SKOV3b and non-infected SKOV3 cells Less than 50 cells were sorted by flow cytometry for each group and the htert mrna expression were detected by real-time PCR The results showed that the Ct value was increased as the cells increased P < 0 05 The htert mrna No * Tel danrongli@ 163 com Received November Accepted January Supported by Natural Science Foundation of Guangxi Zhuang Autonomous Region No * Corresponding author Tel danrongli@ 163 com

2 level of SKOV3h was higher than those of SKOV3b and SKOV3 The method was able to detect the htert expression of a single blastomere thus can be useful in applications of gene diagnosis for oncology research Key words single cell real-time PCR human telomerase reverse transcriptase htert flow cytometry polymerase chain reaction PCR DNA RNA PCR DNA PCR 80 Jeffreys 1 PCR DNA Kumar 2 PCR PCR Elerwin 3 RT- PCR 1 RT-PCR 10 5 ~ mrna RT-PCR SKOV3 SKOV3 h SKOV3 SKOV3 b 3 RNA TPI htert 4 5 BECKMAN AmpliGrid AG480F slide BECKMAN ABI7500 PCR Sensiscript Reverse htert Transcription QIAGEN htert Brilliant II SYBR Green Mix 7-AAD RNA DNA BECKMAN htert mrna NM_ Primer Premier 5 0 htert PCR 20 μl PCR SKOV3 htert SKOV3 htert htert SKOV3 3 BD AmpliSpeed slide cycler SIGMA GAPDH Table 1 Table 2 The sequence of PCR primers for htert and GAPDH Genes Forward primers Reverse primers Amplified fragments / bp htert 5'-CTCCCATTTCATCAGCAAGTTT-3' 5'-CTTGGCTTTCAGGATGGAGTAG-3' 96 GAPDH 5'-GAAGGTGAAGGTCGGAGT-3' 5'-GAAGATGGTGATGGGATTTC-3' 226

3 4 PCR htert mrna SKOV3 10% 100 U / m L 100 m g / ml RPMI % CO 2 2 d PCR 1 80% 1 8 PCR 1 4 SKOV PBS 1mL 0 25% 1 AmpliGrid AG480F slide SKOV3 5 min cdna PCR PCR 1 μl cdna r / min min PBS 2 ~ 3 1 ~ 2 ml PBS 3 ~ 5μL 7-1 PBS PBS AAD 20 min AmpliGrid AG480F slide 3 SPSS ± 5 3 "x ± s SKOV3 h SKOV3 b SKOV3 5 P < cdna QIAGEN 2 1 PCR 1 μl 0 1 μl μl dntp Mix 5 μmol / L each dntp 0 1 μl Oligo dt 10 μmol / L 0 05 μl RNase 10 units / μl 0 05 μl Sensiscript 0 6 μl RNase-free water min AmpliGrid AG480F slide 1 μl 5 μl Ct AmpliSpeed slide cycler Ct 3 μl RNase-free water 3 μl 0 2 ml EP PCR htert mrna 20 μl 10 μl 2 Brilliant II SYBR Green Mix 3 μl cdna 0 2 μl htert sense 0 2 μl htert antisense RNase-free water 2 Brilliant II SYBR Green Mix min s 60 1 min μl 0 5 μl cdna 1 μl 2 μl 3 μl cdna 1 μl 2 μl 3 μl 0 2 μl 0 5 μl μl PCR 1 5% 0 8 μl μl PCR Ct Ct Fig 1 20 μl PCR 10 μl 2 Brilliant II SYBR Green Mix 3 μl cdna 0 2 μl htert sense 0 2 μl htert antisense RNase-free water cdna 2 2 htert Ct cdna PCR cdna F = P = < 0 05 Ct

386 29 Fig 1 Melting curve and amplification curve of different number of cells after real-time quantitative PCR Different number of SKOV3 cells were sorted by flow cytometry onto AmpliGrid AG480F

