Electronic Supplementary Material
DNAmicrospotassayusingsingle-moleculedetectionandrequiring1.8nLsamplesonly
Benhui Sui• Lu Li,Xincang• Li, Jinxing Wang• Xiaoli Zhang• Wenrui Jin
W. Jin*
School of Chemistry and Chemical Engineering,
ShandongUniversity,
27 Shanda Road,
250100 Jinan, China
e-mail: .
Benhui Sui, Xiaoli Zhang
School of Chemistry and Chemical Engineering,
ShandongUniversity,
27 Shanda Road,
250100 Jinan, China
Lu Li,
College of Chemistry, Chemical Engineering and Materials Science,
Key Laboratory of Molecular and Nano Probes,
ShandongNormalUniversity,
88 Wenhua East Road
250014Jinan, China
Xincang Li, Jinxing Wang,
School ofSchool of Life Sciences,
ShandongUniversity,
27 Shanda Road,
250100 Jinan, China
Materials and methods
Reagents and solutions
SA-QDs (2 μmolL-1) with a maximum emission at 655 nm and QD incubation buffer (pH 8.3) from Invitrogen (USA, 3-glycidoxypropyltrimethoxysilane (GOPS, ≥98%), bovine serum albumin (BSA) andTween-20from Sigma (St. Louis, MO, USA, blocking regent from F. Hoffmann-La Roche Ltd., (Germany, Tris, N-lauroyl sarccosine sodium and deionized formamide from Amersco (USA Percoll from Amersham Pharmacia Biotech. Inc., (Piscataway, NJ. USA, diethypyrocarbonate(DEPC) from Bio Basic Inc., (Canada, CellAmpTM whole transcriptome amplification kit, ribonuclease H (RNase H), TaKaRa Ex Taq® (Hot Start Version) and SYBR® Premix Ex Taq™ from TaKaRa Biotech. Co., Ltd. (China, Unizol from Biostar Co., Ltd.(China, and M-MLV reverse transcriptase from Promega (USA, were used in this work.
Thesynthetic DNAs listed in Table S1 andsodium dodecyl sulfate (SDS) were purchased from Sangon Co., Ltd.(China, 1.00× 10-4molL-1 stock solutions for each DNA were prepared by centrifugating for 1 min at 1000 rpm and dissolving in an appropriate volume of TE buffer, and stored at -20 oC. The 1×10-6 molL-1 amino-modified capture DNAs (DNA1s)were obtained by diluting the stock solutions with 0.1 mol L-1 KOH. Otherdilute DNA solutions were obtained by a serial dilution with hybridization buffer.
Other chemicals (analytical grade) were obtained from standard reagent suppliers. To prevent the contamination from the possible repeat sampling, the commercial SA-QD suspension and the stock DNA solutions were divided into several small packs in disinfected plastic vessels. The preparation of the solutions of DNA was performed in a clean bench.
Physiological buffer saline (PBS) consisted of 0.15 molL-1 NaCl, 7.6 × 10-3molL-1 NaH2PO4 and 2.4 × 10-3molL-1 Na2HPO4 (pH 7.4). TE buffer consisted of 0.010 molL-1 Tris-HCl and 0.001 molL-1 Na2EDTA (pH 8.0).PBS-T buffer consisted ofPBSand 0.05% Tween 20. 0.05 molL-1 Tris-HCl buffer (pH=9.0) was prepared by dissolving an appropriate amount of Tris in water and then adjusting the pH value to 9.0 with 0.1 molL-1 HCl. 20×SSC (3 molL-1 NaCl and 0.30 molL-1sodium citrate) was prepared by dissolving an appropriate amount of NaCl and sodium citrate in water and then adjusting the pH value to 7.0 with 0.1 molL-1 HCl.Hybridization buffer consisted of 5×SSC, 50%formamide, 0.02%SDS, 0.1%N-lauroyl sarccosine sodium and 2% (w/v) blocking regent. DEPC treated water was prepared by adding 1 mLof DEPC to 1 L of H2O. After 4 h the DEPC treated water was used.
All aqueous solutions were prepared with doubly distilled water, passed through a 0.22 μm filterand stored at 4 oC.All buffers, disposable plastic wares and disposable micro-pipet tips were disinfected under the pressure of 1.4 kgcm-2 for 20 min in electrothermal-pressure vessel before use, in order to prevent the growth of microorganisms and DNA denaturation. All solutions were prepared in disposable plastic wares using disposable micro-pipet tips.In the experiments of single-cell gene expression, the micropipette tip boxes were immersed in 0.3% H2O2 overnight and the micropipette tips were immersed in DEPC treated water. The glass wares were disinfected for 5 h at 300oC in an oven.
Coverslip silanization
The microspots were fabricated on 0.17-mm-thick glass coverslips (Cole-Parmer Instrument Co.,USA, In order to enhance the adhesive force for amino-modified DNA1, the glass coverslips were silanized with GOPS using the following procedure: The glass coverslips were cleaned by ultrasonication in 30% (v/v) household cleaning liquid (detergent). After removal of the cleaning liquid with tap water, the coversilps were immersed in a chromic acid mixture containing K2Cr2O7 and H2SO4overnight. The coverslips were rinsed with tap water, followed by ultrasonication for 10 min in distilled water, acetone, ethanol, and doubly distilled water, respectively. Then, the coverslips were activated for 10 min in a mixture of 30% H2O2, 37% HCl and doubly distilled H2O (1:1:1 by volume). After cleaning by ultrasonication for 5 min in doubly distilled water three times, the coverslips were placed in a clean box and dried in an oven at 120 oC. The silanization of cleaned coverslips was performed overnight in 10 mmolL-1 GOPS in dry toluene at room temperature. The GOPS-coated coverslips werecleaned by ultrasonication for 5 min in dry toluene, acetone, alcohol and doubly distilled water three times. Finally, the coverslips were dried under a nitrogen flow.
