Reports
Digital PCR For dPCR analysis, half (2.5 µL, in the case of rps18) or all (for all other genes) of the RT reaction mixture having different numbers of synthetic spike-in templates (s_rps18: 5′- TGTGCCGCCGCCA- TGTCCCTAGTGATCCCCGA- GAAGTTTCAGCACATCCTGC- GAGTACTCAACACC-3′), 2 µL TaqMan Assays (inventoried, hspb1: Rn00583001_ g1; hmox1: Rn01536933_m1; slc2a4: Rn01752377_m1; see other probes above; Life Technologies), 10 µL OpenArray Digital PCR Master Mix (Life Technologies) and nuclease free water (2–4.5 µL) were mixed in a total volume of 20 µL. Te mixture was evenly distributed on an OpenArray plate. RT mixes were loaded into 4 wells of a 384-well plate from which the OpenArray autoloader transferred the cDNA master mix by capillary action into 256 nanocap- illary holes (4 subarrays) on an OpenArray plate. Processing of the OpenArray slide and cycling in the OpenArray NT cycler were done as previously described (12, 16). Data analysis was performed using the Biotrove OpenArray Digital PCR Soſtware version 1.0 (download at www.appliedbiosystems. com / Support / OpenArray Digital PCR Soſtware), which uses a proprietary calling algorithm to estimate the quality of each individual threshold cycle (CT calculating a CT
) value by confidence value for the
amplification reaction. For our dPCR protocol amplification, reactions with CT
as reactions having CT
Reverse: 5′-TGTGTGGAATTGTGAGCG- GGCAGCAGAGAGGGAGAAGAG-3′
confidence values below 100 as well values less than 23
or greater than 33 were considered primer dimers or background signals, respectively, and excluded from the data set. Statistical analysis of expression data from hydrogen peroxide treated cells was performed with an unpaired t-test.
Sequencing We sequenced PCR products from individual neurogliaform and pyramidal cells using capillary electrophoresis sequencing on a 3500 Genetic Analyzer (Life Technologies). Aſter single cell PCR, we reamplified 10 µl of the reaction mixture with primers having a universal sequencing tag at the 5′-end of the forward primers and an opseq sequencing tag at the 5′-end of the reverse primers (see italicized letters in the sequences) with the following primers:
rps18
Forward: 5′-GCCAGGGTTTTCCCAGT- CACGACACGCCGCCGCTTGTGCC-3′ Reverse: 5′-TGTGTGGAATTGTGAGCG- GTGGTGTTGAGTACTCGCAGG-3′
gabrd Forward: 5′-GCCAGGGTTTTCCCA- GTCACGACAAGGTCACGAAGC- CAAGG-3′
hspb Forward: 5′-GCCAGGGTTTTCCCA- GTCACGAGGATGAACATGGCTA- CATCTC-3′ Reverse: 5′-TGTGTGGAATTGTGAGCG- GCACCGTGAGTGTGCCCTCAG-3′
hmox-1 Forward: 5′-GCCAGGGTTTTCCCA- GTCACGAGGGTGACAGAAGAG- GCTAAG-3′ Reverse: 5′-TGTGTGGAATTGTGAGCGG- GACTCTGGTCTTTGTGTTCC-3′
slc2a4
Forward:5′-CCGTCGGGTTTCCAGCAG-3′ Reverse: 5′-CAGCAAGGACCAGTGTCCCAG-3′
After purification of the products, we
used different universal and opseq sequencing primers. Sequence homology searches were performed using the BLASTN algorithm (
www.ncbi.nlm.nih.gov/Blast.cgi).
Results and discussion
Figure 1. Application of spike-in templates for dPCR analysis. (A) Synthetic rps18 templates (s_rps18) diluted to a range of 0–81 copies per reaction (see values above the picture, n = 2) was analyzed using the Openarray dPCR platform. Resulting positive calls are represented by orange or red dots (values are shown below the picture). (B) dPCR amplification of slc2a4 was carried out either with the entire cellular contents of one cell (cell#1-cell#4, n = 4) or one third of a single cell RT reaction volume (RT#1-RT#3, n = 3). Spike-in s_rps18 dPCR amplification were done from the same single cell RT reaction from which slc2a4 was amplified (RT#1-RT#3, n = 3). The number of positive reactions from individual dPCRs are shown below the picture. The negative control is labled negc.
Vol. 54 | No. 6 | 2013 330
Analysis of serial dilution of spike-in templates with dPCR To circumvent the sensitivity issues associated with single cell qRT-PCR, we used high- throughput nanocapillary qRT-PCR to record digital signals from mRNA isolated from a single cell in a dPCR setup. To verify sensitivity and dynamic range, we recorded the presence of individual, synthetic target molecules (s_rps18 having the amplicon sequence of rat rps18) in a serial dilution study. Synthetic rps18 template was diluted in parallel experiments (n = 2) to obtain 1–81 molecules in individual reaction mixes containing TaqMan probes for the rps18 gene. Te mixtures were partitioned evenly among hundreds of individual reactions and an absolute readout of total copy number was obtained aſter qRT-PCR in the OpenArray plate format. Te through-hole nanofluidic arrays on the OpenArray plates consist of 3200 subreactions in a 48 subarray format (12, 16). In our study, we divided the plates into 12 sections, each possessing 4 subarrays and 256 individual reactions with different numbers of rps18 template copies compared to a no template negative control. Aſter dPCR, pictures were generated using the OpenArray Digital Software (Life Technologies). We obtained good correlation between the theoret- ically calculated numbers of template molecules in the reaction mixture and the number of positive hits (Figure 1). Moderate differences at the lower end (less than nine molecules) of the dilution spectrum were likely caused by pipetting errors or variability in the spectro- photometric measurements used in the calcu- lations. More pronounced differences at the higher end (more than 27 molecules) of the
www.BioTechniques.com
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68