4 Fig 1 Melting curve and amplification curve of different number of cells after real-time quantitative PCR Different number of SKOV3 cells were sorted by flow cytometry onto AmpliGrid AG480F slide The different number were The cells were reacted with reverse transcription reagents in AmpliSpeed slide cycler Then 3 μl of the reverse transcription products could react as templates for real-time PCR repeated 3 times respectively Melting curve A and amplification curve B showed that only cells were detected by effective amplification The graph represented one of three independent experiments 50 Ct P < Ct P = > Ct P = > 0 05 Table 2 Table 2 Different number of cells of htert gene amplification in Ct value Cell number Ct mean ± ± ± * ± * * P < 0 05 compared with the Ct value of 5 cells PCR GAPDH PCR 2 4 htert mrna 50 Fig 2 Electrophoresis analysis of the real-time PCR products The htert mrna expression of cells were determined by real-time PCR 5 μl PCR products were run on 1 5% agarose gel electrophoresis 1-4 showed htert gene in cells 5-8 showed GAPDH gene in cells M Gradient DNA standard 9 Control 2 3 PCR SKOV3 h htert mrna SKOV3 b SKOV3 cdna PCR 5 μl PCR 1 5% SKOV3 h SKOV SKOV3 b SKOV P < 0 05 PCR Fig 3 Fig htert 2 5 PCR PCR 1 μl cdna μl 0 5 μl 0 6 μl 0 7 μl 0 8 μl 6 Ct F = P = < 0 05 PCR SKOV3 Ct Table 3 htert mrna 0 2 μl Ct PCR P < μl Ct SKOV3 SKOV3 h SKOV3 b 5

5 4 PCR htert mrna 387 Fig 3 Real-time PCR analysis of htert mrna expression in different groups of SKOV3 cells Every 5 cells were sorted by flow cytometry in different groups respectively htert mrna expression was detected by realtime PCR From left to right the SKOV3 untreated group SKOV3 SKOV3 infected with htert vecor group SKOV3h SKOV3 infected with empty vector group SKOV3b htert mrna expression of the htert vector infected group was the highest than that of empty vector infected group The untreated group was the lowest The graph and data represented one of three independent experiments * P < 0 05 vs SKOV3 group Ct P = > μl 2 6 PCR 100% 10 1 Ct % 9 Ct 3 htert RNA DNA 9 htert mrna htert SKOV3 mrna htert PCR 1 1 PCR htert mrna 30 PCR Table 3 Ct value of different primer contents of single blastomere after real-time PCR Primer μl Ct value ± ± * ± * ± * ± * * P < 0 05 compared with the Ct value of 0 2 μl primer PCR 15 ~ 20 μl 1 20 μl PCR fluorescence activated cell sorting FACS PCR laser capture microdissection LCM 14 DNA PCR 2 40 ~ PCR PCR 1

6 DNA 10% PBS cells J tip AmpliGrid AG480F slide PCR RNA RNase 30 min Sci References 1 Jeffreys A J Wilson V Thein S L Hypervariable minisatellite regions in human DNA J Nature Kumar R Barbacid M Oncogene detection at the single cell level J Oncogene Eberwine J Yeh H Miyashiro K et al Analysis of gene expression in single live neurons J Proc Natl Acad Sci U S A Kim N W Piatyszek M A Prowse K R et al Specific association of human telomerase activity with immortal cells and cancer J Science Meeran S M Patel S N Tollefsbol T O Sulforaphane causes epigenetic repression of htert expression in human breast cancer cell lines J PLoS One e Bodnar A G Ouellette M Frolkis M et al Extension of lifespan by introduction of telomerase into normal human cells J Science Counter C M Meyerson M Eaton EN et al Telomerase activity is restored in human cells by ectopic expression of htert hest2 the catalytic subunit of telomerase J Oncogene Wen J Cong Y S Bacchetti S Reconstitution of wild-type or mutant telomerase activity in telomerase-negative immortal human Hum Mol Genet Stampfer M R Yaswen P Human epithelial cell immortalization as a step in carcinogenesis J Cancer Lett J Li J Xie C Pei XT Recent advance of studies on telomerase function in stem cells J Prog Physiol Asami M Nakatsuka T Hayashi T et al Cultivation and characterization of planarian neuronal cells isolated by fluorescence activated cell sorting FACS J Zoolog Sci Anderson S Bankier A Barrell B G et al Sequence and organization of the human mitochondrial genome J Nature Maryanski J L Attuil V Bucher P et al A quantitative single-cell PCR analysis of an antigen-specific TCR repertoire selected during an in vivo CD8 response direct evidence for a wide range of clone sizes with uniform tissue distribution J Mol Immunol Bhattacherjee V Mukhopadhyay P Singh S et al Laser capture microdissection of fluorescently labeled em bryonic cranial neural crest cells J Genesis PCR J Kuang XY Chen F Guo J L et al Laser capture microdissection-single-cell PCR technology applied research J Acta Acad Med Mil Tert Pfaffl M W A new mathematical model for relative quantification in real-time RT PCR J Nucleic acids Res e45

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