Pcr tube

For agent only phantoms, agents were diluted to the required concentration in PBS and the resulting solution was placed into 0.1 mL PCR tubes (Axygen). These tubes were then placed into a cylinder of Agar gel (2%) for imaging. For cell pellet phantoms, cells were washed three times in HBSS, harvested using Accutase, re-suspended in DMEM/F12 for counting and then mixed at the appropriate ratio if applicable before spinning down and re-suspending in a small volume of PBS for transfer into 0.1 mL PCR tubes. These were then spun down to produce a cell pellet, and tubes were placed into Agar as for solutions. In vitro imaging was carried out at room temperature. T₂w images were acquired using Fast Spin Echo Multi Slice (FSEMS: TR = 6.1 s, TE = 70.78 ms, n = 8, 30 × 30 mm FOV, 192 × 192 matrix, 0.5 mm slice thickness).

A total of 120 primers (100 RAPD and 20 ISSR) were used in PCR amplification. RAPD primers used in this study were selected from the study of Singh et al. (2004 (link)), while ISSR primers of UBC series were selected from the report of Aghaei et al. (2012 ). PCR amplification was carried out using 200 µl PCR tubes (Axygen, USA) in thermocyclers (Biometra, Germany). PCR amplification was carried out in a 25 μl reaction volume containing 2.5 μl template DNA (50 ng), 1× Dream Taq PCR buffer with MgCl₂ (Fermentas, USA), 0.4 μl (5 U/μl) Taq polymerase (Fermentas, USA), 0.5 μl (2.5 mM each) dNTPs (Fermentas, USA) and 1 μl (10 pmol/μl) primer (MWG Biotech, Germany). RAPD-PCR was performed at an initial denaturation at 94 °C for 5 min, 38 cycles of 94 °C for 1 min, 38 °C for 1 min, 72 °C for 1.2 min, and final extension at 72 °C for 5 min. The optimal annealing temperature for ISSR primers was found to vary according to the base composition of the primers. Therefore, ISSR-PCR was performed at an initial denaturation temperature of 94 °C for 5 min, 38 cycles of 94 °C for 50 s, 35–58 °C (depending on primer sequence) for 60 s and 72 °C for 1.2 min and a final extension of 72 °C for 10 min.

Dissociated cells from each slice were profiled using microwell-based single-cell RNA-seq [14 (link)] as previously described [9 (link), 16 (link)] with the following modifications: once the RNA-capture step was finished, sealing oil was flushed out of the devices by pipetting 1 mL of wash buffer supplemented with 0.04 U/μl RNase inhibitor (Thermo Fisher Scientific) and then beads were extracted from the device and resuspended in 200 μl of reverse transcription mixture. Bead-suspensions were divided into 50-μl aliquots and placed into PCR tubes (Corning) followed by incubation at 25°C for 30 min and at 42°C for 90 min in a thermocycler. Each cDNA library was barcoded with an Illumina sample index. Libraries with unique Illumina sample indices were pooled for sequencing on (1) an Illumina NextSeq 500 with an 8-base index read, a 21-base read 1 containing cell-identifying barcodes (CB) and unique molecular identifiers (UMIs), and a 63-base read 2 containing the transcript sequence, or (2) an Illumina NovaSeq 6000 with an 8-base index read, a 26-base read 1 containing CB and UMI, and a 91-base read 2 containing the transcript sequence.

Plasma samples were prepared for analysis by the protein precipitation method. An aliquot of 15 µL from each plasma sample was transferred to a PCR tube (Axygen, Union City, CA, USA). Four volumes of acetonitrile containing the analytical internal standard (IS) 4-methylumbelliferone were added and the resulting mixture was vortexed for 10 min on a Multi-Tube vortexer (VWR International, West Chester, PA, USA), and sonicated for 30 min at room temperature. The tube was centrifuged at 12,000× g for 10 min and the supernatant was analyzed for the test substance. Sample analysis was performed by 3200 Q TRAP LC-MS/MS system (Applied Biosystems, Concord, ON, Canada) in a negative MRM mode. The analytical methods are described below.

After each recording, cytoplasm was aspirated into the patch pipette by applying negative pressure, and expelled into a PCR tube (Axygen, Massachusetts) as previously described (Xu et al., 2015 (link)). The presence of mRNAs coding for ChAT, VGluT2, and VGAT was detected by single cell RT-PCR, according to the manufacturer’s instructions (Supplementary Table 1). Then, PCR products were visualized by Safe Gel-stained 1.5% agarose gel electrophoresis.