Preparation of DNA1-modified coverslips
Fifty microlitres of 1×10-6 molL-1NH2-group-modified DNA1 was deposited onto a silanized coverslip. The silanized coverslip was covered with a cleaned coverslip and then incubated overnight at 37 oC in a constant-humidity chamber. After the coverslip modified with DNA1 was washed with PBS-T buffer, PBS and doubly distilled water twice, it was dried under a nitrogen flow. In order to block the modified coverslip, 50 μL of 0.05 molL-1 Tris-HCl buffer (pH 9) containing 0.1% SDS and 0.050 molL-1 ethanolamine was dropped onto the modified coverslip. After the coverslip was covered with a cleaned coverslip, it was incubated for 8 h at 37 oC in a constant-humidity chamber. Then, the coverslip was washed with PBS-T buffer, PBS and doubly distilled water twice. Next, 50 μL of PBScontaining 5% BSA was dropped onto the coverslip. After the coverslip was covered with a cleaned coverslip, it was incubated for 2 h at 37 oC in a constant-humidity chamber. Then, the coverslip was washed with PBS-T buffer, PBS and doubly distilled water twice. The coverslip was dried under a nitrogen flow.
Preparation of QD-DNA2
A solution consisting of 10 μLof 2 ×10-9 molL-1 SA-QD, 10 μL of 1.0×10-7molL-1 DNA2 and 200 μL of PBS (pH 7.4) was incubated for 1 h. The resultant QD-DNA2 conjugates were washed with 100μLof PBS(pH 7.4) thrice by ultrafiltering at 5000gusing Nanosep® Centrifugal Devices(MWCO 10 K, Pall Life Sciences, USA, Then, the QD-DNA conjugates were dispersed in 100μLof PBS(pH 7.4) and stored at 4 °C.
Determination of OPN mRNA in DSC extracts by real-time quantitative reverse transcription polymerase chain reaction
In order to determine the amount of OPN mRNA, the total RNA was isolated from DSCs usingUnizol according to the manufacturer’s instructions. Then 1 U of ribonucleic acid (RNase)-free deoxyribonuclease (DNase)and 1 U of RNase-inhibitor wereused todigest genomic DNA and toblock RNase activity by incubating for 30 min at 37 °C.DNase was inactivated by incubating for 5 min at 75 °C. In order to synthesize cDNA, 3 μLaliquot of 0.17 μgμL-1 RNA sample was transferred to a reaction microtube.After 1 μL of 1 × 10-4 molL-1 reverse transcription OPN primer (5-GTTATGTTTTATTAATTGCTGGACAACCGTG-3) was added, the solution was incubated for 5 min at 70oC, followed by cooling on ice. The synthesis of double-stranded RNA-DNA hybrids consisting of OPN mRNA and corresponding cDNA was initiated by adding 21 μL of the solution consisting of 5 μL of RT buffer, 1.25 μL of 10 mmolL-1 dNTP mixture, 0.625 μL ofrRNAsin (RNase-inhibitor), 1 μL of M-MLV reverse transcriptase and 13.125 μL of RNase-free H2O and incubating for 1 h at 42 oC. The reaction was terminated by a 5-min incubation at 85oC.The cDNA was quantified by real-time quantitative reverse transcription polymerase chain reaction(rtqRT-PCR). To do so, 2 μL of the diluted sample or standard plasmid DNA, 10 μL of 2×SYBR Premix Ex Taq, 4 μL of 1 × 10-6 molL-1forward primer (5-TGACCAGAGTGCTGAAACCCA-3) and4 μL of 1 × 10-6 molL-1 reverse primer (5-CTTTTGGGGTCTACAACCAGCATA-3) were mixed. After a denaturationstep at 95oC for 10 min, the cycle profile used was 15 s at95oC, 20 s at 59 oC, and 20 s at 72oC for 40 cycles of amplification.The solution was used to detect fluorescence intensity to quantify OPN mRNA in the cell extract using Chromo4 real-time PCR detection system (Bio-Rad Laboratories, Inc., USA,
Table S1 Sequence of synthesized single-strandedDNA used in this work.
Single-stranded DNA / SequencesDNA1
tDNA associated with cDNA sequence corresponding to OPN mRNA in DSCs / NH2-5′-T10 GGA CCT GCC AGC AAC CGA-3′
5′-CCTGGACGGTCGTTGGCTTCAAAAGTGAGGTCAACAGGGGT-3′
DNA2 / 5′-TTTTCACTCCAGTTGTCCCCAT-3′-biotin
Reverse transcription OPN mRNA primer in single-cell mRNA analysis / 5-GTTATGTTTTATTAATTGCTGGACAACCGTG-3
Noncomplementary DNAsin the interferenceexperiments / 5′-CTC CAA ATG TAG GAG CTA TCG TT-3′
5′-TAC TTG GAG CCA CTA TCG ACT ACG C-3′
5′-GGC AAG CCG ATA ACG GGA TTA-3′
5′-AAG CCA TGA AGC GGC TTA TGA TTC TTA CCG CCC ACT-3′
5′-GCG AGG ATT TGA CGA AAG CGC ACC TTA AAG-3′
1